U.S. patent application number 10/008029 was filed with the patent office on 2003-07-17 for oligonucleotide analogues.
Invention is credited to Wengel, Jesper.
Application Number | 20030134808 10/008029 |
Document ID | / |
Family ID | 27574410 |
Filed Date | 2003-07-17 |
United States Patent
Application |
20030134808 |
Kind Code |
A1 |
Wengel, Jesper |
July 17, 2003 |
Oligonucleotide analogues
Abstract
Novel oligomers, and synthesis thereof, comprising one or more
bi-, tri, or polycyclic nucleoside analogues are disclosed herein.
The nucleoside analogues have a "locked" structure, termed Locked
Nucleoside Analogues (LNA). LNA's exhibit highly desirable and
useful properties. LNA's are capable of forming nucleobase specific
duplexes and triplexes with single and double stranded nucleic
acids. These complexes exhibit higher thermostability than the
corresponding complexes formed with normal nucleic acids. The
properties of LNA's allow for a wide range of uses such as
diagnostic agents and therapeutic agents in a mammal suffering from
or susceptible to, various diseases.
Inventors: |
Wengel, Jesper; (Odense,
DK) |
Correspondence
Address: |
Dike, Bronstein, Roberts & Cushman
Intellectual Property Practice Group
EDWARDS & ANGELL, LLP
P.O. Box 9169
Boston
MA
02209
US
|
Family ID: |
27574410 |
Appl. No.: |
10/008029 |
Filed: |
November 5, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10008029 |
Nov 5, 2001 |
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09152059 |
Sep 11, 1998 |
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60058541 |
Sep 12, 1997 |
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60068293 |
Dec 19, 1997 |
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60071682 |
Jan 16, 1998 |
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60076591 |
Mar 3, 1998 |
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60083507 |
Apr 29, 1998 |
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60088309 |
Jun 5, 1998 |
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60094355 |
Jul 28, 1998 |
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Current U.S.
Class: |
514/44R ;
514/263.21; 514/269; 514/81; 514/86; 536/23.1; 544/243;
544/244 |
Current CPC
Class: |
C07H 21/00 20130101 |
Class at
Publication: |
514/44 ;
514/263.21; 514/269; 514/81; 514/86; 536/23.1; 544/243;
544/244 |
International
Class: |
A61K 048/00; A61K
031/675; A61K 031/52; C07H 021/04; C07F 009/6512 |
Claims
1. An oligomer (hereinafter termed "LNA modified oligonucleotide")
comprising at least one nucleoside analogue (hereinafter termed
"LNA") of the general formula I 11wherein X is selected from --O--,
--S--, --N(R.sup.N+)--, --C(R.sup.6R.sup.6+)--,
--O--C(R.sup.7R.sup.7+)--, --C(R.sup.6R.sup.6+)--O--,
--S--C(R.sup.7R.sup.7+)--, --C(R.sup.6R.sup.6+)--S--,
--N(R.sup.N+)--C(R.sup.7R.sup.7+)--,
--C(R.sup.6R.sup.6+)--N(R.sup.N+), and
--C(R.sup.6R.sup.6+)--C(R.sup.7R.s- up.7+)--; B is selected from
hydrogen, hydroxy, optionally substituted C.sub.1-4-alkoxy,
optionally substituted C.sub.1-4-alkyl, optionally substituted
C.sub.1-4-acyloxy, nucleobases, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands; P designates the radical position for
an internucleoside linkage to a succeeding monomer, or a
5'-terminal group, such internucleoside linkage or 5'-terminal
group optionally including the substituent R.sup.5; one of the
substituents R.sup.2, R.sup.2+, R.sup.3, and R.sup.3+ is a group P*
which designates an internucleoside linkage to a preceding monomer,
or a 3'-terminal group; one or two pairs of non-geminal
substituents selected from the present substituents of R.sup.1+,
R.sup.4+, R.sup.5, R.sup.5+, R.sup.6, R.sup.6+, R.sup.7, R.sup.7+,
R.sup.N+, and the ones of R.sup.2, R.sup.2+, R.sup.3, and R.sup.3+
not designating P.sup.+ each designates a biradical consisting of
1-8 groups/atoms selected from --C(R.sup.aR.sup.b)--,
--C(R.sup.a).dbd.C(R.sup.a)--, --C(R.sup.a).dbd.N--, --O--,
--Si(R.sup.a).sub.2--, --S--, --SO.sub.2--, --N(R.sup.a)--, and
>C.dbd.Z, wherein Z is selected from --O--, --S--, and
--N(R.sup.a)--, and R.sup.a and R.sup.b each is independently
selected from hydrogen, optionally substituted C.sub.1-12-alkyl,
optionally substituted C.sub.2-12-alkenyl, optionally substituted
C.sub.2-12-alkynyl, hydroxy, C.sub.1-12-alkoxy,
C.sub.2-12-alkenyloxy, carboxy, C.sub.1-2-alkoxycarbonyl,
C.sub.1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,
arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,
heteroarylcarbonyl, amino, mono- and di(C.sub.1-6-alkyl)amino,
carbamoyl, mono- and di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted, and where two geminal substituents R.sup.a
and R.sup.b together may designate optionally substituted methylene
(.dbd.CH.sub.2), and wherein two non-geminal or geminal
substitutents selected from R.sup.a, R.sup.b, and any of the
substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.3+,
R.sup.4+, R.sup.5, R.sup.5+, R.sup.6 and R.sup.6+, R.sup.7, and
R.sup.7+ which are present and not involved in P, P.sup.+ or the
biradical(s) together may form an associated biradical selected
from biradicals of the same kind as defined before; said pair(s) of
non-geminal substituents thereby forming a mono- or bicyclic entity
together with (i) the atoms to which said non-geminal substituents
are bound and (ii) any intervening atoms; and each of the
substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.4+,
R.sup.5, R.sup.5+, R.sup.6 and R.sup.6+, R.sup.7, and R.sup.7+
which are present and not involved in P, P.sup.+ or the
biradical(s), is independently selected from hydrogen, optionally
substituted C.sub.1-12-alkyl, optionally substituted
C.sub.2-12-alkenyl, optionally substituted C.sub.2-12-alkynyl,
hydroxy, C.sub.1-12-alkoxy, C.sub.2-12-alkenyloxy, carboxy,
C.sub.1-12-alkoxycarbonyl, C.sub.1-12-alkylcarbonyl, formyl, aryl,
aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,
heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino,
mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted, and where two geminal substituents together
may designate oxo, thioxo, imino, or optionally substituted
methylene, or together may form a spiro biradical consisting of a
1-5 carbon atom(s) alkylene chain which is optionally interrupted
and/or terminated by one or more heteroatoms/groups selected from
--O--, --S--, and --(NR.sup.N) where R.sup.N is selected from
hydrogen and C.sub.1-4-alkyl, and where two adjacent (non-geminal)
substituents may designate an additional bond resulting in a double
bond; and R.sup.N+, when present and not involved in a biradical,
is selected from hydrogen and C.sub.1-4-alkyl; and basic salts and
acid addition salts thereof; with the proviso that, (i) R.sup.3 and
R.sup.5 do not together designate a biradical selected from
--CH.sub.2--CH.sub.2--, --O--CH.sub.2--, when LNA is a bicyclic
nucleoside analogue; (ii) R.sup.3, R.sup.5, and R.sup.5* do not
together designate a triradical --CH.sub.2--CH(--)--CH.sub.2-- when
LNA is a tricyclic nucleoside analogue; (iii) R.sup.1+ and R.sup.6+
do not together designate a biradical --CH.sub.2-- when LNA is a
bicyclic nucleoside analogue; and (iv) R.sup.4+ and R.sup.6+ do not
together designate a biradical --CH.sub.2-- when LNA is a bicyclic
nucleoside analogue.
2. An oligomer according to claim 1, wherein the one or two pairs
of non-geminal substituents, constituting one or two biradical(s),
respectively, are selected from the present substituents of
R.sup.1+, R.sup.4+, R.sup.6, R.sup.6+, R.sup.7, R.sup.7+, R.sup.N+,
and the ones of R.sup.2, R.sup.2+, R.sup.3, and R.sup.+ not
designating P.sup.+.
3. An oligomer according to claim 1, comprising 1-10000 LNA(s) of
the general formula I and 0-10000 nucleosides selected from
naturally occurring nucleosides and nucleoside analogues, with the
proviso that the sum of the number of nucleosides and the number of
LNA(s) is at least 2.
4. An oligomer according to claim 3, wherein at least one LNA
comprises a nucleobase as the substituent B.
5. An oligomer according to claim 1, wherein one of the
substituents R.sup.3 and R.sup.3+ designates P.sup.+.
6. An oligomer according to claim 1, wherein the LNA(s) has/have
the following formula Ia 12wherein P, P.sup.+, B, X, R.sup.1+,
R.sup.2, R.sup.2+, R.sup.3, R.sup.4+, R.sup.5, and R.sup.5+ are as
defined in claim 1.
7. An oligomer according to claim 6, wherein R.sup.3+ designates
P.sup.+.
8. An oligomer according to claim 1, comprising one biradical
constituted by a pair of (two) non-geminal substituents.
9. An oligomer according to claim 1, wherein X is selected from
--(CR.sup.6R.sup.6+)--, --O--, --S--, and --N(R.sup.N+)--.
10. An oligomer according to claim 1, wherein the biradical(s)
constituted by pair(s) of non-geminal substituents is/are selected
from --(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--Y--,
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--,
--Y--(CR.sup.+R.sup.+).sub.r--Y--(CR- .sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r+s--, --Y--, --Y--Y--, wherein each Y is
independently selected from --O--, --S--, --Si(R.sup.+).sub.2--,
--N(R.sup.+)--, >C.dbd.O, --C(.dbd.O), --N(R.sup.+)--, and
--N(R.sup.+)--C(.dbd.O)--, each R.sup.+ is independently selected
from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto,
amino, mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands,
and/or two adjacent (non-geminal) R.sup.+ may together designate a
double bond, and each of r and s is 0-4 with the proviso that the
sum r+s is 1-5.
11. An oligomer according to claim 10, wherein each biradical is
independently selected from --Y--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--, wherein and each of r and s is
0-3 with the proviso that the sum r+s is 1-4.
12. An oligomer according to claim 11, wherein i) R.sup.2+ and
R.sup.4+ together designate a biradical selected from --Y--,
--(CR.sup.+R.sup.30 ).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s, --Y--; (ii) R.sup.2 and R.sup.3
together designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+)--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; (iii) R.sup.2+ and R.sup.3
together designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; (iv) R.sup.3 and R.sup.4+
together designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; (v) R.sup.3 and R.sup.5
together designate a biradical selected from --Y'--,
--(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R- .sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; (vi) R.sup.1+ and R.sup.4+
together designate a biradical selected from --Y'--,
--(CR.sup.+R.sup.30 ).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.- +R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--NR.sup.+--; or (vii) R.sup.1.sup.+
and R.sup.2.sup.+ together designate a biradical selected from
--Y--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; wherein each of r and s is 0-3
with the proviso that the sum r+s is 1-4, and where Y' is selected
from --NR.sup.+--C(.dbd.O)-- and --C(.dbd.O)--NR.sup.+--.
13. An oligomer according to claim 12, wherein one of the following
criteria applies for at least one LNA: (i) R.sup.2.sup.+ and
R.sup.4.sup.+ together designate a biradical selected from --O--,
--S--, --N(R.sup.+)--, --(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.- r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.- sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).s- ub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O--, --S--(CR.sup.+R.sup.+).sub.r+-
s--O--, --O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.- +).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; (ii) R.sup.2 and
R.sup.3 together designate a biradical selected from --O--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.s- up.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
(iii) R.sup.2+ and R.sup.3 together designate a biradical selected
from --O--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.s- up.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.- s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(C.sup.+R.sup.+).sub.s--;
(iv) R.sup.3 and R.sup.4+ together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
(v) R.sup.3 and R.sup.5 together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
or (vi) R.sup.1+ and R.sup.4+ together designate a biradical
selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
(vii) R.sup.1+ and R.sup.2+ together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
wherein each of r and s is 0-3 with the proviso that the sum r+s is
1-4, and where X is selected from --O--, --S--, and --N(R.sup.H)
where R.sup.H designates hydrogen or C.sub.1-4-alkyl.
14. An oligomer according to claim 13, wherein R.sup.3+ designates
P.sup.+.
15. An oligomer according to claim 14, wherein R.sup.2+ and
R.sup.4+ together designate a biradical.
16. An oligomer according to claim 15, wherein X is 0, R.sup.2 is
selected from hydrogen, hydroxy, and optionally substituted
C.sub.1-6-alkoxy, and R.sup.1+, R.sup.3, R.sup.5, and R.sup.5+
designate hydrogen.
17. An oligomer according to claim 16, wherein the biradical is
selected from --O--, --(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3--.
18. An oligomer according to claim 17, wherein the biradical is
selected from --O--CH.sub.2--, --S--CH.sub.2-- and
--N(R.sup.N)--CH.sub.2--.
19. An oligomer according to claim 15, wherein B is selected from
nucleobases.
20. An oligomer according to claim 19, wherein the oligomer
comprises at least one LNA wherein B is selected from adenine and
guanine and at least one LNA wherein B is selected from thymine,
cytosine and urasil.
21. An oligomer according to claim 16, wherein the biradical is
--(CH.sub.2).sub.2-4--.
22. An oligomer according to claim 14, wherein R.sup.2 and R.sup.3
together designate a biradical.
23. An oligomer according to claim 22, wherein X is O, R.sup.2+ is
selected from hydrogen, hydroxy, and optionally substituted
C.sub.1-6-alkoxy, and R.sup.1+, R.sup.4+, R.sup.5, and R.sup.5+
designate hydrogen.
24. An oligomer according to claim 23, wherein the biradical is
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--.
25. An oligomer according to claim 23, wherein the biradical is
--(CH.sub.2).sub.1-4--.
26. An oligomer according to claim 14, wherein one R.sup.+ is
selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
any remaining substituents R are hydrogen.
27. An oligomer according to claim 14, wherein a group R.sup.+ in
the biradical of at least one LNA is selected from DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands.
28. An oligomer according to claim 14, wherein the LNA(s) has/have
the general formula Ia.
29. An oligomer according to claim 1 of the general formula Ia
13wherein X is --O--; B is selected from nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands; P
designates the radical position for an internucleoside linkage to a
succeeding monomer, or a 5'-terminal group, such internucleoside
linkage or 5'-terminal group optionally including the substituent
R.sup.5; R.sup.3+ is a group P* which designates an internucleoside
linkage to a preceding monomer, or a 3'-terminal group; R.sup.2+
and R.sup.4+ together designate a biradical selected from --O--,
--S--, --N(R.sup.+)--, --(CR.sup.+R.sup.+).sub.r+s+1- --,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--, --O--,
--S--(CR.sup.+R.sup.+).sub.r+s--- O--,
--O--(CR.sup.+R.sup.+).sub.r+s--S--, --N(R.sup.+)
--(CR.sup.+R.sup.+).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup- .+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+- ).sub.r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; wherein each R.sup.+
is independently selected from hydrogen, halogen, azido, cyano,
nitro, hydroxy, mercapto, amino, mono- or di(C.sub.1-6-alkyl)amino,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkyl, DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups,
and ligands, and/or two adjacent (non-geminal) R.sup.+ may together
designate a double bond, and each of r and s is 0-3 with the
proviso that the sum r+s is 1-4; each of the substituents R.sup.1+,
R.sup.2, R.sup.3, R.sup.5, and R.sup.5+ is independently selected
from hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, hydroxy, C.sub.1-6-alkoxy,
C.sub.2-6-alkenyloxy, carboxy, C.sub.1-6-alkoxycarbonyl- ,
C.sub.1-6-alkylcarbonyl, formyl, amino, mono- and
di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-- carbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, azido,
C.sub.1-6-alkanoyloxy, sulphono, sulphanyl, C.sub.1-6-alkylthio,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
halogen, where two geminal substituents together may designate oxo;
and basic salts and acid addition salts thereof.
30. An oligomer according to claim 29, wherein one R.sup.+ is
selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
any remaining substituents R.sup.+ are hydrogen.
31. An oligomer according to claim 29, wherein the biradical is
selected from --O--, (CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--N(R.- sup.N)--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.2-4--.
32. An oligomer according to claim 31, wherein the biradical is
selected from --O--CH.sub.2--, --S--CH.sub.2-- and
--N(R.sup.N)--CH.sub.2--.
33. An oligomer according to claim 29, wherein B is selected from
nucleobases.
34. An oligomer according to claim 33, wherein the oligomer
comprises at least one LNA wherein B is selected from adenine and
guanine and at least one LNA wherein B is selected from thymine,
cytosine and urasil.
35. An oligomer according to claim 29, wherein R.sup.2 is selected
from hydrogen, hydroxy and optionally substituted C.sub.1-6-alkoxy,
and R.sup.1+, R.sup.3, R.sup.5, and R.sup.5+ designate
hydrogen.
36. An oligomer according to claim 1, wherein any internucleoside
linkage of the LNA(s) is selected from linkages consisting of 2 to
4 groups/atoms selected from --CH.sub.2--, --O--, --S--,
--NR.sup.H--, >C.dbd.O, >C.dbd.NR.sup.H, >C.dbd.S,
--Si(R").sub.2--, --SO--, --S(O).sub.2--, --P(O).sub.2--,
--P(O,S)--, --P(S).sub.2--, --PO(R")--, --PO(OCH.sub.3)--, and
--PO(NHR.sup.H)--, where R.sup.H is selected form hydrogen and
C.sub.1-4-alkyl, and R" is selected from C.sub.1-6-alkyl and
phenyl.
37. An oligomer according to claim 36, wherein any internucleoside
linkage of the LNA(s) is selected from
--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CO--CH.sub.2--,
--CH.sub.2--CHOH--CH.sub.2--, --O--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2--, --O--CH.sub.2--CH.dbd.,
--CH.sub.2--CH.sub.2--O--, --NR.sup.H--CH.sub.2--CH.sub.2--,
--CH.sub.2--CH.sub.2--NR.sup.H--, --CH.sub.2--NR.sup.H--CH.sub.2--,
--O--CH.sub.2--CH.sub.2--NR.sup.H--, --NR.sup.H--CO--O--,
--NR.sup.H--CO--NR.sup.H--, --NR.sup.H--CS--NR.sup.H--,
--NR.sup.H--C(.dbd.NR.sup.H)--NR.sup.H--,
NR.sup.H--CO--CH.sub.2--NR.sup.- H--, --O--CO--O--,
--O--CO--CH.sub.2--O--, --O--CH.sub.2--CO--O--,
--CH.sub.2--CO--NR.sup.H--, --O--CO--NR.sup.H--,
--NR.sup.HCO--CH.sub.2--- , --O--CH.sub.2--CO--NR.sup.H--,
--O--CH.sub.2--CH.sub.2--NR.sup.H--, --CH.dbd.N--O--,
--CH.sub.2--NR.sup.H--O--, --CH.sub.2--O--N.dbd.,
--CH.sub.2--O--NR.sup.H--, --CO--NR.sup.H--CH.sub.2--,
--CH.sub.2--NR.sup.H--O--, --CH.sub.2--NR.sup.H--CO--,
--O--NR.sup.H--CH.sub.2--, --O--NR.sup.H--, --O--CH.sub.2--S--,
--S--CH.sub.2--O--, --CH.sub.2--CH.sub.2--S--,
--O--CH.sub.2--CH.sub.2--S- --, --S--CH.sub.2--CH.dbd.,
--S--CH.sub.2--CH.sub.2--, --S--CH.sub.2--CH.sub.2--O--,
--S--CH.sub.2--CH.sub.2--S--, --CH.sub.2--S--CH.sub.2--,
--CH.sub.2--SO--CH.sub.2--, --CH.sub.2--SO.sub.2--CH.sub.2--,
--O--SO--O--, --O--S(O).sub.2--O--, --O--S(O).sub.2--CH.sub.2--,
--O--S(O).sub.2--NR.sup.H--, --NR.sup.H--S(O).sub.2--CH.sub.2--,
--O--S(O).sub.2--CH.sub.2--, --O--P(O).sub.2--O--,
--O--P(O,S)--O--, --O--P(S).sub.2--O--, --S--P(O).sub.2--O--,
--S--P(O,S)--O--, --S--P(S).sub.2--O--, --O--P(O).sub.2--S--,
--O--P(O,S)--S--, --O--P(S).sub.2--S--, --S--P(O).sub.2--S--,
--S--P(O,S)--S--, --S--P(S).sub.2--S--, --O--PO(R")--O--,
--O--PO(OCH.sub.3)--O--, --O--PO(BH.sub.3)--O--,
--O--PO(NHR.sup.N)--O--, --O--P(O).sub.2--NR.sup.H--,
--NR.sup.H--P(O).sub.2--O--, --O--P(O,NR.sup.H)--O--, and
--O--Si(R").sub.2--O--.
38. An oligomer according to claim 37, wherein any internucleoside
linkage of the LNA(s) is selected from --CH.sub.2--CO--NR.sup.H--,
--CH.sub.2--NR.sup.H--O--, --S--CH.sub.2--O--,
--O--P(O).sub.2--O--, --O--P(O,S)--O--, --O--P(S).sub.2--O--,
--NR.sup.H--P(O).sub.2--O--, --O--P(O,NR.sup.H)--O--,
--O--PO(R")--O--, --O--PO(CH.sub.3)--O--, and
--O--PO(NHR.sup.N)--O--, where R.sup.H is selected form hydrogen
and C.sub.1-4-alkyl, and R" is selected from C.sub.1-6-alkyl and
phenyl.
39. An oligomer according to claim 1, wherein each of the
substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.3+,
R.sup.4+, R.sup.5, R.sup.5+, R.sup.6, R.sup.6+, R.sup.7, and
R.sup.7+ of the LNA(s), which are present and not involved in P,
P.sup.+ or the biradical(s), is independently selected from
hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, hydroxy, C.sub.1-6-alkoxy,
C.sub.2-6-alkenyloxy, carboxy, C.sub.1-6-alkoxycarbonyl- ,
C.sub.1-6-alkylcarbonyl, formyl, amino, mono- and
di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-- carbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, azido,
C.sub.1-6-alkanoyloxy, sulphono, sulphanyl, C.sub.1-6-alkylthio,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
halogen, where two geminal substituents together may designate oxo,
and where R.sup.N+, when present and not involved in a biradical,
is selected from hydrogen and C.sub.1-4-alkyl.
40. An oligomer according to claim 1, wherein X is selected from
--O--, --S--, and --NR.sup.N+, and each of the substituents
R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.3+, R.sup.4+, R.sup.5,
R.sup.5+, R.sup.6, R.sup.6+, R.sup.7, and R.sup.7+ of the LNA(s),
which are present and not involved in P, P.sup.+ or the
biradical(s), designate hydrogen.
41. An oligomer according to claim 1, wherein P is a 5'-terminal
group selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkyl, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkylcarbonyloxy, optionally
substituted aryloxy, monophosphate, diphosphate, triphosphate, and
--W--A', wherein W is selected from --O--, --S--, and
--N(R.sup.H)-- where R.sup.H is selected from hydrogen and
C.sub.1-6-alkyl, and where A' is selected from DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands.
42. An oligomer according to claim 1, wherein P* is a 3'-terminal
group selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkylcarbonyloxy, optionally substituted aryloxy, and
--W--A', wherein W is selected from --O--, --S--, and
--N(R.sup.H)-- where R.sup.H is selected from hydrogen and
C.sub.1-6-alkyl, and where A' is selected from DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands.
43. An oligomer according to claim 1, having the following formula
V:G--[Nu--L].sub.n{0}--{[LNA--L].sub.m(q)--[Nu--L].sub.n(q)}.sub.q--G*
Vwherein q is 1-50; each of n(0), . . . , n(q) is independently
0-10000; each of m(1), . . . , m(q) is independently 1-10000; with
the proviso that the sum of n(0),. . . , n(q) and m(1), . . . ,
m(q) is 2-15000; G designates a 5'-terminal group; each Nu
independently designates a nucleoside selected from naturally
occurring nucleosides and nucleoside analogues; each LNA
independently designates a nucleoside analogue; each L
independently designates an internuc:leoside linkage between two
groups selected from Nu and LNA, or L together with G* designates a
3'-terminal group; and each LNA--L independently designates a
nucleoside analogue of the general formula I: 14wherein the
substituents B, P, P.sup.+, R.sup.1+, R.sup.2, R.sup.2+, R.sup.3,
R.sup.4+, R.sup.5, and R.sup.5+, and X are as defined in claim
1.
44. An oligomer according to claim 1, further comprising a PNA
mono- or oligomer segment of the formula 15wherein B is a defined
above for the formula I, AASC designates hydrogen or an amino acid
side chain, t is 1-5, and w is 1-50.
45. An oligomer according to claim 1, which has an increased
specificity towards complementary ssRNA or ssDNA compared to the
native oligonucleotide.
46. An oligomer according to claim 1, which has an increased
affinity towards complementary ssRNA or ssDNA compared to the
native oligonucleotide.
47. An oligomer according to claim 1, which is capable of binding
to a target sequence in a dsDNA or dsRNA molecule by way of "strand
displacement" or by triple helix formation.
48. An oligomer according to claim 1, which is more resistant to
nucleases than the native oligonucleotide.
49. An oligomer according to claim 1, which has nucleic acid
catalytic activity (LNA modified ribozymes).
50. An oligomer comprising at least one nucleoside analogue which
imparts to the oligomer a T.sub.m with a complementary DNA
oligonucleotide which is at least 2.5.degree. C. higher than that
of the corresponding unmodified reference oligonucleotide which
does not comprise any nucleoside analogue.
51. An oligomer according to claim 50, wherein the T.sub.m is at
least 2.5.times.N .degree.C. higher, where N is the number of
nucleoside analogues.
52. An oligomer comprising at least one nucleoside analogue which
imparts to the oligomer a T.sub.m with a complementary RNA
oligonucleotide which is at least 4.0.degree. C. higher than that
of the corresponding unmodified reference oligonucleotide which
does not comprise any nucleoside analogue.
53. An oligomer according to claim 52, wherein the T.sub.m is at
least 4.0.times.N .degree.C. higher, where N is the number of
nucleoside analogues.
54. An oligomer according to claim 50 or 52, wherein the oligomer
is as defined in claim 1, where the at least one nucleoside
analogue has the formula I where B is a nucleobase.
55. An oligomer according to claim 50, wherein said oligomer, when
hybridised with a partially complementary DNA oligonucleotide
having one or more mismatches with said oligomer, exhibits a
reduction in T.sub.m, as a result of said mismatches, which is
equal to or greater than the reduction which would be observed with
the corresponding unmodified reference oligonucleotide which does
not comprise any nucleoside analogues.
56. An oligomer according to claim 52, wherein said oligomer, when
hybridised with a partially complementary RNA oligonucleotide
having one or more mismatches with said oligomer, exhibits a
reduction in T.sub.m, as a result of said mismatches, which is
equal to or greater than the reduction which would be observed with
the corresponding unmodified reference oligonucleotide which does
not comprise any nucleoside analogues.
57. An oligomer according to claim 50 or 52, which has
substantially the same sensitivity of T.sub.m to the ionic strength
of the hybridisation buffer as that of the corresponding unmodified
reference oligonucleotide.
58. An oligomer according to claim 50 or 52, which is at least 30%
modified.
59. An oligomer according to claim 50 or 52, which has
substantially higher 3'-exonucleolytic stability than the
corresponding unmodified reference oligonucleotide.
60. A nucleoside analogue (hereinafter LNA) of the general formula
II 16wherein the substituent B is selected from nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands; X is
selected from --O--, --S--, --N(R.sup.N+), and
--C(R.sup.6R.sup.6+)--; one of the substituents R.sup.2, R.sup.2+,
R.sup.3, and R.sup.3+is a group Q*; each of Q and Q* is
independently selected from hydrogen, azido, halogen, cyano, nitro,
hydroxy, Prot-O--, Act-O--, mercapto, Prot-S--, Act-S--,
C.sub.1-6-alkylthio, amino, Prot-N(R.sup.H)--, Act-N(R.sup.H)--,
mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, optionally substituted
C.sub.2-6-alkenyloxy, optionally substituted C.sub.2-6-alkynyl,
optionally substituted C.sub.2-6-alkynyloxy, monophosphate,
diphosphate, triphosphate, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, ligands, carboxy, sulphono, hydroxymethyl,
Prot-O--CH.sub.2--, Act-O--CH.sub.2--, aminomethyl,
Prot-N(R.sup.H)--CH.sub.2--, Act-N(R.sup.H)--CH.sub.2--,
carboxymethyl, sulphonomethyl, where Prot is a protection group for
--OH, --SH, and --NH(R.sup.H), respectively, Act is an activation
group for --OH, --SH, and --NH(R.sup.H), respectively, and R.sup.H
is selected from hydrogen and C.sub.1-6-alkyl; (i) R.sup.2+ and
R.sup.4+ together designate a biradical selected from --O--,
--(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O--,
--S--(CR.sup.+R.sup.+).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; (ii) R.sup.2 and
R.sup.3 together designate a biradical selected from --O--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.s- up.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
(iii) R.sup.2+ and R.sup.3 together designate a biradical selected
from --O--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.s- up.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.- s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
(iv) R.sup.3 and R.sup.4+ together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
(v) R.sup.3 and R.sup.5 together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
or (vi) R.sup.1+ and R.sup.4+ together designate a biradical
selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.s--; (vii) R.sup.1+ and R.sup.2+ together
designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup- .+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.r-- -, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
wherein each R.sup.+ is independently selected from hydrogen,
halogen, azido, cyano, nitro, hydroxy, mercapto, amino, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands, and/or two adjacent
(non-geminal) R.sup.+ may together designate a double bond, and
each of r and s is 0-3 with the proviso that the sum r+s is 1-4;
each of the substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3,
R.sup.4+, R.sup.5, and R.sup.5+, which are not involved in Q,
Q.sup.+ or the biradical, is independently selected from hydrogen,
optionally substituted C.sub.1-12-alkyl, optionally substituted
C.sub.2-12-alkenyl, optionally substituted C.sub.2-12-alkynyl,
hydroxy, C.sub.1-12-alkoxy, C.sub.2-12-alkenyloxy, carboxy,
C.sub.1-12-alkoxycarbonyl, C.sub.1-12-alkylcarbonyl, formyl, aryl,
aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,
heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino,
mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-16-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted, and where two geminal substituents together
may designate oxo, thioxo, imino, or optionally substituted
methylene, or together may form a spiro biradical consisting of a
1-5 carbon atom(s) alkylene chain which is optionally interrupted
and/or terminated by one or more heteroatoms/groups selected from
--O--, --S--, and --(NR.sup.N) where R.sup.N is selected from
hydrogen and C.sub.1-4-alkyl, and where two adjacent (non-geminal)
substituents may designate an additional bond resulting in a double
bond; and R.sup.N+, when present and not involved in a biradical,
is selected from hydrogen and C.sub.1-4-alkyl; and basic salts and
acid addition salts thereof; with the first proviso that, (i)
R.sup.3 and R.sup.5 do not together designate a biradical selected
from --CH.sub.2--CH.sub.2--, --O--CH.sub.2--, and
--O--Si(.sup.iPr).sub.2--O--- Si(.sup.iPr).sub.2--O--; and with the
second proviso that any chemical group (including any nucleobase),
which is reactive under the conditions prevailing in
oligonucleotide synthesis, is optionally functional group
protected.
61. A nucleoside analogue according to claim 60, wherein the group
B is selected from nucleobases and functional group protected
nucleobases.
62. A nucleoside analogue according to claim 60, wherein X is
selected from --O--, --S--, and --N(R.sup.n+).
63. A nucleoside analogue according to claim 60, wherein each of
the substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.3+,
R.sup.4+, R.sup.5, and R.sup.5+, which are present and not involved
in Q, Q.sup.+ or the biradical, is independently selected from
hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, hydroxy, C.sub.1-6-alkoxy,
C.sub.2-6-alkenyloxy, carboxy, C.sub.1-6-alkoxycarbonyl,
C.sub.1-6-alkylcarbonyl, formyl, amino, mono- and
di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-carbonyl, C.sub.1-6-alkyl-carbonylamino,
carbamido, azido, C.sub.1-6-alkanoyloxy, sulphono, sulphanyl,
C.sub.1-6-alkylthio, DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups, ligands, and halogen, where two geminal substituents
together may designate oxo, and where R.sup.N+, when present and
not involved in a biradical, is selected from hydrogen and
C.sub.1-4-alkyl, with the proviso that any hydroxy, amino,
mono(C.sub.1-6-alkyl)amino, sulfanyl, and carboxy is optionally
protected.
64. A nucleotide analogue according to claim 60, each of the
substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.3+,
R.sup.4+, and R.sup.5, R.sup.5+, R.sup.6, R.sup.6+, which are
present and not involved in Q.sup.+ or the biradical, designate
hydrogen.
65. A nucleoside analogue according to claim 60, wherein R.sup.3+
designates P.sup.+.
66. A nucleoside analogue according to claim 60, wherein Q is
independently selected from hydrogen, azido, halogen, cyano, nitro,
hydroxy, Prot-O--, mercapto, Prot-S--, C.sub.1-6-alkylthio, amino,
Prot-N(R.sup.H)--, mono- or di(C.sub.1-6-alkyl)amino, optionally
substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkyl, optionally substituted C.sub.2-6-alkenyl,
optionally substituted C.sub.2-6-alkenyloxy, optionally substituted
C.sub.2-6-alkynyl, optionally substituted C.sub.2-6-alkynyloxy,
monophosphate, diphosphate, triphosphate, DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, ligands, carboxy, sulphono,
hydroxymethyl, Prot-O--CH.sub.2--, aminomethyl,
Prot-N(R.sup.H)--CH.sub.2--, carboxymethyl, sulphonomethyl, where
Prot is a protection group for --OH, --SH, and --NH(R.sup.H),
respectively, and R.sup.H is selected from hydrogen and
C.sub.1-6-alkyl; and Q.sup.+ is selected from hydrogen, azido,
halogen, cyano, nitro, hydroxy, Act-O--, mercapto, Act-S--,
C.sub.1-6-alkylthio, amino, Act-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups, ligands, carboxy, sulphono, where Act is an activation
group for --OH, --SH, and --NH(R.sup.H), respectively, and R.sup.H
is selected from hydrogen and C.sub.1-6-alkyl.
67. A nucleotide analogue according to claim 60, having the general
formula IIa 17wherein the substituents Q, B, R.sup.1+, R.sup.2,
R.sup.2+, R.sup.3, R.sup.3+, R.sup.4+, R.sup.5, and R.sup.5+ are as
defined in claim 60.
68. A nucleoside analogue according to claim 67, wherein R.sup.3+
designates P.sup.+.
69. A nucleoside analogue according to claim 68, wherein R.sup.2+
and R.sup.4+ together designate a biradical.
70. A nucleoside analogue according to claim 69, wherein X is O,
R.sup.2 selected from hydrogen, hydroxy, and optionally substituted
C.sub.1-6-alkoxy, and R.sup.1+, R.sup.3, R.sup.5, and R.sup.5+
designate hydrogen.
71. A nucleoside analogue according to claim 70, wherein the
biradical is selected from --O--,
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3--.
72. A nucleoside analogue according to claim 71, wherein the
biradical is selected from --O--CH.sub.2--, --S--CH.sub.2-- and
--N(R.sup.N)--CH.sub.2--.
73. A nucleoside analogue according to claim 69, wherein B is
selected from nucleobases.
74. A nucleoside analogue according to claim 73, wherein the
oligomer comprises at least one LNA wherein B is selected from
adenine and guanine and at least one LNA wherein B is selected from
thymine, cytosine and urasil.
75. A nucleoside analogue according to claim 70, wherein the
biradical is --(CH.sub.2).sub.2-4--.
76. A nucleoside analogue according to claim 68, wherein R.sup.2
and R.sup.3 together designate a biradical.
77. A nucleoside analogue according to claim 76, wherein X is O,
R.sup.2+ is selected from hydrogen, hydroxy, and optionally
substituted C.sub.1-6-alkoxy, and R.sup.1+, R.sup.4+, R.sup.5, and
R.sup.5+ designate hydrogen.
78. A nucleoside analogue according to claim 77, wherein the
biradical is --(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--.
79. A nucleoside analogue according to claim 77, wherein the
biradical is --(CH.sub.2).sub.1-4--.
80. A nucleoside analogue according to claim 68, wherein one
R.sup.+ is selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
any remaining substituents R.sup.+ are hydrogen.
81. A nucleoside analogue according to claim 68, wherein a group
R.sup.+ in the biradical of at least one LNA is selected from DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands.
82. A nucleoside analogue according to claim 68, wherein the LNA(s)
has/have the general formula Ia.
83. A nucleoside analogue according to claim 60 of the general
formula IIa 18wherein X is --O--; B is selected from nucleobases,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands;
R.sup.3+ is a group Q*; each of Q and Q.sup.+ is independently
selected from hydrogen, azido, halogen, cyano, nitro, hydroxy,
Prot-O--, Act-O--, mercapto, Prot-S--, Act-S--,
C.sub.1-6-alkylthio, amino, Prot-N(R.sup.H)--, Act-N(R.sup.H)--,
mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, optionally substituted
C.sub.2-6-alkenyloxy, optionally substituted C.sub.2-6-alkynyl,
optionally substituted C.sub.2-6-alkynyloxy, monophosphate,
diphosphate, triphosphate, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, ligands, carboxy, sulphono, hydroxymethyl,
Prot-O--CH.sub.2--, Act-O--CH.sub.2--, aminomethyl,
Prot-N(R.sup.H)--CH.sub.2--, Act-N(R.sup.H)--CH.sub.2--,
carboxymethyl, sulphonomethyl, where Prot is a protection group for
--OH, --SH, and --NH(R.sup.H), respectively, Act is an activation
group for --OH, --SH, and --NH(R.sup.H), respectively, and R.sup.H
is selected from hydrogen and C.sub.1-6-alkyl; R.sup.2+ and
R.sup.4+ together designate a biradical selected from --O--, --S,
--N(R.sup.+)--, --(CR.sup.+R.sup.+).sub.r+s+1--- ,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O--,
--S--(CR.sup.+R.sup.+).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; wherein each R.sup.+
is independently selected from hydrogen, halogen, azido, cyano,
nitro, hydroxy, mercapto, amino, mono- or di(C.sub.1-6-alkyl)amino,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkyl, DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups,
and ligands, and/or two adjacent (non-geminal) R.sup.+ may together
designate a double bond, and each of r and s is 0-3 with the
proviso that the sum r+s is 1-4; each of the substituents R.sup.1+,
R.sup.2, R.sup.3, R.sup.5, and R.sup.5+ is independently selected
from hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, hydroxy, C.sub.1-6-alkoxy,
C.sub.2-6-alkenyloxy, carboxy, C.sub.1-6-alkoxycarbonyl- ,
C.sub.1-6-alkylcarbonyl, formyl, amino, mono- and
di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-- carbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, azido,
C.sub.1-6-alkanoyloxy, sulphono, sulphanyl, C.sub.1-6-alkylthio,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
halogen, where two geminal substituents together may designate oxo;
and basic salts and acid addition salts thereof; and with the
proviso that any chemical group (including any nucleobase), which
is reactive under the conditions prevailing in oligonucleotide
synthesis, is optionally functional group protected.
84. A nucleotide analogue according to claim 83, wherein one
R.sup.+ is selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
any remaining substituents R.sup.+ are hydrogen.
85. A nucleotide analogue according to claim 83, wherein the
biradical is selected from --O--,
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--N(R.- sup.N)--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.2-4--.
86. A nucleoside analogue according to claim 85, wherein the
biradical is selected from --O--CH.sub.2--, --S--CH.sub.2-- and
--N(R.sup.N)--CH.sub.2--.
87. A nucleoside analogue according to claim 83, wherein B is
selected from nucleobases.
88. A nucleoside analogue according to claim 87, wherein the
oligomer comprises at least one LNA wherein B is selected from
adenine and guanine and at least one LNA wherein B is selected from
thymine, cytosine and urasil.
89. A nucleoside analogue according to claim 83, wherein B
designates a nucleobase, X is --O--, R.sup.2+ and R.sup.4+ together
designate a biradical selected from
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3-- where
R.sup.N is selected from hydrogen and C.sub.1-4-alkyl, Q designates
Prot-O--, R.sup.3+ is Q.sup.+ which designates Act-OH, and
R.sup.1+, R.sup.2, R.sup.3, R.sup.5, and R.sup.5+ each designate
hydrogen, wherein Act and Prot are as defined in claim 58.
90. A nucleoside analogue according to claim 83, wherein B
designates a nucleobase, X is --O--, R.sup.2+ and R.sup.4+ together
designate a biradical selected from
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3-- where
R.sup.N is selected from hydrogen and C.sub.1-4-alkyl, Q is
selected from hydroxy, mercapto, C.sub.1-6-alkylthio, amino, mono-
or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyloxy, monophosphate,
diphosphate, and triphosphate, R.sup.3+ is Q.sup.+ which is
selected from hydrogen, azido, halogen, cyano, nitro, hydroxy,
mercapto, C.sub.1-6-alkylthio, amino, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, and optionally
substituted C.sub.2-6-alkynyloxy, R.sup.3 is selected from
hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, and optionally substituted
C.sub.2-6-alkynyl, and R.sup.1+, R.sup.2, R.sup.5, and R.sup.5+
each designate hydrogen.
91. A nucleoside analogue according to claim 83, wherein B
designates a nucleobase, X is --O--, R.sup.2 and R.sup.3 together
designate a biradical selected from
--(CH.sub.2).sub.0-1--O--CH.dbd.CH--,
--(CH.sub.2).sub.0-1--S--CH.dbd.CH--, and
--(CH.sub.2).sub.0-1--N(R.sup.N- )--CH.dbd.CH-- where R.sup.N is
selected from hydrogen and C.sub.1-4-alkyl, Q is selected from
hydroxy, mercapto, C.sub.1-6-alkylthio, amino, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.2-6-alkenyloxy, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate, diphosphate, and triphosphate,
R.sup.3+ is Q.sup.+ which is selected from hydrogen, azido,
halogen, cyano, nitro, hydroxy, mercapto, C.sub.1-6-alkylthio,
amino, mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, optionally substituted
C.sub.2-6-alkenyloxy, optionally substituted C.sub.2-6-alkynyl, and
optionally substituted C.sub.2-6-alkynyloxy, and R.sup.1+,
R.sup.2+, R.sup.4+, R.sup.5, and R.sup.5+ each designate
hydrogen.
92. A nucleoside analogue according to claim 60, which is selected
from
(1R,3R,4R,7S)-7-(2-cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4'-dime-
thoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,
(1R,3R,4R,7S)-7-hydroxy-1-(4,4'-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-
-2,5-dioxabicyclo[2.2.1]heptane-7-O-(2-chlorophenylphosphate), and
(1R,3R,4R,7S)-7-hydroxy-1-(4,4'-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-
-2,5-dioxabicyclo[2.2.1]heptane-7-O-(H-phosphonate) and the
3-(cytosin-1-yl), 3-(urasil-1-yl), 3-(adenin-1-yl) and
3-(guanin-1-yl) analogues thereof
93. A method of using an LNA as defined in claim 60 for the
preparation of an LNA modified oligonucleotide (an oligomer) as
defined in claim 1.
94. A method according to claim 93, wherein the LNA modified
oligonucleotide comprises normal nucleosides as well as modified
nucleosides different from those defined in claim 60.
95. A method according to claim 93, wherein the incorporation of
LNA modulates the ability of the oligonucleotide to act as a
substrate for nucleic acid active enzymes.
96. A method of using of an LNA as defined in claim 60 for the
preparation of a conjugate of an LNA modified oligonucleotide and a
compound selected from proteins, amplicons, enzymes,
polysaccharides, antibodies, haptens, peptides, and PNA.
97. A conjugate of an LNA modified oligonucleotide (an oligomer) as
defined in claim 1 and a compound selected from proteins,
amplicons, enzymes, polysaccharides, antibodies, haptens, peptides,
and PNA.
98. A method of using an LNA as defined in claim 60 as a substrate
for enzymes active on nucleic acids.
99. A method according to claim 98, wherein the substituent Q in
the formula I in claim 60 designates a triphosphate,
100. A method according to claim 98, wherein the LNA is used as a
substrate for DNA and RNA polymerases.
101. A method of using an LNA as defined in claim 60 as a
therapeutic agent.
102. A method of using an LNA as defined in claim 60 for diagnostic
purposes.
103. A solid support material having immobilised thereto an
optionally nucleobase protected and optionally 5'-OH protected
LNA.
104. A method of using one or more LNA as defined in claim 60 in
the construction of solid surface onto which LNA modified
oligonucleotides of different sequences are attached.
105. A method according to claim 113, wherein the LNA modified
oligonucleotides are attached in a predetermined pattern.
106. A method according to claim 113, wherein the LNAs are used to
equalise the T.sub.m of the corresponding unmodified reference
oligonucleotides.
107. A method according to claim 113, wherein the LNA modified
oligonucleotides have an increased affinity toward complementary
ssDNA or ssRNA compared to native oligonucleotide.
108. A method according to claim 113, wherein the LNA modified
oligonucleotides have an increased specificity toward complementary
ssDNA or ssRNA compared to native oligonucleotide.
109. A method of using LNA modified oligomers (ribozymes) as
defined in claim 1 in the sequence specific cleavage of target
nucleic acids.
110. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 in therapy.
111. A method according to claim 110, wherein the LNA modified
oligonucleotide recruits RNAseH.
112. A method of using complexes of more than one LNA modified
oligonucleotide (an oligomer) as defined in claim 1 in therapy.
113. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 as an aptamer in therapeutic
applications.
114. A method according to claim 119 , wherein the LNA modified
oligonucleotide comprises at least one internucleoside linkage not
being a phosphate diester linkage.
115. A method of using an LNA modified oli(gonucleotide (an
oligomer) as defined in claim 1 in diagnostics.
116. A method according to claim 115, wherein the oligonucleotide
comprises a photochemically active group, a thermochemically active
group, a chelating group, a reporter group, or a ligand that
facilitates the direct of indirect detection of the oligonucleotide
or the immobilisation of the oligonucleotide onto a solid
support.
117. A method according to claim 116, wherein the photochemically
active group, the thermochemically active group, the chelating
group, the reporter group, or the ligand includes a spacer (K),
said spacer comprising a chemically cleavable group.
118. A method according to claim 116, wherein the photochemically
active group, the thermochemically active group, the chelating
group, the reporter group, or the ligand is attached via the
biradical (i.e. as R.sup.+) of at least one of the LNA(s) of the
oligonucleotide.
119. A method according to claim 115 for capture and detection of
naturally occurring or synthetic double stranded or single stranded
nucleic acids.
120. A method according to claim 115 for purification of naturally
occurring double stranded or single stranded nucleic acids.
121. A method according to claim 115 as a probe in in-situ
hybridisation, in Southern hydridisation, Dot blot hybridisation,
reverse Dot blot hybridisation, or in Northern hybridisation.
122. A method according to claim 115 in the construction of an
affinity pair.
123. A method according to claim 115 as a primer in a nucleic acid
sequencing reaction or primer extension reactions.
124. A method according to claim 115 as a primer in a nucleic acid
amplification reaction.
125. A method according to claim 124, wherein the primer is so
adapted that the amplification reaction is an essentially linear
reaction.
126. A method according to claim 124, wherein the primer is so
adapted that the amplification reaction is an essentially
exponential reaction.
127. A method according to claim 124, wherein the nucleic acid
amplification reaction results in a double stranded DNA product
comprising at least one single stranded end.
128. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 as an aptamer in molecular
diagnostics.
129. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 as an aptamer in RNA mediated
catalytic processes.
130. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 as an aptamer in specific binding
of antibiotics, drugs, amino acids, peptides, structural proteins,
protein receptors, protein enzymes, saccharides, polysaccharides,
biological cofactors, nucleic acids, or triphosphates.
131. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 as an aptamer in the separation of
enantiomers from racemic mixtures by stereospecific binding.
132. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 for the labelling of cells.
133. A method according to claim 132, wherein the label allows the
cells to be separated from unlabelled cells.
134. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 to hybridise to non-protein coding
cellular RNAs in vivo or in-vitro.
135. A method of using an LNA modified oli(gonucleotide (an
oligomer) as defined in claim 1 in the construction of an
oligonucleoltide containing a fluorophor and a quencher, positioned
in such a way that the hybridised state of the oligonucleotide can
be distinguished from the unbound state of the oligonucleotide by
an increase in the fluorescent signal from the probe.
136. A method of using an LNA modified oligonucleotide (an
oligomer) as defined in claim 1 in the construction of Taqman
probes or Molecular Beacons.
137. A kit for the isolation, purification, amplification,
detection, identification, quantification, or capture of natural or
synthetic nucleic acids, the kit comprising a reaction body and one
or more LNA modified oligonucleotides (oligomer) as defined in
claim 1.
138. A kit according to claim 137, wherein the LNA modified
oligonucleotides are immobilised onto said reactions body.
139. A kit for the isolation, purification, amplification,
detection, identification, quantification, or capture of natural or
synthetic nucleic acids, the kit comprising a reaction body and one
or more LNAs as defined in claim 60.
140. A kit according to claim 139, wherein the LNAs are immobilised
onto said reactions body.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of bi- and
tricyclic nucleoside analogues and to the synthesis of such
nucleoside analogues which are useful in the formation of synthetic
oligonucleotides capable of forming nucleobase specific duplexes
and triplexes with single stranded and double stranded nucleic
acids. These complexes exhibit higher thermostability than the
corresponding complexes formed with normal nucleic acids. The
invention also relates to the field of bi- and tricyclic nucleoside
analogues and the synthesis of such nucleosides which may be used
as therapeutic drugs and which may be incorporated in
oligonucleotides by template dependent nucleic acid
polymerases.
BACKGROUND OF THE INVENTION
[0002] Synthetic oligonucleotides are widely used compounds in
disparate fields such as molecular biology and DNA-based
diagnostics and therapeutics.
[0003] Therapeutics
[0004] In therapeutics, e.g., oligonucleotides have been used
successfully to block translation in vivo of specific mRNAs thereby
preventing the synthesis of proteins which are undesired or harmful
to the cell/organism. This concept of oligonucleotide mediated
blocking of translation is known as the "antisense" approach.
Mechanistically, the hybridising oligonucleotide is thought to
elicit its effect by either creating a physical block to the
translation process or by recruiting cellular enzymes that
specifically degrades the mRNA part of the duplex (RNAseH).
[0005] More recently, oligoribonucleotides and
oligodeoxyribonucleotides and analogues thereof which combine RNAse
catalytic activity with the ability to sequence specifically
interact with a complementary RNA target (ribozymes) have attracted
much interest as antisense probes. Thus far ribozymes have been
reported to be effective in cell cultures against both viral
targets and oncogenes.
[0006] To completely prevent the synthesis of a given protein by
the antisense approach it is necessary to block/destroy all mRNAs
that encode that particular protein and in many cases the number of
these mRNA are fairly large. Typically, the mRNAs that encode a
particular protein are transcribed from a single or a few genes.
Hence, by targeting the gene ("antigene" approach) rather than its
mRNA products it should be possible to either block production of
its cognate protein more efficiently or to achieve a significant
reduction in the amount of oligonucleotides necessary to elicit the
desired effect. To block transcription, the oligonucleotide must be
able to hybridise sequence specifically to double stranded DNA. In
1953 Watson and Crick showed that deoxyribo nucleic acid (DNA) is
composed of two strands (Nature, 1953, 171, 737) which are held
together in a helical configuration by hydrogen bonds formed
between opposing complementary nucleobases in the two strands. The
four nucleobases, commonly found in DNA are guanine (G), adenine
(A), thymine (T) and cytosine (C) of which the G nucleobase pairs
with C, and the A nucleobase pairs with T. In RNA the nucleobase
thymine is replaced by the nucleobase uracil (U) which similarly to
the T nucleobase pairs with A. The chemical groups in the
nucleobases that participate in standard duplex formation
constitute the Watson-Crick face. In 1959, Hoogsteen showed that
the purine nucleobases (G and A) in addition to their Watson-Crick
face have a Hoogsteen face that can be recognised from the outside
of a duplex, and used to bind pyrimidine oligonucleotides via
hydrogen bonding, thereby forming a triple helix structure.
Although making the "antigene" approach conceptually feasible the
practical usefulness of triple helix forming oligomers is currently
limited by several factors including the requirement for homopurine
sequence motifs in the target gene and a need for unphysiologically
high ionic strength and low pH to stabilise the complex.
[0007] The use of oligonucleotides known as aptamers are also being
actively investigated. This promising new class of therapeutic
oligonucleotides are selected in vitro to specifically bind to a
given target with high affinity, such as for example ligand
receptors. Their binding characteristics are likely a reflection of
the ability of oligonucleotides to form three dimensional
structures held together by intramolecular nucleobase pairing.
[0008] Likewise, nucleosides and nucleoside analogues have proven
effective in chemotherapy of numerous viral infections and
cancers.
[0009] Also, various types of double-stranded RNAs have been shown
to effectively inhibit the growth of several types of cancers.
[0010] Diagnostics
[0011] In molecular biology, oligonucleotides are routinely used
for a variety of purposes such as for example (i) as hybridisation
probes in the capture, identification and quantification of target
nucleic acids (ii) as affinity probes in the purification of target
nucleic acids (iii) as primers in sequencing reactions and target
amplification processes such as the polymerase chain reaction (PCR)
(iv) to clone and mutate nucleic acids and (vi) as building blocks
in the assembly of macromolecular structures.
[0012] Diagnostics utilises many of the oligonucleotide based
techniques mentioned above in particular those that lend themselves
to easy automation and facilitate reproducible results with high
sensitivity. The objective in this field is to use oligonucleotide
based techniques as a means to, for example (i) tests humans,
animals and food for the presence of pathogenic micro-organisms
(ii) to test for genetic predisposition to a disease (iii) to
identify inherited and acquired genetic disorders, (iv) to link
biological deposits to suspects in crime trials and (vl to validate
the presence of micro-organisms involved in the production of foods
and beverages.
[0013] General Considerations
[0014] To be useful in the extensive range of different
applications outlined above, oligonucleotides have to satisfy a
large number of different requirements. In antisense therapeutics,
for instance, a useful oligonucleotide must be able to penetrate
the cell membrane, have good resistance to extra- and intracellular
nucleases and preferably have the ability to recruit endogenous
enzymes like RNAseH. In DNA-based diagnostics and molecular biology
other properties are important such as, e.g., the ability of
oligonucleotides to act as efficient substrates for a wide range of
different enzymes evolved to act on natural nucleic acids, such as
e.g. polymerases, kinases, ligases and phosphatases. The
fundamental property of oligonucleotides, however, which underlies
all uses is their ability to recognise and hybridise sequence
specifically to complementary single stranded nucleic acids
employing either Watson-Crick hydrogen bonding (A-T and G-C) or
other hydrogen bonding schemes such as the Hoogsteen mode. The are
two important terms affinity and specificity are commonly used to
characterise the hybridisation properties of a given
oligonucleotide. Affinity is a measure of the binding strength of
the oligonucleotide to its complementary target sequence (expressed
as the thermostability (T.sub.m) of the duplex). Each nucleobase
pair in the duplex adds to the thermostability and thus affinity
increases with increasing size (No. of nucleobases) of the
oligonucleotide. Specificity is a measure of the ability of the
oligonucleotide to discriminate between a fully complementary and a
mismatched target sequence. In other words, specificity is a
measure of the loss of affinity associated with mismatched
nucleobase pairs in the target. At constant oligonucleotide size
the specificity increases with increasing number of mismatches
between the oligonucleotide and its targets (i.e. the percentage of
mismatches increases). Conversely, specificity decreases when the
size of the oligonucleotide is increased at a constant number of
mismatches (i.e. the percentage of mismatches decreases). Stated
another way, an increase in the affinity of an oligonucleotide
occurs at the expense of specificity and vice-versa.
[0015] This property of oligonucleotides creates a number of
problems for their practical use. In lengthy diagnostic procedures,
for instance, the oligonucleotide needs to have both high affinity
to secure adequate sensitivity of the test and high specificity to
avoid false positive results. Likewise, an oligonucleotide used as
antisense probes needs to have both high affinity for its target
mRNA to efficiently impair its translation and high specificity to
avoid the unintentional blocking of the expression of other
proteins. With enzymatic reactions, like, e.g., PCR amplification,
the affinity of the oligonucleotide primer must be high enough for
the primer/target duplex to be stable in the temperature range
where the enzymes exhibits activity, and specificity needs to be
high enough to ensure that only the correct target sequence is
amplified.
[0016] Given the shortcomings of natural oligonucleotides, new
approaches for enhancing specificity and affinity would be highly
useful for DNA-based therapeutics, diagnostics and for molecular
biology techniques in general.
[0017] Conformationally Restricted Nucleosides
[0018] It is known that oligonucleotides undergo a conformational
transition in the course of hybridising to a target sequence, from
the relatively random coil structure of the single stranded state
to the ordered structure of the duplex state.
[0019] A number of conformationally restricted oligonucleotides
including bicyclic and tricyclic nucleoside analogues (FIGS. 1A and
1B in which B=nucleobase) have been synthesised, incorporated into
oligonucleotide and oligonucleotide analogues and tested for their
hybridisation and other properties.
[0020] Bicyclo[3.3.0] nucleosides (bcDNA) with an additional C-3',
C-5 '-ethano-bridge (A and B) have been synthesised with all five
nucleobases (G, A, T, C and U) whereas (C) has been synthesised
only with T and A nucleobases (M. Tarkoy, M. Bolli, B. Schweizer
and C. Leumann, Helv. Chim. Acta, 1993, 76, 481; Tarkoy and C.
Leumann, Angew. Chem., Int. Ed, Engl., 1993, 32, 1432; M. Egli, P.
Lubini, M. Dobler and C. Leumann, J. Am. Chem. Soc., 1993, 115,
5855; M. Tarkoy, M. Bolli and C. Leumann, Helv. Chim. Acta, 1994,
77, 716; M. Bolli and C. Leumann, Angew. Chem., Int. Ed. Engl.,
1995, 34, 694; M. Bolli, P. Lubini and C. Leumann, Helv. Chim.
Acta, 1995, 78, 2077; J. C. Litten, C. Epple and C. Leumann,
Bioorg. Med. Chem. Lett., 1995, 5, 1231; J. C. Litten and C.
Leumann, Helv. Chim. Acta, 1996, 79, 1129; M. Bolli, J. C. Litten,
R. Schultz and C. Leumann, Chem. Biol., 1996, 3, 197; M. Bolli, H.
U. Trafelet and C. Leumann, Nucleic Acids Res., 1996, 24, 4660).
DNA oligonucleotides containing a few, or being entirely composed,
of these analogues are in most cases able to form Watson-Crick
bonded duplexes with complementary DNA and RNA oligonucleotides.
The thermostability of the resulting duplexes, however, is either
distinctly lower (C), moderately lower (A) or comparable to (B) the
stability of the natural DNA and RNA counterparts. All bcDNA
oligomers exhibited a pronounced increase in sensitivity to the
ionic strength of the hybridisation media compared to the natural
counterparts. The .alpha.-bicyclo-DNA (,B) is more stable towards
the 3'-exonuclease snake venom phosphordiesterase than the
.beta.-bicyclo-DNA (A) which is only moderately more stable than
unmodified oligonucleotides.
[0021] Bicarbocyclo[3.1.0]nucleosides with an additional C-1',
C-6'- or C-6', C-4'-methano-bridge on a cyclopentane ring (D and E,
respectively) have been synthesised with all five nucleobases (T,
A, G, C and U). Only the T-analogues, however, have been
incorporated into oligomers. Incorporation of one or ten monomers D
in a mixed poly-pyrimidine DNA oligonucleotide resulted in a
substantial decrease in the affinity towards both DNA and RNA
oligonucleotides compared to the unmodified reference
oligonucleotide. The decrease was more pronounced with ssDNA than
with ssRNA. Incorporation of one monomer E in two different
poly-pyrimidine DNA oligonucleotides induced modest increases in
T.sub.m's of 0.8.degree. C. and 2.1.degree. C. for duplexes towards
ssRNA compared with unmodified reference duplexes. When ten
T-analogues were incorporated into a 15 mer oligonucleotide
containing exclusively phosphorothioate internucleoside linkages,
the T.sub.m against the complementary RNA oligonucleotide was
increased approximately 1.3.degree. C. per modification compared to
the same unmodified phosphorothioate sequence. Contrary to the
control sequence the oligonucleotide containing the bicyclic
nucleoside E failed to mediate RNAseH cleavage. The hybridisation
properties of oligonucleotides containing the G, A, C and
U-analogues of E have not been reported. Also, the chemistry of
this analogue does not lend itself to further intensive
investigations on completely modified oligonucleotides (K.-H.
Altmann, R. Kesselring, E. Francotte and G. Rihs, Tetrahedron
Lett., 1994, 35, 2331; K.-H. Altmann, R. Imwikelried, R. Kesselring
and G. Rihs, Tetrahedron Lett., 1994, 35, 7625; V. E. Marquez, M.
A. Siddiqui, A. Ezzitouni, P. Russ, J. Wang, R. W. Wagner and M. D.
Matteucci, J. Med. Chem., 1996, 39, 3739; A. Ezzitouni and V. E.
Marquez, J. Chem. Soc., Perkin Trans. 1, 1997, 1073).
[0022] A bicyclo[3.3.0] nucleoside containing an additional C-2',
C-3'-dioxalane ring has been synthesised as a dimer with an
unmodified nucleoside where the additional ring is part of the
internucleoside linkage replacing a natural phosphodiester linkage
(F). This analogue was only synthesised as either thymine-thymine
or thymine-5-methylcytosine blocks. A 15-mer polypyrimidine
sequence containing seven of these dimeric blocks and having
alternating phosphordiester- and riboacetal-linkages, exhibited a
substantially decreased T.sub.m against complementary ssRNA
compared to a control sequence with exclusively natural
phosphordiester internucleoside linkages (R. J. Jones, S.
Swaminathan, J. F. Millagan, S. Wadwani, B. S. Froehler and M.
Matteucci, J. Am. Chem. Soc., 1993, 115, 9816).
[0023] The two dimers (G and H) with additional C-2', C-3'-dioxane
rings forming bicyclic[4.3.0]-systems in acetal-type
internucleoside linkages have been synthesised as T-T dimers and
incorporated once in the middle of 12 mer polypyrimidine
oligonucleotides. Oligonucleotides containing either G or H both
formed significantly less stable duplexes with complementary ssRNA
and ssDNA compared with the unmodified control oligonucleotide (J.
Wang and M. D. Matteucci, Bioorg. Med. Chem. Lett., 1997, 7,
229).
[0024] Dimers containing a bicyclo[3.1.0]nucleoside with a C-2',
C-3'-methano bridge as part of amide- and sulfonamide-type (I and
J) internucleoside linkages have been synthesised and incorporated
into oligonucleotides. Oligonucleotides containing one ore more of
these analogues showed a significant reduction in T.sub.m compared
to unmodified natural oligonucleotide references (C. G.
Yannopoulus, W. Q. Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G.
Just, Synlett, 1997, 378).
[0025] A trimer with formacetal internucleoside linkages and a
bicyclo[3.3.0] glucose-derived nucleoside analogue in the middle
(K) has been synthesised and connected to the 3'-end of an
oligonucleotide. The T.sub.m against complementary ssRNA was
decreased by 4.degree. C., compared to a control sequence, and by
1.5.degree. C. compared to a sequence containing two
2',5'-formacetal linkages in the 3'-end (C. G. Yannopoulus, W. Q.
Zhou, P. Nower, D. Peoch, Y. S. Sanghvi and G. Just, Synlett, 1997,
378).
[0026] Very recently oligomers composed of tricyclic
nucleoside-analogues (L) have been reported to show increased
duplex stability compared to natural DNA (R. Steffens and C.
Leumann (Poster SB-B4), Chimia, 1997, 51, 436).
[0027] An attempt to make the bicyclic uridine nucleoside analogue
Q planned to contain an additional O-2',C-4'-five-membered ring,
starting from 4'-C-hydroxymethyl nucleoside P, failed (K. D.
Nielsen, Specialerapport (Odense University, Denmark), 1995).
[0028] Until now the pursuit of conformationally restricted
nucleosides useful in the formation of synthetic oligonucleotides
with significantly improved hybridisation characteristics has met
with little success. In the majority of cases, oligonucleotides
containing these analogues form less stable duplexes with
complementary nucleic acids compared to the unmodified
oligonucleotides. In other cases, where moderate improvement in
duplex stability is observed, this relates only to either a DNA or
an RNA target, or it relates to fully but not partly modified
oligonucleotides or vice versa. An appraisal of most of the
reported analogues are further complicated by the lack of data on
analogues with G, A and C nucleobases and lack of data indicating
the specificity and mode of hybridisation. In many cases, synthesis
of the reported monomer analogues is very complex while in other
cases the synthesis of fully modified oligonucleotides is
incompatible with the widely used phosphoramidite chemistry
standard.
SUMMARY OF THE INVENTION
[0029] In view of the shortcomings of the previously known
nucleoside analogues, the present inventors have now provided novel
nucleoside analogues (LNAs) and oligonucleotides have included LNA
nucleoside analogues therein. The novel LNA nucleoside analogues
have been provided with all commonly used nucleobases thereby
providing a full set of nucleoside analogues for incorporation in
oligonucleotides. As will be apparent from the following, the LNA
nucleoside analogues and the LNA modified oligonucleotide provides
a wide range of improvements for oligonucleotides used in the
fields of diagnostics and therapy. Furthermore, the LNA nucleoside
analogues and the LNA modified oligonucleotide also provides
completely new perspectives in nucleoside and oligonucleolide based
diagnostics and therapy.
[0030] Thus, the present invention relates to oligomers comprising
at least one nucleoside analogue (hereinafter termed "LNA") of the
general formula I 1
[0031] wherein
[0032] X is selected from --O--, --S--, --N(R.sup.N+)--,
--C(R.sup.6R.sup.6+)--, --O--C(R.sup.7R.sup.7+)--,
--C(R.sup.6R.sup.6+)--O--, --S--C(R.sup.7R.sup.7+)--,
--C(R.sup.6R.sup.6+)--S--,
--N(R.sup.N+)--C(R.sup.7R.sup.7+)--,--C(R.sup.-
6R.sup.6+)--N(R.sup.N+)--, and
--C(R.sup.6R.sup.6+)--C(R.sup.7R.sup.7+)--;
[0033] B is selected from hydrogen, hydroxy, optionally substituted
C.sub.1-4-alkoxy, optionally substituted C.sub.1-4-alkyl,
optionally substituted C.sub.1-4-acyloxy, nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands;
[0034] P designates the radical position for an internucleoside
linkage to a succeeding monomer, or a 5'-terminal group, such
internucleoside linkage or 5'-terminal group optionally including
the substituent R.sup.5;
[0035] one of the substituents R.sup.2, R.sup.2+, R.sup.3, and
R.sup.3+ is a group P* which designates an internucleoside linkage
to a preceding monomer, or a 3'-terminal group;
[0036] one or two pairs of non-geminal substituents selected from
the present substituents of R.sup.1+, R.sup.4+, R.sup.5, R.sup.5+,
R.sup.6, R.sup.6+, R.sup.7, R.sup.7+, R.sup.N+, and the ones of
R.sup.2, R.sup.2+, R.sup.3, and R.sup.3+ not designating P.sup.+
each designates a biradical consisting of 1-8 groups/atoms selected
from --C(R.sup.aR.sup.b)--, --C(R.sup.a).dbd.C(R.sup.a)--,
--C(R.sup.a).dbd.N--, --O--, --Si(R.sup.a).sub.2--, --S--,
--SO.sub.2--, --N(R.sup.a)--, and >C.dbd.Z,
[0037] wherein Z is selected from --O--, --S--, and --N(R.sup.a)--,
and R.sup.a and R.sup.b each is independently selected from
hydrogen, optionally substituted C.sub.1-12-alkyl, optionally
substituted C.sub.2-12-alkenyl, optionally substituted
C.sub.2-12-alkynyl, hydroxy, C.sub.1-12-alkoxy,
C.sub.2-12-alkenyloxy, carboxy, C.sub.1-12-alkoxycarbonyl,
C.sub.1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,
arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy,
heteroarylcarbonyl, amino, mono- and di(C.sub.1-6-alkyl)amino,
carbamoyl, mono- and di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted, and where two geminal substituents R.sup.a
and R.sup.b together may designate optionally substituted methylene
(.dbd.CH.sub.2), and wherein two non-geminal or geminal
substitlutents selected from R.sup.a, R.sup.b, and any of the
substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.3+,
R.sup.4+, R.sup.5, R.sup.5+, R.sup.6 and R.sup.6+, R.sup.7, and
R.sup.7+ which are present and not involved in P, P.sup.+ or the
biradical(s) together may form an associated biradical selected
from biradicals of the same kind as defined before; said pair(s) of
non-geminal substituents thereby forming a mono- or bicyclic entity
together with (i) the atoms to which said non-geminal substituents
are bound and (ii) any intervening atoms; and
[0038] each of the substituents R.sup.1+, R.sup.2, R.sup.2+,
R.sup.3, R.sup.4+, R.sup.5, R.sup.5+, R.sup.6 and R.sup.6+,
R.sup.7+ which are present and not involved in P, P.sup.+ or the
biradical(s), is independently selected from hydrogen, optionally
substituted C.sub.1-12-alkyl, optionally substituted
C.sub.2-12-alkenyl, optionally substituted C.sub.2-12-alkynyl,
hydroxy, C.sub.1-12-alkoxy, C.sub.2-12-alkenyloxy, carboxy,
C.sub.1-12-alkoxycarbonyl, C.sub.1-12-alkylcarbonyl, formyl, aryl,
aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,
heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino,
mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-aminocarbonyl, amino-C.sub.1-6-alkyl-aminoc-
arbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted, and where two geminal substituents together
may designate oxo, thioxo, imino, or optionally substituted
methylene, or together may form a spiro biradical consisting of a
1-5 carbon atom(s) alkylene chain which is optionally interrupted
and/or terminated by one or more heteroatoms/groups selected from
--O--, --S--, and --(NR.sup.N) where R.sup.N is selected from
hydrogen and C.sub.1-4-alkyl, and where two adjacent (non-geminal)
substituents may designate an additional bond resulting in a double
bond; and R.sup.N+, when present and not involved in a biradical,
is selected from hydrogen and C.sub.1-4-alkyl;
[0039] and basic salts and acid addition salts thereof;
[0040] with the proviso that,
[0041] (i) R.sup.3 and R.sup.5 do not together designate a
biradical selected from --CH.sub.2--CH.sub.2--, --O--CH.sub.2--,
when LNA is a bicyclic nucleoside analogue;
[0042] (ii) R.sup.3, R.sup.5, and R.sup.5* do not together
designate a triradical --CH.sub.2--CH(-)--CH.sub.2-- when LNA is a
tricyclic nucleoside analogue;
[0043] (iii) R.sup.1+ and R.sup.6+ do not together designate a
biradical --CH.sub.2-- when LNA is a bicyclic nucleoside analogue;
and
[0044] (iv) R.sup.4+ and R.sup.6+ do not together designate a
biradical --CH.sub.2-- when LNA is a bicyclic nucleoside
analogue.
[0045] The present invention furthermore relates to nucleoside
analogues (hereinafter LNAs) of the general formula II 2
[0046] wherein the substituent B is selected from nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands;
[0047] X is selected from --O--, --S--, --N(R.sup.N+)--, and
--C(R.sup.6R.sup.6+)--;
[0048] one of the substituents R.sup.2, R.sup.2+, R.sup.3, and
R.sup.3+ is a group Q*;
[0049] each of Q and Q.sup.+ is independently selected from
hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O--, Act-O--,
mercapto, Prot-S--, Act-S--, C.sub.1-6-alkylthio, amino,
Prot-N(R.sup.H)--, Act-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2.sub.6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, ligands, carboxy,
sulphono, hydroxymethyl, Prot-O--CH.sub.2--, Act-O--CH.sub.2--,
aminomethyl, Prot-N(R.sup.H)--CH.sub.2--,
Act-N(R.sup.H)--CH.sub.2--, carboxymethyl, sulphonomethyl, where
Prot is a protection group for --OH, --SH, and --NH(R.sup.H),
respectively, Act is an activation group for --OH, --SH, and
--NH(R.sup.H), respectively, and R.sup.H is selected from hydrogen
and C.sub.1-6-alkyl;
[0050] (i) R.sup.2+ and R.sup.4+ together designate a biradical
selected from --O--, --(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--O- --(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.- +).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s- --,
--O--(CR.sup.+R.sup.+).sub.r+s--O--,
--S--(CR.sup.+R.sup.+).sub.r+s--O- --,
--O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).s- ub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--;
[0051] (ii) R.sup.2 and R.sup.3 together designate a biradical
selected from --O--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--- (CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+)- .sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub-
.s--;
[0052] (iii) R.sup.2 + and R.sup.3 together designate a biradical
selected from --O--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--- (CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+)- .sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub-
.s--;
[0053] (iv) R.sup.3 and R.sup.4+ together designate a biradical
selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
[0054] (v) R.sup.3 and R.sup.5 together designate a biradical
selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.g--;
or
[0055] (vi) R.sup.1+ and R.sup.4+ together designate a biradical
selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
[0056] (vii) R.sup.1+ and R.sup.2+ together designate a biradical
selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
[0057] wherein each R.sup.+ is independently selected from
hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto, amino,
mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands,
and/or two adjacent (non-geminal) R.sup.+ may together designate a
double bond, and each of r and s is 0-3 with the proviso that the
sum r+s is 1-4;
[0058] each of the substituents R.sup.1+, R.sup.2, R.sup.2+,
R.sup.3, R.sup.4+, R.sup.5, and R.sup.5+, which are not involved in
Q, Q.sup.+ or the biradical, is independently selected from
hydrogen, optionally substituted C.sub.1-12-alkyl, optionally
substituted C.sub.2-12-alkenyl, optionally substituted
C.sub.2-12-alkynyl, hydroxy, C.sub.1-12-alkoxy,
C.sub.2-12-alkenyloxy, carboxy, C.sub.1-12-alkoxycarbonyl,
C.sub.1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,
arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,
heteroarylcarbonyl, amino, mono- and di(C.sub.1-6-alkyl)amino,
carbamoyl, mono- and di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-amino- carbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl- ,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands, where aryl and heteroaryl may be
optionally substituted, and where two geminal substituents together
may designate oxo, thioxo, imino, or optionally substituted
methylene, or together may form a spiro biradical consisting of a
1-5 carbon atom(s) alkylene chain which is optionally interrupted
and/or terminated by one or more heteroatoms/groups selected from
--O--, --S--, and --NR.sup.N)-- where R.sup.N is selected from
hydrogen and C.sub.1-4-alkyl, and where two adjacent (non-geminal)
substituents may designate an additional bond resulting in a double
bond; and R.sup.N+, when present and not involved in a biradical,
is selected from hydrogen and C.sub.1-4-alkyl;
[0059] and basic salts and acid addition salts thereof;
[0060] with the first proviso that,
[0061] (i) R.sup.3 and R.sup.5 do not together designate a
biradical selected from --CH.sub.2--CH.sub.2--, --O--CH.sub.2--,
and --O--Si(.sup.iPr).sub.2--O--Si(.sup.iPr).sub.2--O--;
[0062] and with the second proviso that any chemical group
(including any nucleobase), which is reactive under the conditions
prevailing in oligonucleotide synthesis, is optionally functional
group protected.
[0063] The present invention also relates to the use of the
nucleoside analogues (LNAs) for the preparation of oligomers, and
the use of the oligomers as well as the nucleoside analogues (LNAs)
in diagnostics, molecular biology research, and in therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] FIGS. 1A and 1B illustrate known conformationally restricted
nucleotides.
[0065] FIG. 2 illustrates nucleotide/nucleoside analogues of the
invention.
[0066] FIG. 3 illustrates the performance of LNA modified
oligonucleotides in the sequence specific capture of PCR
amplicons.
[0067] FIGS. 4A and 4B illustrate that LNA modified
oligonucleotides are able to capture its cognate PCR amplicon by
strand invasion.
[0068] FIG. 5 illustrates that LNA modified oligonucleotides,
immobilised on a solid surface, function efficiently in the
sequence specific capture of a PCR amplicon.
[0069] FIG. 6 illustrates that LNA modified oligonucleotides can
act as substrates for T4 polynucleotide kinase.
[0070] FIG. 7 illustrates that LNA modified oligonucleotides can
function as primers for nucleic acid polymerases.
[0071] FIG. 8 illustrates that LNA modified oligonucleotides can
functions as primers in target amplification processes.
[0072] FIG. 9 illustrates that LNA modified oligonucleotides
carrying a 5' anthraquinone can be covalently immobilised on a
solid support by irradiation and that the immobilised oligomer is
efficient in the capture of a complementary DNA oligo.
[0073] FIG. 10 illustrates that LNA-thymidine-5'-triphosphate
(LNA-TTP) can act as a substrate for terminal deoxynucleotidyl
transferase (TdT).
[0074] FIG. 11 illustrates hybridisation and detection on an array
with different LNA modified Cy3-labelled 8 mers.
[0075] FIGS. 12 and 13 illustrate hybridisation and detection of
end mismatches on an array with LNA modified Cy3-labelled 8
mers.
[0076] FIG. 14 illustrates blockade by LNA of
[D-Ala2]deltorphin-induced antinociception in the warm water tail
flick test in conscious rats.
[0077] FIGS. 15A, 15B, and 15C illustrate Hybridization and
detection of end mismatches on an array with AT and all LNA
modified Cy3-labelled 8 mers.
[0078] FIGS. 16 and 17 illustrate that LNA can be delivered to
living human MCF-7 breast cancer cells.
[0079] FIGS. 18 and 19 illustrate the use of [.alpha..sup.33P]
ddNTP's and ThermoSequenase.TM. DNA Polymerase to sequence DNA
templates containing LNA T monomers.
[0080] FIGS. 20 and 21 illustrate that exonuclease free Klenow
fragment DNA polymerase I can incorporate LNA Adenosine, Cytosine,
Guanosine and Uridine-5'-triphosphates into a DNA strand.
[0081] FIG. 22 illustrates the ability of terminal deoxynucleotidyl
transferase (TdT) to tail LNA modified oligonucleotides.
[0082] FIGS. 23A and 23B illustrate that fully mixed LNA monomers
can be used to significantly increase the performance of
immobilised biotinylated-DNA oligos in the sequence specific
capture of PCR amplicons.
[0083] FIGS. 24 to 41 illustrates possible synthetic routes towards
the LNA monomers of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0084] When used herein, the term "LNA" (Locked Nucleoside
Analogues) refers to the bi- and tri-cyclic nucleoside analogues of
the invention, either incorporated in the oligomer of the invention
(general formula I) or as discrete chemical species (general
formula II). The term "monomeric LNA" specifically refers to the
latter case.
[0085] Oligomers and Nucleoside Analogues
[0086] As mentioned above, the present invention i.a. relates to
novel oligomers (oligonucleotides) comprising one or more bi-,
tri-, or polycyclic nucleoside analogues (hereinafter termed
"LNA"). It has been found that the incorporation of such LNAs in
place of, or as a supplement to, e.g., known nucleosides confer
interesting and highly useful properties to an oligonucleotide. Bi-
and tricyclic, especially bicyclic, LNAs seem especially
interesting within the scope of the present invention.
[0087] Each of the possible LNAs incorporated in an oligomer
(oligonucleotide) has the general formula I 3
[0088] wherein X is selected from --O-- (the furanose motif),
--S--, --N(R.sup.N+)--, C(R.sup.6R.sup.6+)--,
--O--C(R.sup.7R.sup.7+)--, --C(R.sup.6R.sup.6+)--O--,
--S--C(R.sup.7R.sup.7+)--, --C(R.sup.6R.sup.6+)--S--,
--N(R.sup.N+)--C(R.sup.7R.sup.7+)--,
--C(R.sup.6R.sup.6+)--N(R.sup.N+)--, and
--C(R.sup.6R.sup.6+)--C(R.sup.7R- .sup.7+)--, where R.sup.6,
R.sup.6+, R.sup.7, R.sup.7+, and R.sup.N+ are as defined further
below. Thus, the LNAs incorporated in the oligomeDr may comprise an
either 5- or 6-membered ring as an essential part of the bi-, tri-,
or polycyclic structure. It is believed that 5-membered rings
(X.dbd.--O--, --S--, N(R.sup.N+)--, --C(R.sup.6R.sup.6+)--) are
especially interesting in that they are able to occupy essentially
the same conformations (however locked by the introduction of one
or more biradicals (see below)) as the native furanose ring of a
naturally occurring nucleoside. Among the possible 5-membered
rings, the situations where X designates --O--, --S--, and
N(R.sup.N+)-- seem especially interesting , and the situation where
X is --O-- appears to be particularly interesting.
[0089] The substituent B may designate a group which, when the
oligomer is complexing with DNA or RNA, is able to interact (e.g.
by hydrogen bonding or covalent bonding or electronic interaction)
with DNA or RNA, especially nucleobases of DNA or RNA.
Alternatively, the substituent B may designate a group which acts
as a label or a reporter, or the substituent B may designate a
group (e.g. hydrogen) which is expected to have little or no
interactions with DNA or RNA. Thus, the substituent B is preferably
selected from hydrogen, hydroxy, optionally substituted
C.sub.1-4-alkoxy, optionally substituted C.sub.1-4-alkyl,
optionally substituted C.sub.1-4-acyloxy, nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and liganids.
[0090] In the present context, the terms "nucleobase" covers
naturally occurring nucleobases as well as non-naturally occurring
nucleobases. It should be clear to the person skilled in the art
that various nucleobases which previously have been considered
"non-naturally occurring" have subsequently been found in nature.
Thus, "nucleobase" includes not only the known purine and
pyrimidine heterocycles, but also heterocyclic analogues and
tautomers thereof. Illustrative examples of nucleobases are
adenine, guanine, thymine, cytosine, uracil, purine, xanthine,
diaminopurine, 8-oxo-N.sup.6-methyladenine, 7-deazaxanthine,
7-deazaguanine, N.sup.4,N.sup.4-ethanocytosin,
N.sup.6,N.sup.6-ethano-2,6- -diaminopurine, 5-methylcytosine,
5-(C.sup.3-C.sup.6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,
pseudoisocytosine, 2-hydroxy-5-methyl-4-tr- iazolopyridin,
isocytosine, isoguanin, inosine and the "non-naturally occurring"
nucleobases described in Benner et al., U.S. Pat. No. 5,432,272.
The term "nucleobase" is intended to cover every and all of these
examples as well as analogues and tautomers thereof. Especially
interesting nucleobases are adenine, guanine, thymine, cytosine,
and uracil, which are considered as the naturally occurring
nucleobases in relation to therapeutic and diagnostic application
in humans.
[0091] When used herein, the term "DNA intercalator" means a group
which can intercalate into a DNA or RNA helix, duplex or triplex.
Examples of functional parts of DNA intercalators are acridines,
anthracene, quinones such as anthraquinone, indole, quinoline,
isoquinoline, dihydroquinones, anthracyclines, tetracyclines,
methylene blue, anthracyclinone, psoralens, coumarins,
ethidium-halides, dynemicin, metal complexes such as
1,10-phenanthroline-copper,
tris(4,7-diphenyl-1,10-phenanthroline)ruth- enium-cobalt-enediynes
such as calcheamicin, porphyrins, distamycin, netropcin, viologen,
daunomycin. Especially interesting examples are acridines, quinones
such as anthraquinone, methylene blue, psoralens, coumarins, and
ethidium-halides.
[0092] In the present context, the term "photochemically active
groups" covers compounds which are able to undergo chemical
reactions upon irradiation with light. Illustrative examples of
functional groups hereof are quinones, especially
6-methyl-1,4-naphtoquinone, anthraquinone, naphtoquinone, and
1,4-dimethyl-anthraquinone, diazirines, aromatic azides,
benzophenones, psoralens, diazo compounds, and diazirino
compounds.
[0093] In the present context "thermochemically reactive group" is
defined as a functional group which is able to undergo
thermochemically-induced covalent bond formation with other groups.
Illustrative examples of functional parts thermochemically reactive
groups are carboxylic acids, carboxylic acid esters such as
activated esters, carboxylic acid halides such as acid fluorides,
acid chlorides, acid bromide, and acid iodides, carboxylic acid
azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid
esters, sulfonic acid halides, semicarbazides, thiosemicarbazides,
aldehydes, ketones, primary alkohols, secondary alkohols, tertiary
alkohols, phenols, alkyl halides, thiols, disulphides, primary
amines, secondary amines, tertiary amines, hydrazines, epoxides,
maleimides, and boronic acid derivatives.
[0094] In the present context, the term "chelating group" means a
molecule that contains more than one binding site and frequently
binds to another molecule, atom or ion through more than one
binding site at the same time. Examples of functional parts of
chelating groups are iminodiacetic acid, nitrilotriacetic acid,
ethylenediamine tetraacetic acid (EDTA), aminophosphonic acid,
etc.
[0095] In the present context, the term "reporter group" means a
group which is detectable either by itself or as a part of an
detection series. Examples of functional parts of reporter groups
are biotin, digoxigenin, fluorescent groups (groups which are able
to absorb electromagnetic radiation, e.g. light or X-rays, of a
certain wavelength, and which subsequently reemits the energy
absorbed as radiation of longer wavelength; illustrative examples
are dansyl (5-dimethylamino)-1-naphthal- enesulfonyl), DOXYL
(N-oxyl-4,4-dimethyloxazolidine), PROXYL
(N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO
(N-oxyl-2,2,6,6-tetramethy- lpiperidine), dinitrophenyl, acridines,
coumarins, Cy3 and Cy5 (trademarks for Biological Detection
Systems, Inc.), erytrosine, coumaric acid, umbelliferone, texas
red, rhodamine, tetramethyl rhodamine, Rox,
7-nitrobenzo-2-oxa-1-diazole (NBD), pyrene, fluorescein, Europium,
Ruthenium, Samarium, and other rare earth metals), radioisotopic
labels, chemiluminescence labels (labels that are detectable via
the emission of light during a chemical reaction), spin labels (a
free radical (e.g. substituted organic nitroxides) or other
paramagnetic probes (e.g. Cu.sup.2+, Mg.sup.2+) bound to a
biological molecule being detectable by the use of electron spin
resonance spectroscopy), enzymes (such as peroxidases, alkaline
phosphatases, .crclbar.-galactosidases, and glycose oxidases),
antigens, antibodies, haptens (groups which are able to combine
with an antibody, but which cannot initiate an immune response by
itself, such as peptides and steroid hormones), carrier systems for
cell membrane penetration such as: fatty acid residues, steroid
moieties (cholesteryl), vitamin A, vitamin D, vitamin E, folic acid
peptides for specific receptors, groups for mediating endocytose,
epidermal growth factor (EGF), bradykinin, and platelet derived
growth factor (PDGF). Especially interesting examples are biotin,
fluorescein, Texas Red, rhodamine, dinitrophenyl, digoxigenin,
Ruthenium, Europium, Cy5, Cy3, etc.
[0096] In the present context "ligand" means something which binds.
Ligands can comprise functional groups such as: aromatic groups
(such as benzene, pyridine, naphtalene, anthracene, and
phenanthrene), heteroarormatic groups (such as thiophene, furan,
tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic
acids, carboxylic acid esters, carboxylic acid halides, carboxylic
acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic
acid esters, sulfonic acid halides, semicarbazides,
thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary
alcohols, tertiary alcohols, phenols, alkyl halides, thiols,
disulphides, primary amines, secondary amines, tertiary amines,
hydrazines, epoxides, maleimides, C.sub.1-C.sub.20 alkyl groups
optionally interrupted or terminated with one or more heteroatoms
such as oxygen atoms, nitrogen atoms, and/or sulphur atoms,
optionally containing aromatic or monopolyunsaturated hydrocarbons,
polyoxyethylene such as polyethylene glycol, oligo/polyamides such
as poly-.beta.-alanine, polyglycine, polylysine, peptides,
oligo/polysaccharides, oligo/polyphosphates, toxins, antibiotics,
cell poisons, and steroids, and also "affinity ligands", i.e.
functional groups or biomolecules that have a specific affinity for
sites on particular proteins, antibodies, poly- and
oligosaccharides, and other biomolecules.
[0097] It will be clear for the person skilled in the art that the
above-mentioned specific examples under DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands correspond to the
"active/functional" part of the groups in question. For the person
skilled in the art it is furthermore clear that DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands are typically
represented in the form M-K- where M is the "active/functional"
part of the group in question and where K is a spacer through which
the "active/functional" part is attached to the 5- or 6-membered
ring. Thus, it should be understood that the group B, in the case
where B is selected from DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups, and ligands, has the form M-K-, where M is the
"active/functional" part of the DNA intercalator, photochemically
active group, thermochemically active group, chelating group,
reporter group, and ligand, respectively, and where K is an
optional spacer comprising 1-50 atoms, preferably 1-30 atoms, in
particular 1-15 atoms, between the 5- or 6-membered ring and the
"active/functional" part.
[0098] In the present context, the term "spacer" means a
thermochemically and photochemically non-active distance-making
group and is used to join two or more different moieties of the
types defined above. Spacers are selected on the basis of a variety
of characteristics including their hydrophobicity, hydrophilicity,
molecular flexibility and length (e.g. see Hermanson et. al.,
"Immobilized Affinity Ligand Techniques", Academic Press, San
Diego, Calif. (1992), p. 137-ff). Generally, the length of the
spacers are less than or about 400 .ANG., in some applications
preferably less than 100 .ANG.. The spacer, thus, comprises a chain
of carbon atoms optionally interrupted or terminated with one or
more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or
sulphur atoms. Thus, the spacer K may comprise one or more amide,
ester, amino, ether, and/or thioether functionalities, and
optionally aromatic or mono/polyunsaturated hydrocarbons,
polyoxyethylene such as polyethylene glycol, oligo/polyamides such
as poly-.beta.-alanine, polyglycine, polylysine, and peptides in
general, oligosaccharides, oligo/polyphosphates. Moreover the
spacer may consist of combined units thereof. The length of the
spacer may vary, taking into consideration the desired or necessary
positioning and spatial orientation of the "active/functional" part
of the group in question in relation to the 5- or 6-membered ring.
In particularly interesting embodiments, the spacer includes a
chemically cleavable group. Examples of such chemically cleavable
groups include disulphide groups cleavable under reductive
conditions, peptide fragments cleavable by peptidases, etc.
[0099] In one embodiment of the present invention, K designates a
single bond so that the "active/functional" part of the group in
question is attached directly to the 5- or 6-membered ring.
[0100] In a preferred embodiment, the substituent 13 in the general
formulae I and II is preferably selected from nucleobases, in
particular from adenine, guanine, thymine, cytosine and urasil.
[0101] In the oligomers of the present invention (formula I), P
designates the radical position for an internucleoside linkage to a
succeeding monomer, or a 5'-terminal group. The first possibility
applies when the LNA in question is not the 5'-terminal "monomer",
whereas the latter possibility applies when the LNA in question is
the 5'-terminal "monomer". It should be understood (which also will
be clear from the definition of internucleoside linkage and
5'-terminal group further below) that such an internucleoside
linkage or 5'-terminal group may include the substituent R.sup.5
(or equally applicable: the substituent R.sup.5+) thereby forming a
double bond to the group P. (5'-Terminal refers to the position
corresponding to the 5' carbon atom of a ribose moiety in a
nucleoside.)
[0102] On the other hand, an internucleoside linkage to a preceding
monomer or a 3'-terminal group (P.sup.+) may originate from the
positions defined by one of the substituents R.sup.2, R.sup.2+,
R.sup.3, and R.sup.3+, preferably from the positions defined by one
of the substituents R.sup.3 and R.sup.3+. Analogously, the first
possibility applies where the LNA in question is not the
3'-terminal "monomer", whereas the latter possibility applies when
the LNA in question is the 3'-terminal "monomer". (3'-Terminal
refers to the position corresponding to the 3' carbon atom of a
ribose moiety in a nucleoside.)
[0103] In the present context, the term "monomer" relates to
naturally occurring nucleosides, non-naturally occurring
nucleosides, PNAs, etc. as well as LNAs. Thus, the term "succeeding
monomer" relates to the neighbouring monomer in the 5'-terminal
direction and the "preceding monomer" relates to the neighbouring
monomer in the 3'-terminal direction. Such succeeding and preceding
monomers, seen from the position of an LNA monomer, may be
naturally occurring nucleosides or non-naturally occurring
nucleosides, or even further LNA monomers,
[0104] Consequently, in the present context (as can be derived from
the definitions above), the term "oligomer" means an
oligonucleotide modified by the incorporation of one or more
LNA(s).
[0105] The crucial part of the present invention is the presence of
one or more rings fused to the 5- or 6-membered ring illustrated
with the general formula I. Thus, one or two pairs of non-geminal
substituents selected from the present substituents of R.sup.1+,
R.sup.4+, R.sup.5, R.sup.5+, R.sup.6, R.sup.6+, R.sup.7, R.sup.7+,
R.sup.N+, and the ones of R.sup.2, R.sup.2+, R.sup.3, and R.sup.3+
not designating P.sup.+ each designates a biradical consisting of
1-8 groups/atoms, preferably 1-4 groups/atoms, independently
selected from --C(R.sup.aR.sup.b)--, --C(R.sup.a).dbd.C(R.sup.a)--,
--C(R.sup.a).dbd.N--, --O--, --Si(R.sup.a).sub.2--, --S--,
--SO.sub.2--, --N(R.sup.a)--, and >C.dbd.Z. (The term "present"
indicates that the existence of some of the substituents, i.e.
R.sup.6, R.sup.6+, R.sup.7, R.sup.7+, R.sup.N+, is dependent on
whether X includes such substituents.)
[0106] In the groups constituting the biradical(s), Z is selected
from --O--, --S--, and --N(R.sup.a)--, and R.sup.a and R.sup.b each
is independently selected from hydrogen, optionally substituted
C.sub.1-12-alkyl, optionally substituted C.sub.2-12-alkenyl,
optionally substituted C.sub.2-12-alkynyl, hydroxy,
C.sub.1-12-alkoxy, C.sub.2-12-alkenyloxy, carboxy,
C.sub.1-12-alkoxycarbonyl, C.sub.1-12-alkylcarbonyl, formyl, aryl,
aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,
heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino,
mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-carbonyl, amino-C.sub.1-6-alkyl-amino-
carbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl- ,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands (where the latter groups may include a
spacer as defined for the substituent B), where aryl and heteroaryl
may be optionally substituted. Moreover, two geminal substituents
R.sup.a and R.sup.b together may designate optionally substituted
methylene (.dbd.CH.sub.2 optionally substituted one or two times
with substituents as defined as optional substituents for aryl),
and two non-geminal or geminal substituents selected from R.sup.a,
R.sup.b, and any of the substituents R.sup.1+, R.sup.2, R.sup.2+,
R.sup.3, R.sup.3+, R.sup.4+, R.sup.5, R.sup.5+, R.sup.6 and
R.sup.6+, R.sup.7, and R.sup.7+ which are present and not involved
in P, P.sup.+ or the biradical(s) may together form an associated
biradical selected from biradicals of the same kind as defined
before. It will be clear that each of the pair(s) of non-geminal
substituents thereby forms a mono- or bicyclic entity together with
(i) the atoms to which the non-geminal substituents are bound and
(ii) any intervening atoms.
[0107] It is believed that biradicals which are bound to the ring
atoms of the 5- or 6-membered rings are preferred in that inclusion
of the substituents R.sup.5 and R.sup.5+ may cause an undesired
sterical interaction with internucleoside linkage. Thus, it is
preferred that the one or two pairs of non-geminal substituents,
which are constituting one or two biradical(s), respectively, are
selected from the present substituents of R.sup.1+, R.sup.4+,
R.sup.6, R.sup.6+, R.sup.7, R.sup.7+, R.sup.N+, and the ones of
R.sup.2, R.sup.2+, R.sup.3, and R.sup.3+ not designating
P.sup.+.
[0108] Preferably, the LNAs incorporated in the oliqomers comprise
only one biradical constituted by a pair of (two) non-geminal
substituents. In particular, it is preferred that R.sup.3+
designates P.sup.+ and that the biradical is formed between
R.sup.2+ and R.sup.4+ or R.sup.2 and R.sup.3.
[0109] This being said, it should be understood (especially with
due consideration of the known bi- and tricyclic nucleoside
analogues--see "Background of the Invention") that the present
invention does not relate to oligomers comprising the following bi-
or tricyclic nucleosides analogues:
[0110] (i) R.sup.2 and R.sup.3 together designate a biradical
selected from --O--CH.sub.2--CH.sub.2-- and
--O--CH.sub.2--CH.sub.2--CH.sub.2-- when LNA is a bicyclic
nucleoside analogue;
[0111] (ii) R.sup.3 and R.sup.5 together designate a biradical
selected from --CH.sub.2--CH.sub.2--, --O--CH.sub.2--, when LNA is
a bicyclic nucleoside analogue;
[0112] (iii) R.sup.3, R.sup.5, and R.sup.5* together designate a
triradical --CH.sub.2--CH(-)--CH.sub.2-- when LNA is a tricyclic
nucleoside analogue;
[0113] (iv) R.sup.1+ and R.sup.6+ together designate a biradical
--CH.sub.2-- when LNA is a bicyclic nucleoside analogue; or
[0114] (v) R.sup.4+ and R.sup.6+ together designate a biradical
--CH.sub.2-- when LNA is a bicyclic nucleoside analogue;
[0115] except where such bi- or tricyclic nucleoside analogues are
combined with one or more of the novel LNAs defined herein.
[0116] In the present context, i.e. in the present description and
claims, the orientation of the biradicals are so that the left-hand
side represents the substituent with the lowest number and the
right-hand side represents the substituent with the highest number,
thus, when R.sup.3 and R.sup.5 together designate a biradical
"--O--CH.sub.2--", it is understood that the oxygen atom represents
R.sup.3, thus the oxygen atom is e.g. attached to the position of
R.sup.3, and the methylene group represents R.sup.5.
[0117] Considering the numerous interesting possibilities for the
structure of the biradical(s) in LNA(s) incorporated in oligomers
according to the invention, it is believed that the biradical(s)
constituted by pair(s) of non-geminal substituents preferably
is/are selected from
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--Y--,
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--,
--Y--(CR.sup.+R.sup.+).sub.r--Y--(CR- .sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r+s--, --Y--, --Y--Y--, wherein each Y is
independently selected from --O--, --S--, --Si(R.sup.+).sub.2--,
--N(R.sup.+)--, >C.dbd.O, --C(.dbd.O)--N(R.sup.+)--, and
--N(R.sup.+)--C(.dbd.O)--, each R.sup.+ is independently selected
from hydrogen, halogen, azido, cyano, nitro, hydroxy, mercapto,
amino, mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands,
and/or two adjacent (non-geminal) R.sup.+ may together designate a
double bond; and each of r and s is 0-4 with the proviso that the
sum r+s is 1-5. Particularly interesting situations are those
wherein each biradical is independently selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--, wherein and each of r and s is
0-3 with the proviso that the sum r+s is 1-4.
[0118] Considering the positioning of the biradical in the LNA(s),
it is believed (based on the preliminary findings (see the
examples)) that the following situations are especially
interesting, namely where: R.sup.2+ and R.sup.4+ together designate
a biradical selected from --Y--, --(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R- .sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; R.sup.2 and R.sup.3 together
designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; R.sup.2+ and R.sup.3 together
designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; R.sup.3 and R.sup.4+ together
designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+).sub.s--, and
--Y--(CR.sup.+R.sup.+ ).sub.r+s--Y--; R.sup.3 and R.sup.5 together
designate a biradical selected from --Y'--,
--(CR.sup.+R.sup.+).sub.r+s+1--, --(CR.sup.+R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; R.sup.1+ and R.sup.4+ together
designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s+1-- -,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.sup.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--NR.sup.+; or where R.sup.1+ and
R.sup.2+ together designate a biradical selected from --Y--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--Y--(CR.sup.+R.s- up.+).sub.s--, and
--Y--(CR.sup.+R.sup.+).sub.r+s--Y--; wherein each of r and s is 0-3
with the proviso that the sum r+s is 1-4, Y is as defined above,
and where Y' is selected from --NR.sup.+--C(.dbd.O)-- and
--C(.dbd.O)--NR.sup.+--.
[0119] Particularly interesting oligomers are those wherein one of
the following criteria applies for at least one LNA in an oligomer:
R.sup.2+ and R.sup.4+ together designate a biradical selected from
--O--, --S--, --N(R.sup.+)--, --(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.- r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.- sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).s- ub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O--, --S--(CR.sup.+R.sup.+).sub.r+-
s--O--, --O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.- +).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; R.sup.2 and R.sup.3
together designate a biradical selected from --O--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.s- up.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.2+ and R.sup.3 together designate a biradical selected from
--O--, --CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.su- p.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.3 and R.sup.4+ together designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.3 and R.sup.5 together designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,--(CR.sup.+R.sup.-
+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R- .sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.1+ and R.sup.4+ together designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup- .+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s-- -, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
or R.sup.1+ and R.sup.2+ together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
wherein each of r and s is 0-3 with the prcoviso that the sum r+s
is 1-4, and where R.sup.H designates hydrogen or
C.sub.1-4-alkyl.
[0120] It is furthermore preferred that one R is selected from
hydrogen, hydroxy, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands, and any remaining
substituents R.sup.+ are hydrogen.
[0121] In one preferred embodiment, one group R.sup.+ in the
biradical of at least one LNA is selected from DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands (where the latter
groups may include a spacer as defined for the substituent B).
[0122] With respect to the substituents R.sup.1+, R.sup.2,
R.sup.2+, R.sup.3, R.sup.4+, R.sup.5, R.sup.5+, R.sup.6 and
R.sup.6+, R.sup.7, and R.sup.7+, which are present and not involved
in P, P.sup.+ or the biradical(s), these are independently selected
from hydrogen, optionally substituted C.sub.1-12-alkyl, optionally
substituted C.sub.2-12-alkenyl, optionally substituted
C.sub.2-12-alkynyl, hydroxy, C.sub.1-12-alkoxy,
C.sub.2-12-alkenyloxy, carboxy, C.sub.1-12-alkoxycarbonyl,
C.sub.1-12-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,
arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,
heteroarylcarbonyl, amino, mono- and di(C.sub.1-6-alkyl)amino,
carbamoyl, mono- and di(C.sub.1-6-alkyl)-amino-carbonyl,
amino-C.sub.1-6-alkyl-amino- carbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl- ,
C.sub.1-6-alkyl-carbonylamino, carbamido, C.sub.1-6-alkanoyloxy,
sulphono, C.sub.1-6-alkylsulphonyloxy, nitro, azido, sulphanyl,
C.sub.1-6-alkylthio, halogen, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, and ligands (where the latter groups may include a
spacer as defined for the substituent B), where aryl and heteroaryl
may be optionally substituted, and where two geminal substituents
together may designate oxo, thioxo, imino, or optionally
substituted methylene, or together may form a spiro biradical
consisting of a 1-5 carbon atom(s) alkylene chain which is
optionally interrupted and/or terminated by one or more
heteroatoms/groups selected from --O--, --S--, and --NR.sup.N)--
where R.sup.N is selected from hydrogen and C.sub.1-4-alkyl, and
where two adjacent (non-geminal) substituents may designate an
additional bond resulting in a double bond; and R.sup.N+, when
present and not involved in a biradical, is selected from hydrogen
and C.sub.1-4-alkyl.
[0123] Preferably, each of the substituents R.sup.1+, R.sup.2,
R.sup.2+, R.sup.3, R.sup.3+, R.sup.4+, R.sup.5, R.sup.5+, R.sup.6,
R.sup.6+, R.sup.7, and R.sup.7+ of the LNA(s), which are present
and not involved in P, P.sup.+ or the biradical(s), is
independently selected from hydrogen, optionally substituted
C.sub.1-6-alkyl, optionally substituted C.sub.2-6-alkenyl, hydroxy,
C.sub.1-6-alkoxy, C.sub.2-6-alkenyloxy, carboxy,
C.sub.1-6-alkoxycarbonyl, C.sub.1-6-alkylcarbonyl, formyl, amino,
mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-carbonyl, C.sub.1-6-alkyl-carbonylamino,
carbamido, azido, C.sub.1-6-alkanoyloxy, sulphono, sulphanyl,
C.sub.1-6-alkylthio, DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups, and ligands, and halogen, where two geminal substituents
together may designate oxo, and where R.sup.N+, when present and
not involved in a biradical, is selected from hydrogen and
C.sub.1-4-alkyl.
[0124] In a preferred embodiment of the present invention, X is
selected from --O--, --S--, and --NR.sup.N+, in particular --O--,
and each of the substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3,
R.sup.3+, R.sup.4+, R.sup.5, R.sup.5+, R.sup.6, R.sup.6+, R.sup.7,
and R.sup.7+ of the LNA(s), which are present and not involved in
P, P.sup.+ or the biradical(s), designate hydrogen.
[0125] In an even more preferred embodiment of the present
invention, R.sup.2+ and R.sup.4+ of an LNA incorporated into an
oligomer together designate a biradical. Preferably, X is O,
R.sup.2 selected from hydrogen, hydroxy, and optionally substituted
C.sub.1-6-alkoxy, and R.sup.1+, R.sup.3, R.sup.5, and R.sup.5+
designate hydrogen, and, more specifically, the biradical is
selected from --O--, --(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(- CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3-- -, and
--(CH.sub.2).sub.2-4--, in particular from --O--CH.sub.2--,
--S--CH.sub.2--, and --NR.sup.H--CH.sub.2--. Generally, with due
regard to the results obtained so far, it is preferred that the
biradical constituting R.sup.2+ and R.sup.4+ forms a two carbon
atom bridge, i.e. the biradical forms a five membered ring with the
furanose ring (X.dbd.O).
[0126] In another embodiment of the present invention, R.sup.2 and
R.sup.3 of an LNA incorporated into an oligomer together designate
a biradical. Preferably, X is O, R.sup.2+ is selected from
hydrogen, hydroxy, and optionally substituted C.sub.1-6-alkoxy, and
R.sup.1+, R.sup.4+, R.sup.5, and R.sup.5+ designate hydrogen, and,
more specifically, the biradical is selected from
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--N(R.- sup.H)--(CH.sub.2).sub.1-3-- and
--(CH.sub.2).sub.1-4--, in particular from --O--CH.sub.2--,
--S--CH.sub.2--, --N(R.sup.H)--CH.sub.2--. In the latter case, the
amino and thio variants appears to be particularly interesting.
[0127] In a further embodiment of the present invention, R.sup.2+
and R.sup.3 of an LNA incorporated into an oligomer together
designate a biradical. Preferably, X is O, R.sup.2 is selected from
hydrogen, hydroxy, and optionally substituted C.sub.1-6-alkoxy, and
R.sup.1+, R.sup.4+, R.sup.5, and R.sup.5+ designate hydrogen, and,
more specifically, the biradical is selected from
--(CH.sub.2).sub.0-1--O--(CH- .sub.2).sub.1-3-- and
--(CH.sub.2).sub.2-4--.
[0128] In a further embodiment of the present invention, R.sup.3
and R.sup.4+ of an LNA incorporated into an oligomer together
designate a biradical. Preferably, X is O, R.sup.2+ selected from
hydrogen, hydroxy, and optionally substituted C.sub.1-6-alkoxy, and
R.sup.1+, R.sup.2, R.sup.5, and R.sup.5+ designate hydrogen, and,
more specifically, the biradical is
--(CH.sub.2).sub.0-2--O--(CH.sub.2).sub.0-2--.
[0129] In a further embodiment of the present invention, R.sup.3
and R.sup.5+ of an LNA incorporated into an oligomer together
designate a biradical. Preferably, X is O, R.sup.2+ selected from
hydrogen, hydroxy, and optionally substituted C.sub.1-6-alkoxy, and
R.sup.1+, R.sup.2, R.sup.4, and R.sup.5 designate hydrogen, and,
more specifically, the biradical is selected from
--O--(CHR.sup.+).sub.2-3-- and
--(CHR.sup.+).sub.1-3--O--(CHR.sup.+).sub.0-3--.
[0130] In a further embodiment of the present invention, R.sup.1+
and R.sup.4+ of an LNA incorporated into an oligomer together
designate a biradical. Preferably, X is O, R.sup.2+ selected from
hydrogen, hydroxy, and optionally substituted C.sub.1-6-alkoxy, and
R.sup.2, R.sup.3, R.sup.5, and R.sup.5+ designate hydrogen, and,
more specifically, the biradical is
--(CH.sub.2).sub.0-2--O--(CH.sub.2).sub.0-2--.
[0131] In these embodiments, it is furthermore preferred that at
least one LNA incorporated in an oligomer includes a nucleobase
(substituent B) selected from adenine and guanine. In particular,
it is preferred that an oligomer have LNA incorporated therein both
include at least one nucleobase selected from thymine, urasil and
cytosine and at least one nucleobase selected from adenine and
guanine. For LNA monomers, it is especially preferred that the
nucleobase is selected from adenine and guanine.
[0132] For these interesting embodiments, it is also preferred that
the LNA(s) has/have the general formula Ia (see below).
[0133] Within a variant of these interesting embodiments, all
monomers of a oligonucleotide are LNA monomers.
[0134] As it will be evident from the general formula I (LNA(s) in
an oligomer) (and the general formula II (monomeric LNA)--see
below) and the definitions associated therewith, there may be one
or several asymmetric carbon atoms present in the oligomers (and
monomeric LNAs) depending on the nature of the substituents and
possible biradicals, cf. below. The oligomers prepared according to
the method of the invention, as well as the oligomers per se, are
intended to include all stereoisomers arising from the presence of
any and all isomers of the individual monomer fragments as well as
mixtures thereof, including racemic mixtures. When considering the
5- or 6-membered ring, it is, however, believed that certain
stereochemical configurations will be especially interesting, e.g.
the following 4
[0135] where the wavy lines represent the possibility of both
diastereomers arising from the interchange of the two substituents
in question.
[0136] An especially interesting stereoisomeric representation is
the case where the LNA(s) has/have the following formula Ia 5
[0137] Also interesting as a separate aspect of the present
invention is the variant of formula Ia where B is in the
".alpha.-configuration".
[0138] In these cases, as well as generally, R.sup.3+ preferably
designates P.sup.+.
[0139] The oligomers according to the invention typically comprise
1-10000 LNA(s) of the general formula I (or of the more detailed
general formula Ia) and 0-10000 nucleosides selected from naturally
occurring nucleosides and nucleoside analogues. The sum of the
number of nucleosides and the number of LNA(s) is at least 2,
preferably at least 3, in particular at least 5, especially at
least 7, such as in the range of 2-15000, preferably in the range
of 2-100, such as 3-100, in particular in the range of 2-50, such
as 3-50 or 5-50 or 7-50.
[0140] Preferably at least one LNA comprises a nucleobase as the
substituent B.
[0141] In the present context, the term "nucleoside" means a
glycoside of a heterocyclic base. The term "nucleoside" is used
broadly as to include non-naturally occurring nucleosides,
naturally occurring nucleosides as well as other nucleoside
analogues. Illustrative examples of nucleosides are ribonucleosides
comprising a ribose moiety as well as deoxyribonuclesides
comprising a deoxyribose moiety. With respect to the bases of such
nucleosides, it should be understood that this may be any of the
naturally occurring bases, e.g. adenine, guanine, cytosine,
thymine, and uracil, as well as any modified variants thereof or
any possible unnatural bases.
[0142] When considering the definitions and the known nucleosides
(naturally occurring and non-naturally occurring) and nucleoside
analogues (including known bi- and tricyclic analogues), it is
clear that an oligomer may comprise one or more LNA(s) (which may
be identical or different both with respect to the selection of
substituent and with respect to selection of biradical) and one or
more nucleosides and/or nucleoside analogues. In the present
context "oligonucleotide" means a successive chain of nucleosides
connected via internucleoside linkages, however, it should be
understood that a nucleobase in one or more nucleotide units
(monomers) in an oligomer (oligonucleotide) may have been modified
with a substituent B as defined above.
[0143] The oligomers may be linear, branched or cyclic. In the case
of a branched oligomer, the branching points may be located in a
nucleoside, in an internucleoside linkage or, in an intriguing
embodiment, in an LNA. It is believed that in the latter case, the
substituents R.sup.2, R.sup.2+, R.sup.3, and R.sup.3+ may designate
two groups P* each designating an internucleoside linkage to a
preceding monorner, in particular, one of R.sup.2 and R.sup.2+
designate P.sup.+ and one or R.sup.3 and R.sup.3+ designated a
further P.sup.+.
[0144] As mentioned above, the LNA(s) of an oligomer are connected
with other monomers via an internucleoside linkage. In the present
context, the term "internucleoside linkage" means a linkage
consisting of 2 to 4, preferably 3, groups/atoms selected from
--CH.sub.2--, --O--, --S--, --NR.sup.H--, >C.dbd.O,
>C.dbd.NR.sup.H, >C.dbd.S, --Si(R").sub.2--, --SO--,
--S(O).sub.2--, --P(O).sub.2--, --PO(BH.sub.3)--, --P(O,S)--,
--P(S).sub.2--, --PO(R")--, --PO(OCH.sub.3)--, and
--PO(NHR.sup.H)--, where R.sup.H is selected form hydrogen and
C.sub.1-4-alkyl, and R" is selected from C.sub.1-6-alkyl and
phenyl. Illustrative examples of such internucleoside linkages are
--CH.sub.2--CH.sub.2--CH.sub.2--, --CH.sub.2--CO--CH.sub.2--,
--CH.sub.2--CHOH--CH.sub.2--, --O--CH.sub.2--O--,
--O--CH.sub.2--CH.sub.2- --, --O--CH.sub.2--CH.dbd. (including
R.sup.5 when used as a linkage to a succeeding monomer),
--CH.sub.2--CH.sub.2--O--, --NR.sup.H--CH.sub.2--CH.- sub.2--,
--CH.sub.2--CH.sub.2--NR.sup.H--, --CH.sub.2--NR.sup.H--CH.sub.2--
-, --O--CH.sub.2--CH.sub.2--NR.sup.H--, NR.sup.H--CO--O--,
--NR.sup.H--CO--NR.sup.H--, --NR.sup.H--CS--NR.sup.H--,
--NR.sup.H--C(.dbd.NR.sup.H)--NR.sup.H--,
--NR.sup.H--CO--CH.sub.2--NR.su- p.H--, --O--CO--O--,
--O--CO--CH.sub.2--O--, --O--CH.sub.2--CO--O--,
--CH.sub.2--CO--NR.sup.H--, --O--CO--NR.sup.H--,
--NR.sup.H--CO--CH.sub.2- --, --O--CH.sub.2--CO--NR.sup.H--,
--O--CH.sub.2--CH.sub.2--NR.sup.H--, --CH.dbd.N--O--,
--CH.sub.2--NR.sup.H--O--, --CH.sub.2--O--N.dbd. (including R.sup.5
when used as a linkage to a succeeding monomer),
--CH.sub.2--O--NR.sup.H--, --CO--NR.sup.H--CH.sub.2--,
--CH.sub.2--NR.sup.H--O--, --CH.sub.2--NR.sup.H--CO--,
--O--NR.sup.H--CH.sub.2--, --O--NR.sup.H--, --O--CH.sub.2--S--,
--S--CH.sub.2--O--, --CH.sub.2--CH.sub.2--S--,
--O--CH.sub.2--CH.sub.2--S- --, --S--CH.sub.2--CH.dbd. (including
R.sup.5 when used as a linkage to a succeeding monomer),
--S--CH.sub.2--CH.sub.2--, --S--CH.sub.2--CH.sub.2--- O--,
--S--CH.sub.2--CH.sub.2--S--, --CH.sub.2--S--CH.sub.2--,
--CH.sub.2--SO--CH.sub.2--, --CH.sub.2--SO.sub.2--CH.sub.2--,
--O--SO--O--, --O--S(O).sub.2--O--, --O--, S(O).sub.2--NR.sup.H--,
--NR.sup.H--S(O).sub.2--CH.sub.2--, --O--S(O).sub.2--CH.sub.2--,
--O--P(O).sub.2--O--, --O--P(O,S)--O--, --O--P(S).sub.2--O--,
--S--P(O).sub.2--O--, --S--P(O,S)--O--, --S--P(S).sub.2--O--,
--O--P(O).sub.2--S--, --O--P(O,S)--S--, --O--P(S).sub.2--S--,
--S--P(O).sub.2--S--, --S--P(O,S)--S--, --S--P(S).sub.2--S--,
--O--PO(R")--O--, --O--PO(OCH.sub.3)--O--,
--O--PO(OCH.sub.2CH.sub.3)--O-- -,
--O--PO(OCH.sub.2CH.sub.2S--R)--O--, --O--PO(BH.sub.3)--O--,
--O--PO(NHR.sup.N)--O--, --O--P(O).sub.2--NR.sup.H--,
--NR.sup.H--P(O).sub.2--O--, --O--P(O,NR.sup.H)--O--,
--CH.sub.2--P(O).sub.2--O--, --O--P(O).sub.2--CH.sub.2--, and
--O--Si(R").sub.2--O--; among which --CH.sub.2--CO--NR.sup.H--,
--CH.sub.2--NR.sup.H--O--, --S--CH.sub.2--O--,
--O--P(O).sub.2--O--, --O--P(O,S)--O--, --O--P(S).sub.2--O--,
--NR.sup.H--P(O).sub.2--O--, --O--P(O,NR.sup.H)--O--,
--O--PO(R")--O--, --O--PO(CH.sub.3)--O--, and
--O--PO(NHR.sup.N)--O--, where R.sup.H is selected form hydrogen
and C.sub.1-4-alkyl, and R' is selected from C.sub.1-6-alkyl and
phenyl, are especially preferred. Further illustrative examples are
given in Mesmaeker et. al., Current Opinion in Structural Biology
1995, 5, 343-355. The left-hand side of the internucleoside linkage
is bound to the 5- or 6-membered ring as substituent P.sup.+,
whereas the right-hand side is bound to the 5'-position of a
preceding monomer.
[0145] It is also clear from the above that the group P may also
designate a 5'-terminal group in the case where the LNA in question
is the 5'-terminal monomer. Examples of such 5'-terminal groups are
hydrogen, hydroxy, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkylcarbonyloxy, optionally substituted aryloxy,
monophosphate, diphosphate, triphosphate, and -W-A', wherein W is
selected from --O--, --S--, and --N(R.sup.H)-- where R.sup.H is
selected from hydrogen and C.sub.1-6-alkyl, and where A' is
selected from DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups,
and ligands (where the latter groups may include a spacer as
defined for the substituent B).
[0146] In the present description and claims, the terms
"monophosphate", "diphosphate", and "triphosphate" mean groups of
the formula: --O--P(O).sub.2--O.sup.-,
--O--P(O).sub.2--O--P(O).sub.2--O.sup.-, and
--O--P(O).sub.2--O--P(O).sub.2--O--P(O).sub.2--O.sup.-,
respectively.
[0147] In a particularly interesting embodiment, the group P
designates a 5'-terminal groups selected from monophosphate,
diphosphate and triphosphate. Especially the triphosphate variant
is interesting as a substrate.
[0148] Analogously, the group P* may designate a 3'-terminal group
in the case where the LNA in question is the 3'-terminal monomer.
Examples of such 3'-terminal groups are hydrogen, hydroxy,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkylcarbonyloxy, optionally substituted aryloxy, and
-W-A', wherein W is selected from --O--, --S--, and --N(R.sup.H)--
where R.sup.H is selected from hydrogen and C.sub.1-6-alkyl, and
where A' is selected from DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups, and ligands (where the latter groups may include a spacer
as defined for the substituent B).
[0149] In a preferred embodiment of the present invention, the
oligomer has the following formula V:
G--[Nu--L].sub.n(0)--{[LNA--L].sub.m(q)--[Nu--L].sub.n(q)}.sub.q--G*
V
[0150] wherein
[0151] q is 1-50;
[0152] each of n(0), . . . , n(q) is independently 0-10000;
[0153] each of m(1), . . . , m(q) is independently 1-10000;
[0154] with the proviso that the sum of n(0), . . . , n(q) and
m(1), . . . , m(q) is 2-15000;
[0155] G designates a 5'-terminal group;
[0156] each Nu independently designates a nucleoside selected from
naturally occurring nucleosides and nucleoside analogues;
[0157] each LNA independently designates a nucleoside analogue;
[0158] each L independently designates an internucleoside linkage
between two groups selected from Nu and LNA, or L together with G*
designates a 3'-terminal group; and
[0159] each LNA-L independently designates a nucleoside analogue of
the general formula I as defined above, or preferably of the
general formula Ia as defined above.
[0160] Within this embodiment, as well as generally, the present
invention provides the intriguing possibility of including LNAs
with different nucleobases, in particular both nucleobases selected
from thymine, cytosine and urasil and nucleobases selected from
adenine and guanine.
[0161] In another embodiment of the present invention, the oligomer
further comprises a PNA mono- or oligomer segment of the formula
6
[0162] wherein B is a defined above for the formula I, AASC
designates hydrogen or an amino acid side chain, t is 1-5, and w is
1-50.
[0163] In the present context, the term "amino acid side chain"
means a group bound to the .alpha.-atom of an .alpha.-amino acids,
i.e. corresponding to the .alpha.-amino acid in question without
the glycine moiety, preferably an either naturally occurring or a
readily available .alpha.-amino acid. Illustrative examples are
hydrogen (glycine itself), deuterium (deuterated glycine), methyl
(alanine), cyanomethyl (.beta.-cyano-alanine), ethyl, 1-propyl
(norvaline), 2-propyl (valine), 2-methyl-1-propyl (leucine),
2-hydroxy-2-methyl-1-propyl (.beta.-hydroxy-leucine), 1-butyl
(norleucine), 2-butyl (isoleucine), methylthioethyl (methionine),
benzyl (phenylalanine), p-amino-benzyl (p-amino-phenylalanine),
p-iodo-benzyl (p-iodo-phenylalanine), p-fluoro-benzyl
(p-fluoro-phenylalanine), p-bromo-benzyl (p-bromo-phenylalanine),
p-chloro-benzyl (p-chloro-phenylalanine), p-nitro-benzyl
(p-nitro-phenylalanine), 3-pyridylmethyl
(.beta.-(3-pyridyl)-alanine), 3,5-diiodo-4-hydroxy-benzyl
(3,5-diiodo-tyrosine), 3,5-dibromo-4-hydroxy-benzyl
(3,5-dibromo-tyrosine), 3,5-dichloro-4-hydroxy-benzyl
(3,5-dichloro-tyrosine), 3,5-difluoro-4-hydroxy-benzyl
(3,5-difluoro-tyrosine), 4-methoxy-benzyl (O-methyl-tyrosine),
2-naphtylmethyl (.beta.-(2-naphtyl)-alanine), 1-naphtylmethyl
(.beta.-(1-naphtyl)-alanine), 3-indolylmethyl (tryptophan),
hydroxymethyl (serine), 1-hydroxyethyl (threonine), mercaptomethyl
(cysteine), 2-mercapto-2-propyl (penicillamine), 4-hydroxybenzyl
(tyrosine), aminocarbonylmethyl (asparagine), 2-aminocarbonylethyl
(glutamine), carboxymethyl (aspartic acid), 2-carboxyethyl
(glutamic acid), aminomethyl (.alpha.,.beta.-diaminopropionic
acid), 2-aminoethyl (.alpha.,.gamma.-diaminobutyric acid),
3-amino-propyl (ornithine), 4-amino-1-butyl (lysine),
3-guanidino-1-propyl (arginine), and 4-imidazolylmethyl
(histidine).
[0164] PNA mono- or oligomer segment may be incorporated in a
oligomer as described in EP 0672677 A2.
[0165] The oligomers of the present invention are also intended to
cover chimeric oligomers. "Chimeric oligomers" means two or more
oligomers with monomers of different origin joined either directly
or via a spacer. Illustrative examples of such oligomers which can
be combined are peptides, PNA-oligomers, oligomers containing
LNA's, and oligonucleotide oligomers.
[0166] Apart from the oligomers defined above, the present
invention also provides monomeric LNAs useful, e.g., in the
preparation of oligomers, as substrates for, e.g., nucleic acid
polymerases, polynucleotide kinases, terminal transferases, and as
therapeutical agents, see further below. The monomeric LNAs
correspond in the overall structure (especially with respect to the
possible biradicals) to the LNAs defined as constituents in
oligomers, however with respect to the groups P and P.sup.+, the
monomeric LNAs differ slightly as will be explained below.
Furthermore, the monomeric LNAs may comprise functional group
protecting groups, especially in the cases where the monomeric LNAs
are to be incorporated into oligomers by chemical synthesis.
[0167] An interesting subgroup of the possible monomeric LNAs
comprises bicyclic nucleoside analogues (LNAs) of the general
formula II 7
[0168] wherein the substituent B is selected from nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands; X is
selected from --O--, --S--, --N(R.sup.N+)--, and
--C(R.sup.6R.sup.6+)--, preferably from --O--, --S--, and
--N(R.sup.N+)--; one of the substituents R.sup.2, R.sup.2+,
R.sup.3, and R.sup.3+ is a group Q*;
[0169] each of Q and Q.sup.+ is independently selected from
hydrogen, azido, halogen, cyano, nitro, hydroxy, Prot-O--, Act-O--,
mercapto, Prot-S--, Act-S--, C.sub.1-6-alkylthio, amino,
Prot-N(R.sup.H)--, Act-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate, diphosphzate, triphosphate,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, ligands, carboxy,
sulphono, hydroxymethyl, Prot-O--CH.sub.2--, Act-O--CH.sub.2--,
aminomethyl, Prot-N(R.sup.H)--CH.sub.2--,
Act-N(R.sup.H)--CH.sub.2--, carboxymethyl, sulphonomethyl, where
Prot is a protection group for --OH, --SH, and --NH(R.sup.H),
respectively, Act is an activation group for --OH, --SH, and
--NH(R.sup.H), respectively, and R.sup.H is selected from hydrogen
and C.sub.1-6-alkyl;
[0170] R.sup.2+ and R.sup.4+ together designate a biradical
selected from --O--, --S--, --N(R.sup.+)--,
--(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O--,
--S--(CR.sup.+R.sup.+).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; R.sup.2 and R.sup.3
together designate a biradical selected from --O--,
--(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.s- up.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.2+ and R.sup.3 together designate a biradical selected from
--O--, --(CR.sup.+R.sup.+).sub.r+s--,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.s- up.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.3 and R.sup.4+ together designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.3 and R.sup.5 together designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
R.sup.1+ and R.sup.4 + together designate a biradical selected from
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
or R.sup.1+ and R.sup.2+ together designate a biradical selected
from --(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--, and
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--;
wherein R.sup.+ is as defined above for the oligomers; and each of
the substituents R.sup.1+, R.sup.2, R.sup.2+, R.sup.3, R.sup.4+,
R.sup.5, and R.sup.5+, which are not involved in Q, Q.sup.+ or the
biradical, are as defined above for the oligomers.
[0171] It should furthermore be understood, with due consideration
of the known bicyclic nucleoside analogues, that R.sup.3 and
R.sup.5 do not together designate a biradical selected from
--CH.sub.2--CH.sub.2--, --O--CH.sub.2--, and
--O--Si(.sup.iPr).sub.2--O--Si(.sup.iPr).sub.2--O--.
[0172] The monomeric LNAs also comprise basic salts and acid
addition salts thereof. Furthermore, it should be understood that
any chemical group (including any nucleobase), which is reactive
under the conditions prevailing in chemical oligonucleotide
synthesis, is optionally functional group protected as known in the
art. This means that groups such as hydroxy, amino, carboxy,
sulphono, and mercapto groups, as well as nucleobases, of a
monomeric LNA are optionally functional group protected. Protection
(and deprotection) is performed by methods known to the person
skilled in the art (see, e.g., Greene, T. W. and Wuts, P. G. M.,
"Protective Groups in Organic Synthesis", 2.sup.nd ed., John Wiley,
N.Y. (1991), and M. J. Gait, Oligonucleotide Synthesis, IRL Press,
1984).
[0173] Illustrative examples of hydroxy protection groups are
optionally substituted trityl, such as 4,4'-dimethoxytrityl (DMT),
4-monomethoxytrityl (MMT), and trityl, optionally substituted
9-(9-phenyl)xanthenyl (pixyl), optionally substituted
ethoxycarbonyloxy, p-phenylazophenyloxycarbonyloxy,
tetraahydropyranyl (thp), 9-fluorenylmethoxycarbonyl (Fmoc),
methoxytetrahydropyranyl (mthp), silyloxy such as trimethylsilyl
(TMS), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS),
triethylsilyl, and phenyldimethylsilyl, benzyloxycarbonyl or
substituted benzyloxycarbonyl ethers such as 2-bromo
benzyloxycarbonyl, tert-butylethers, alkyl ethers such as methyl
ether, acetals (including two hydroxy groups), acyloxy such as
acetyl or halogen substituted acetyls, e.g. chloroacetyl or
fluoroacetyl, isobutyryl, pivaloyl, benzoyl and substituted
benzoyls, methoxymethyl (MOM), benzyl ethers or substituted benzyl
ethers such as 2,6-dichlorobenzyl (2,6-Cl.sub.2Bzl). Alternatively,
the hydroxy group may be protected by attachment to a solid support
optionally through a linker.
[0174] Illustrative examples of amino protection groups are Fmoc
(fluorenylmethoxycarbonyl), BOC (tert-butyloxycarbonyl),
trifluoroacetyl, allyloxycarbonyl (alloc, AOC), benzyloxycarbonyl
(Z, Cbz), substituted benzyloxycarbonyls such as 2-chloro
benzyloxycarbonyl (2-CIZ), monomethoxytrityl (MMT), dimethoxytrityl
(DMT), phthaloyl, and 9-(9-phenyl)xanthenyl (pixyl).
[0175] Illustrative examples of carboxy protection groups are allyl
esters, methyl esters, ethyl esters, 2-cyanoethylesters,
trimethylsilylethylesters, benzyl esters (Obzl), 2-adamantyl esters
(O-2-Ada), cyclohexyl esters (OcHex), 1,3-oxazolines, oxazoler,
1,3-oxazolidines, amides or hydrazides.
[0176] Illustrative examples of mercapto protecting groups are
trityl (Trt), acetamidomethyl (acm), trimethylacetamidomethyl
(Tacm), 2,4,6-trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu),
9-fluorenylmethyl (Fm), 3-nitro-2-pyridinesulfenyl (Npys), and
4-methylbenzyl (Meb).
[0177] Furthermore, it may be necessary or desirable to protect any
nucleobase included in an monomeric LNA, especially when the
monomeric LNA is to be incorporated in an oligomer according to the
invention. In the present context, the term "protected nucleobases"
means that the nucleobase in question is carrying a protection
group selected among the groups which are well-known for a man
skilled in the art (see e.g. Protocols for Oligonucleotides and
Analogs, vol 20, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa,
N.J.; S. L. Beaucage and R. P. Iyer, Tetrahedoron, 1993, 49, 6123;
S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223; and E.
Uhlmann and A. Peyman, Chem. Rev., 90, 543.). Illustrative examples
are benzoyl, isobutyryl, tert-butyl, tert-butyloxycarbonyl,
4-chloro-benzyloxycarbonyl, 9-fluorenylmethyl,
9-fluorenylmethyloxycarbonyl, 4-methoxybenzoyl,
4-methoxytriphenylmethyl, optionally substituted triazolo,
p-toluenesulphonyl, optionally substituted sulphonyl, isopropyl,
optionally substituted amidines, optionally substituted trityl,
phenoxyacetyl, optionally substituted acyl, pixyl,
tetrahydropyranyl, optionally substituted silyl ethers, and
4-methoxybenzyloxycarbonyl. Chapter 1 in "Protocols for
oligonucleotide conjugates", Methods in Molecular Biology, vol 26,
(Sudhir Agrawal, ed.), Humana Press, 1993, Totowa, N.J. and S. L.
Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223 disclose
further suitable examples.
[0178] In a preferred embodiment, the group B in a monomeric LNA is
preferably selected from nucleobases and protected nucleobases.
[0179] In an embodiment of the monomeric LNAs according to the
present invention, one of Q and Q.sup.+, preferably Q.sup.+,
designates a group selected from Act-O--, Act-S--,
Act-N(R.sup.H)--, Act-O--CH.sub.2--, Act-S--CH.sub.2--,
Act-N(R.sup.H)--CH.sub.2--, and the other of Q and Q.sup.+,
preferably Q, designates a group selected from hydrogen, azido,
halogen, cyano, nitro, hydroxy, Prot-O--, mercapto, Prot-S--,
C.sub.1-6-alkylthio, amino, Prot-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, ligands, carboxy,
sulphono, hydroxymethyl, Prot-O--CH.sub.2--, aminomethyl,
Prot-N(R.sup.H)--CH.sub.2- --, carboxymethyl, sulphonomethyl, and
R.sup.H is selected from hydrogen and C.sub.1-6-alkyl.
[0180] In the case described above, the group Prot designates a
protecting group for --OH, --SH, and --NH(R.sup.H), respectively.
Such protection groups are selected from the same as defined above
for hydroxy protection groups, mercapto protection group, and amino
protection groups, respectively, however taking into consideration
the need for a stable and reversible protection group. However, it
is preferred that any protection group for --OH is selected from
optionally substituted trityl, such as dimethoxytrityl (DMT),
monomethoxytrityl (MMT), and trityl, and 9-(9-phenyl)xanthenyl
(pixyl), optionally substituted, tetrahydropyranyl (thp) (further
suitable hydroxy protection groups for phosphoramidite
oligonucleotide synthesis are described in Agrawal, ed. "Protocols
for Oligonucleotide Conjugates", Methods in Molecular Biology, vol.
26, Humana Press, Totowa, N.J. (1994) and Protocols for
Oligonucleotides and Analogs, vol 20, (Sudhir Agrawal, ed.), Humana
Press, 1993, Totowa, N.J.), or protected as acetal; that any
protection group for --SH is selected from trityl, such as
dimethoxytrityl (DMT), monomethoxytrityl (MMT), and trityl, and
9-(9-phenyl)xanthenyl (pixyl), optionally substituted,
tetrahydropyranyl (thp) (further suitable mercapto protection
groups for phosphoramidite oligonucleotide synthesis are also
described in Agrawal (see above); and that any protecting group for
--NH(R.sup.H) is selected from trityl, such as dimethoxytrityl
(DMT), monomethoxytrityl (MMT), and trityl, and
9-(9-phenyl)xanthenyl (pixyl), optionally substituted,
tetrahydropyranyl (thp) (further suitable amino protection groups
for phosphoramidite oligonucleotide synthesis are also described in
Agrawal (see above).
[0181] In the embodiment above, as well as for any monomeric LNAs
defined herein, Act designates an activation group for --OH, --SH,
and --NH(R.sup.H), respectively. Such activation groups are, eg.,
selected from optionally substituted O-phosphoramidite, optionally
substituted O-phosphortriester, optionally substituted
O-phosphordiester, optionally substituted H-phosphonate, and
optionally substituted O-phosphonate.
[0182] In the present context, the term "phosphoramidite" means a
group of the formula --P(OR.sup.x)--N(R.sup.y).sub.2, wherein
R.sup.x designates an optionally substituted alkyl group, e.g.
methyl, 2-cyanoethyl, or benzyl, and each of R.sup.y designate
optionally substituted alkyl groups, e.g. ethyl or isopropyl, or
the group --N(R.sup.y).sub.2 forms a morpholino group
(--N(CH.sub.2CH.sub.2).sub.2O). R.sup.x preferably designates
2-cyanoethyl and the two R.sup.y are preferably identical and
designate isopropyl. Thus, an especially relevant phosphoramidite
is N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.
[0183] It should be understood that the protecting groups used
herein for a single monomeric LNA or several monomeric LNAs may be
selected so that when this/these LNA(s) are incorporated in an
oligomer according to the invention, it will be possible to perform
either a simultaneous deprotection or a sequential deprotection of
the functional groups. The latter situation opens for the
possibility of regioselectively introducing one or several
"active/functional" groups such as DNA intercalators,
photochemically active groups, thermochemically active groups,
chelating groups, reporter groups, and ligands, where such groups
may be attached via a spacer as described above.
[0184] In a preferred embodiment, Q is selected from hydrogen,
azido, halogen, cyano, nitro, hydroxy, Prot-O--, mercapto,
Prot-S--, C.sub.1-6-alkylthio, amino, Prot-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate, diphosphate, triphosphate, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, ligands, carboxy,
sulphono, hydroxymethyl, Prot-O--CH.sub.2--, aminomethyl,
Prot-N(R.sup.H)--CH.sub.2- --, carboxymethyl, sulphonomethyl, where
Prot is a protection group for --OH, --SH, and --NH(R.sup.H),
respectively, and R.sup.H is selected from hydrogen and
C.sub.1-6-alkyl; and Q.sup.+ is selected from hydrogen, azido,
halogen, cyano, nitro, hydroxy, Act-O--, mercapto, Act-S--,
C.sub.1-6-alkylthio, amino, Act-N(R.sup.H)--, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, optionally substituted
C.sub.2-6-alkynyloxy, DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups, ligands, carboxy, sulphono, where Act is an activation
group for --OH, --SH, and --NH(R.sup.H), respectively, and R.sup.H
is selected from hydrogen and C.sub.1-6)alkyl.
[0185] The monomeric LNAs of the general formula II may, as the
LNAs incorporated into oligomers, represent various stereoisomers.
Thus, the stereochemical variants described above for the LNAs
incorporated into oligomers are believed to be equally applicable
in the case of monomeric LNAs (however, it should be noted that P
should then be replaced with Q).
[0186] In a preferred embodiment of the present invention, the
monomeric LNA has the general formula IIa 8
[0187] wherein the substituents are defined as above.
[0188] Furthermore, with respect to the definitions of
substituents, biradicals, R.sup.+, etc. the same preferred
embodiments as defined above for the oligomer according to the
invention also apply in the case of monomeric LNAs.
[0189] In a particularly interesting embodiment of the monomeric
LNAs of the present nvention, B designates a nucleobase, preferably
a nucleobase selected from thymine, cytosine, urasil, adenine and
guanine (in particular adenine and guanine), X is --O--, R.sup.2+
and R.sup.4+ together designate a biradical selected from
--(CH.sub.2).sub.0-1--O--(CH- .sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3--, in
particular --O--CH.sub.2--, --S--CH.sub.2-- and
--R.sup.N--CH.sub.2--, where R.sup.N is selected from hydrogen and
C.sub.1-4-alkyl, Q designates Prot-O--, R.sup.3+ is Q.sup.+ which
designates Act-OH, and R.sup.1+, R.sup.2, R.sup.3, R.sup.5, and
R.sup.5+ each designate hydrogen. In this embodiment, R.sup.N may
also be selected from DNA intercalators, photochemically active
groups, thermochemically active groups, chelating groups, reporter
groups and ligands.
[0190] In a further particularly interesting embodiment of the
monomeric LNAs of the present invention, B designates a nucleobase,
preferably a nucleobase selected from thymine, cytosine, urasil,
adenine and guanine (in particular adenine and guanine), X is
--O--, R.sup.2+ and R.sup.4+ together designate a biradical
selected from --(CH.sub.2).sub.0-1--O--(CH- .sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3--, in
particular --O--CH.sub.2--, --S--CH.sub.2-- and
--R.sup.N--CH.sub.2--, where R.sup.N is selected from hydrogen and
C.sub.1-4-alkyl, Q is selected from hydroxy, mercapto,
C.sub.1-6-alkylthio, amino, mono- or di(C.sub.1-6-alkyl)amino,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.2-6-alkenyloxy, optionally substituted C.sub.2-6-alkynyloxy,
monophosphate, diphosphate, and triphosphate, R.sup.3+ is Q.sup.+
which is selected from hydrogen, azido, halogen, cyano, nitro,
hydroxy, mercapto, C.sub.1-6-alkylthio, amino, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted C.sub.2-6-alkenyloxy,
optionally substituted C.sub.2-6-alkynyl, and optionally
substituted C.sub.2-6-alkynyloxy, R.sup.3 is selected from
hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, and optionally substituted
C.sub.2-6-alkynyl, and R.sup.1+, R.sup.2, R.sup.5, and R.sup.5+
each designate hydrogen. Also here, R.sup.N may also be selected
from DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups
and ligands.
[0191] In a further particularly interesting embodiment of the
monomeric LNAs of the present invention, B designates a nucleobase,
X is --O--, R.sup.2 and R.sup.3 together designate a biradical
selected from --(CH.sub.2).sub.0-1--O--CH.dbd.CH--,
--(CH.sub.2).sub.0-1--S--CH.dbd.CH-- -, and
--(CH.sub.2).sub.0-1--N(R.sup.N)--CH.dbd.CH-- where R.sup.N is
selected from hydrogen and C.sub.1-4-alkyl, Q is selected from
hydroxy, mercapto, C.sub.1-6-alkylthio, amino, mono- or
di(C.sub.1-6-alkyl)amino, optionally substituted C.sub.1-6-alkoxy,
optionally substituted C.sub.2-6-alkenyloxy, optionally substituted
C.sub.2-6-alkynyloxy, monophosphate, diphosphate, and triphosphate,
R.sup.3+ is Q.sup.+ which is selected from hydrogen, azido,
halogen, cyano, nitro, hydroxy, mercapto, C.sub.1-6-alkylthio,
amino, mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, optionally substituted
C.sub.2-6-alkenyloxy, optionally substituted C.sub.2-6-alkynyl, and
optionally substituted C.sub.2-6-alkynyloxy, and R.sup.1+,
R.sup.2+, R.sup.4+, R.sup.5, and R.sup.5+ each designate
hydrogen.
[0192] One aspect of the invention is to provide various
derivatives of LNAs for solid-phase and/or solution phase
incorporation into an oligomer. As an illustrative example,
monomers suitable for incorporation of
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyc-
lo[2.2.1]heptane,
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(cytosin-1-yl)-
-2,5-dioxabicyclo[2.2.1 ]heptane, (1S,3R,4R,
7S)-7-hydroxy-1-hydroxymethyl-
-3-(urasil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,
(1S,3R,4R,7S)-7-hydroxy-1-
-hydroxymethyl-3-(guanin-1-yl)-2,5-dioxabicyclo-[2.2.1]heptane, and
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(adenin-1-yl)-2,5-dioxabicyclo[-
2.2.1]heptane using the phosphoramidite approach, the
phosphortriester approach, and the H-phosphonate approach,
respectively, are
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4'-dime-
thoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,
(1R,3R,4R,7S)-7-hydroxy-1-(4,4'-di-methoxytrityloxymethyl)-3-(thymin-1-yl-
)-2,5-dioxabicyclo[2.2.1 ]heptane-7-O-(2-chlorophenylphosphate),
and
(1R,3R,4R,7S,)-7-hydroxy-1-(4,4'-dimethoxytrityloxymethyl)-3-(thymin-1-yl-
)-2,5-dioxabicyclo[2.2.1]heptane-7-O-(H-phosphonate) and the
3-(cytosin-1-yl), 3-(urasil-1-yl), 3-(adenin-1-yl) and
3-(guanin-1-yl) analogues thereof, respectively. Furthermore, the
analogues where the methyleneoxy biradical of the monomers is
substituted with a methylenethio, a methyleneamino, or a
1,2-ethylene biradical are also expected to constitute particularly
interesting variants within the present invention. The
methylenethio and methyleneamino analogues are believed to equally
applicable as the methyleneoxy analogue and therefore the specific
reagents corresponding to those mentioned for incorporation of
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxa-bicy-
clo[2.2.1]heptane,
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(cytosin-1yl)-
-2,5-dioxabicyclo[2.2.1]heptane,
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-
-(urasil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane,
(1S,3R,4R,7S)-7-hydroxy-1-h-
ydroxymethyl-3-(guanin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane, and
(1S,3R,4R,7S)-7-hydroxy-1-hydroxymethyl-3-(adenin-1-yl)-2,5-dioxabicyclo[-
2.2.1]heptane should also be considered as particularly interesting
reactive monomers within the present invention. For the
methyleneamine analogue, it should be noted that the secondary
amine may carry a substituent selected from optionally substituted
C.sub.1-6-alkyl such as methyl and benzyl, optionally substituted
C.sub.1-6-alkylcarbonyl such as trifluoroacetyl, optionally
substituted arylcarbonyl and optionally substituted
heteroarylcarbonyl.
[0193] In a particularly interesting embodiment, the present
invention relates to an oligomer comprising at least one LNA of the
general formula Ia 9
[0194] wherein X is --O--; B is selected from nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands; P
designates the radical position for an internucleoside linkage to a
succeeding monomer, or a 5'-terminal group, such internucleoside
linkage or 5'-terminal group optionally including the substituent
R.sup.5; R.sup.3+ is a group P* which designates an internucleoside
linkage to a preceding monomer, or a 3'-terminal group; R.sup.2+
and R.sup.4+ together designate a biradical selected from --O--,
--S, --N(R.sup.+)--, --(CR.sup.+R.sup.+).sub.r+s+1--- ,
--(CR.sup.+R.sup.+).sub.r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).sub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O,
--S--(CR.sup.+R.sup.+).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--,
--S--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.- r+s--N(R.sup.+)--,
--N(R.sup.+)--(CR.sup.+R.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; wherein each R.sup.+
is independently selected from hydrogen, halogen, azido, cyano,
nitro, hydroxy, mercapto, amino, mono- or di(C.sub.1-6-alkyl)amino,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkyl, DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups,
and ligands, and/or two adjacent (non-geminal) R.sup.+ may together
designate a double bond, and each of r and s is 0-3 with the
proviso that the sum r+s is 1-4; each of the substituents R.sup.1+,
R.sup.2, R.sup.3, R.sup.5, and R.sup.5+ is independently selected
from hydrogen, optionally substituted C.sub.1-6-alkyl, optionally
substituted C.sub.2-6-alkenyl, hydroxy, C.sub.1-6-alkoxy,
C.sub.2-6-alkenyloxy, carboxy, C.sub.1-6-alkoxycarbonyl- ,
C.sub.1-6-alkylcarbonyl, formyl, amino, mono- and
di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-- carbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, azido,
C.sub.1-6-alkanoyloxy, sulphono, sulphanyl, C.sub.1-6-alkylthio,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
halogen, where two geminal substituents together may designate oxo;
and basic salts and acid addition salts thereof. In particular, one
R.sup.+ is selected from hydrogen, hydroxy, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
any remaining substituents R.sup.+ are hydrogen. Especially, the
biradical is selected from --O--,
--(CH.sub.2).sub.0-1--O--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--S--(- CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.2-4-- -.
[0195] In a further particularly interesting embodiment, the
present invention relates to an LNA of the general formula IIa
10
[0196] wherein X is --O--; B is selected from nucleobases, DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands;
R.sup.3+ is a group Q*; each of Q and Q.sup.+ is independently
selected from hydrogen, azido, halogen, cyano, nitro, hydroxy,
Prot-O--, Act-O--, mercapto, Prot-S--, Act-S--,
C.sub.1-6-alkylthio, amino, Prot-N(R.sup.H)--, Act-N(R.sup.H)--,
mono- or di(C.sub.1-6-alkyl)amino, optionally substituted
C.sub.1-6-alkoxy, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, optionally substituted
C.sub.2-6-alkenyloxy, optionally substituted C.sub.2-6-alkynyl,
optionally substituted C.sub.2-6-alkynyloxy, monophosphate,
diphosphate, triphosphate, DNA intercalators, photochemically
active groups, thermochemically active groups, chelating groups,
reporter groups, ligands, carboxy, sulphono, hydroxymethyl,
Prot-O--CH.sub.2--, Act-O--CH.sub.2--, aminomethyl,
Prot-N(R.sup.H)--CH.sub.2--, Act-N(R.sup.H)--CH.sub.2--,
carboxymethyl, sulphonomethyl, where Prot is a protection group for
--OH, --SH, and --NH(R.sup.H), respectively, Act is an activation
group for --OH, --SH, and --NH(R.sup.H), respectively, and R.sup.H
is selected from hydrogen and C.sub.1-6-alkyl; R.sup.2+ and
R.sup.4+ together designate a biradical selected from --O--, --S,
--N(R.sup.+)--, --(CR.sup.+R.sup.+).sub.r+s+1--,
--(CR.sup.+R.sup.+).sub.- r--O--(CR.sup.+R.sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--S--(CR.sup.+R.- sup.+).sub.s--,
--(CR.sup.+R.sup.+).sub.r--N(R.sup.+)--(CR.sup.+R.sup.+).s- ub.s--,
--O--(CR.sup.+R.sup.+).sub.r+s--O--, --S--(CR.sup.+R.sup.+).sub.r+-
s--O--, --O--(CR.sup.+R.sup.+).sub.r+s--S--,
--N(R.sup.+)--(CR.sup.+R.sup.- +).sub.r+s--O--,
--O--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--, --S--(CR.sup.30
R.sup.+).sub.r+s--S--, --N(R.sup.+)--(CR.sup.+R.sup.+).su-
b.r+s--N(R.sup.+)--, --N(R.sup.+)--(CR.sup.+).sub.r+s--S--, and
--S--(CR.sup.+R.sup.+).sub.r+s--N(R.sup.+)--; wherein each R.sup.+
independently selected from hydrogen, halogen, azido, cyano, nitro,
hydroxy, mercapto, amino, mono- or di(C.sub.1-6-alkyl)amino,
optionally substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkyl, DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups,
and ligancis, and/or two adjacent (non-geminal) R.sup.+ may
together designate a double bond, and each of r and s is 0-3 with
the proviso that the sum r+s is 1-4; each of the substituents
R.sup.1+, R.sup.2, R.sup.3, R.sup.5, and R.sup.5+ is independently
selected from hydrogen, optionally substituted C.sub.1-6-alkyl,
optionally substituted C.sub.2-6-alkenyl, hydroxy,
C.sub.1-6-alkoxy, C.sub.2-6-alkenyloxy, carboxy,
C.sub.1-6-alkoxycarbonyl- , C.sub.1-6-alkylcarbonyl, formyl, amino,
mono- and di(C.sub.1-6-alkyl)amino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-amino-- carbonyl,
C.sub.1-6-alkyl-carbonylamino, carbamido, azido,
C.sub.1-6-alkanoyloxy, sulphono, sulphanyl, C.sub.1-6-alkylthio,
DNA intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands, and
halogen, where two geminal substituents together may designate oxo;
and basic salts and acid addition salts thereof; and with the
proviso that any chemical group (including any nucleobase), which
is reactive under the conditions prevailing in oligonucleotide
synthesis, is optionally functional group protected. Preferably,
one R.sup.+ is selected from hydrogen, hydroxy, optionally
substituted C.sub.1-6-alkoxy, optionally substituted
C.sub.1-6-alkyl, DNA intercalators, photochemically active groups,
thermochemically active groups, chelating groups, reporter groups,
and ligands, and any remaining substituents R.sup.+ are hydrogen.
Especially, the biradical is selected from --O--,
--(CH.sub.2).sub.0-1--O- --(CH.sub.2).sub.1-3--,
--(CH.sub.3).sub.0-1--S--(CH.sub.2).sub.1-3--,
--(CH.sub.2).sub.0-1--N(R.sup.N)--(CH.sub.2).sub.1-3--, and
--(CH.sub.2).sub.2-4--.
[0197] Generally, the present invention provides oligomers having
surprisingly good hybridisation properties with respect to affinity
and specificity. Thus, the present invention provides an oligomer
comprising at least one nucleoside analogue which imparts to the
oligomer a T.sub.m with a complementary DNA oligonucleotide which
is at least 2.5.degree. C. higher, preferably at least 3.5.degree.
C. higher, in particular at least 4.0.degree. C. higher, especially
at least 5.0.degree. C. higher, than that of the corresponding
unmodified reference oligonucleotide which does not comprise any
nucleoside analogue. In particular, the T.sub.m of the oligomer is
at least 2.5.times.N .degree.C. higher, preferably at least
3.5.times.N .degree.C. higher, in particular at least 4.0.times.N
.degree.C. higher, especially at least 5.0.times.N .degree.C.
higher, where N is the number of nucleoside analogues.
[0198] In the case of hybridisation with a complementary RNA
oligonucleotide, the at least one nucleoside analogue imparts to
the oligomer a T.sub.m with the complementary DNA oligonucleotide
which is at least 4.0.degree. C. higher, preferably at least
5.0.degree. C. higher, in particular at least 6.0.degree. C.
higher, especially at least 7.0.degree. C. higher, than that of the
corresponding unmodified reference oligonucleotide which does not
comprise any nucleoside analogue. In particular, the T.sub.m of the
oligomer is at least 4.0.times.N .degree.C higher, preferably at
least 5.0.times.N.degree. C. higher, in particular at least
6.0.times.N .degree.C. higher, especially at least 7.0.times.N
.degree.C. higher, where N is the number of nucleoside
analogues.
[0199] The term "corresponding unmodified reference
oligonucleotide" is intended to mean an oligonucleotide solely
consisting of naturally occurring nucleotides which represents the
same nucleobases in the same absolute order (and the same
orientation).
[0200] The T.sub.m is measured under one of the following
conditions (i.e. essentially as illustrated in Example 129):
[0201] a) 10 mM Na.sub.2HPO.sub.4, pH 7.0, 100 mM NaCl, 0.1 mM
EDTA;
[0202] b) 10 mM Na.sub.2HPO.sub.4, pH 7.0, 0.1 mM EDTA; or
[0203] c) 3M tetrametylammoniumchlorid (TMAC), 10 mM
Na.sub.2HPO.sub.4, pH 7.0, 0.1 mM EDTA;
[0204] preferably under conditions a), at equimolar amounts
(typically 1.0 .mu.M) of the oligomer and the complementary DNA
oligonucleotide.
[0205] The oligomer is preferably as defined above, where the at
least one nucleoside analogue has the formula I where B is a
nucleobase. In particular interesting is the cases where at least
one nucleoside analogue includes a nucleobase selected from adenine
and guanine.
[0206] Furthermore, with respect to specificity and affinity, the
oligomer, when hybridised with a partially complementary DNA
oligonucleotide, or a partially complementary RNA oligonucleotide,
having one or more mismatches with said oligomer, should exhibit a
reduction in T.sub.m, as a result of said mismatches, which is
equal to or greater than the reduction which would be observed with
the corresponding unmodified reference oligonucleotide which does
not comprise any nucleoside analogues. Also, the oligomer should
have substantially the same sensitivity of T.sub.m to the ionic
strength of the hybridisation buffer as that of the corresponding
unmodified reference oligonucleotide.
[0207] Oligomers defined herein are typically at least 30%
modified, preferably at least 50% modified, in particular 70%
modified, and in some interesting applications 100% modified.
[0208] The oligomers of the invention has substantially higher
3'-exonucleolytic stability than the corresponding unmodified
reference oligonucleotide. This important property can be examined
as described in Example 136.
[0209] Definitions
[0210] In the present context, the term "C.sub.1-12-alkyl" means a
linear, cyclic or branched hydrocarbon group having 1 to 12 carbon
atoms, such as methyl, ethyl, propyl, iso-propyl, cyclopropyl,
butyl, tert-butyl, iso-butyl, cyclobutyl, pentyl, cyclopentyl,
hexyl, cyclohexyl, and dodecyl. Analogously, the term
"C.sub.1-6-alkyl" means a linear, cyclic or branched hydrocarbon
group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl,
iso-propyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the term
"C.sub.1-4-alkyl" is intended to cover linear, cyclic or branched
hydrocarbon groups having 1 to 4 carbon atoms, e.g. methyl, ethyl,
propyl, iso-propyl, cyclopropyl, butyl, iso-butyl, tert-butyl,
cyclobutyl.
[0211] Preferred examples of "C.sub.1-6-alkyl" are methyl, ethyl,
propyl, iso-propyl, butyl, tert-butyl, iso-butyl, pentyl,
cyclopentyl, hexyl, cyclohexyl, in particular methyl, ethyl,
propyl, iso-propyl, tert-butyl, iso-butyl and cyclohexyl. Preferred
examples of "C.sub.1-4-alkyl" are methyl, ethyl, propyl,
iso-propyl, butyl, tert-butyl, and iso-butyl.
[0212] Similarly, the term "C.sub.2-12-alkenyl" covers linear,
cyclic or branched hydrocarbon groups having 2 to 12 carbon atoms
and comprising one unsaturated bond. Examples of alkenyl groups are
vinyl, allyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,
dodecaenyl. Analogously, the term "C.sub.2-6-alkenyl" is intended
to cover linear, cyclic or branched hydrocarbon groups having 2 to
6 carbon atoms and comprising one unsaturated bond. Preferred
examples of alkenyl are vinyl, allyl, butenyl, especially
allyl.
[0213] Similarly, the term "C.sub.2-12-alkynyl" means a linear or
branched hydrocarbon group having 2 to 12 carbon atoms and
comprising a triple bond. Examples hereof are ethynyl, propynyl,
butynyl, octynyl, and dodecanyl.
[0214] In the present context, ie. in connection with the terms
"alkyl", "alkenyl", and "alkynyl", the term "optionally
substituted" means that the group in question may be substituted
one or several times, preferably 1-3 times, with group(s) selected
from hydroxy (which when bound to an unsaturated carbon atom may be
present in the tautomeric keto form), C.sub.1-6-alkoxy (i.e.
C.sub.1-6-alkyl-oxy), C.sub.2-6-alkenyloxy, carboxy, oxo (forming a
keto or aldehyde functionality), (C.sub.1-6-alkoxycarbonyl,
C.sub.1-6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl, aryloxy,
arylcarbonyl, heteroaryl, heteroaryloxycarbonyl, heteroaryloxy,
heteroarylcarbonyl, amino, amino- and di(C.sub.1-6-alkyl)amino;
carbamoyl, mono- and di(C.sub.1-6-alkyl)aminocarbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkylcarbonylamino, cyano, guanidino, carbamido,
C.sub.1-6-alkanoyloxy, sulphono, C.sub.1-6-alkylsulphonyloxy,
nitro, sulphanyl, C.sub.1-6-alkylthio, halogen, where any aryl and
heteroaryl may be substituted as specifically describe below for
"optionally substituted aryl and heteroaryl".
[0215] Preferably, the substituents are selected from hydroxy,
C.sub.1-6-alkoxy, carboxy, C.sub.1-6-alkoxycarbonyl,
C.sub.1-6-alkylcarbonyl, formyl, aryl, aryloxycarbonyl,
arylcarbonyl, heteroaryl, amino, mono- and
di(C.sub.1-6-alkyl)aimino, carbamoyl, mono- and
di(C.sub.1-6-alkyl)-aminocarbonyl,
amino-C.sub.1-6-alkyl-aminocarbony- l, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkylcarbonylamirio, cyano, carbamido, halogen, where
aryl and heteroaryl may be substituted 1-5 times, preferably 1-3
times, with C.sub.1-4-alkyl, C.sub.1-4-alkoxy, nitro, cyano, amino
or halogen. Especially preferred examples are hydroxy,
C.sub.1-6-alkoxy, carboxy, aryl, heteroaryl, amino, mono- and
di(C.sub.1-6-alkyl)amino, and halogen, here aryl and heteroaryl may
be substituted 1-3 times with C.sub.1-4-alkyl, C.sub.1-4-alkoxy,
nitro, cyano, amino or halogen.
[0216] In the present context the term "aryl" means a fully or
partially aromatic carbocyclic ring or ring system, such as phenyl,
naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracyl, phenanthracyl,
pyrenyl, benzopyrenyl, fluorenyl and xanthenyl, among which phenyl
is a preferred example.
[0217] The term "heteroaryl" means a fully or partially aromatic
carbocyclic ring or ring system where one or more of the carbon
atoms have been replaced with heteroatoms, e.g. nitrogen (.dbd.N--
or --NH), sulphur, and/or oxygen atoms. Examples of such heteroaryl
groups are oxazolyl, isoxazolyl, thiiazolyl, isothiazolyl,
pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl,
piperidinyl, coumaryl, furyl, quinolyl, benzothiazolyl,
benzotriazolyl, benzodiazolyl, benzooxozolyl, phthalazinyl,
phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl,
carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl,
phenoxazonyl.
[0218] In the present context, i.e. in connection with the terms
"aryl" and "heteroaryl", the term "optionally substituted" means
that the group in question may be substituted one or several times,
preferably 1-5 times, in particular 1-3 times) with group(s)
selected from hydroxy (which when present in an enol system may be
represented in the tautomeric keto form), C.sub.1-6-alkyl,
C.sub.1-6-alkoxy, oxo (which may be represented in the tautomeric
enol form), carboxy, C.sub.1-6-alkoxycarbonyl,
C.sub.1-6-alkylcarbonyl, formyl, aryl, aryloxy, aryloxycarbonyl,
arylcarbonyl, heteroaryl, amino, mono- and
di(C.sub.1-6-alkyl)amino; carbamoyl, mono- and
di(C.sub.1-6-alkyl)aminoca- rbonyl,
amino-C.sub.1-6-alkyl-aminocarbonyl, mono- and
di(C.sub.1-6-alkyl)amino-C.sub.1-6-alkyl-aminocarbonyl,
C.sub.1-6-alkylcarbonylamino, cyano, guanidino, carbamido,
C.sub.1-6-alkanoyloxy, sulphono, C.sub.1-6-alkylsulphonyloxy,
nitro, sulphanyl, dihalogen-C.sub.1-4-alkyl,
trihalogen-C.sub.1-4-alkyl, halogen, where aryl and heteroaryl
representing substituents may be substituted 1-3 times with
C.sub.1-4-alkyl, C.sub.1-4-alkoxy, nitro, cyano, amino or halogen.
Preferred examples are hydroxy, C.sub.1-6-alkyl, C.sub.1-6-alkoxy,
carboxy, C.sub.1-6-alkoxycarbonyl, C.sub.1-6-alkylcarbonyl, aryl,
amino, mono- and di(C.sub.1-6-alkyl)amino, and halogen, wherein
aryl may be substituted 1-3 times with C.sub.1-4-alkyl,
C.sub.1-4-alkoxy, nitro, cyano, amino or halogen.
[0219] "Halogen" includes fluoro, chloro, bromo, and iodo.
[0220] It should be understood that oligomers (wherein LNAs are
incorporated) and LNAs as such include possible salts thereof, of
which pharmaceutically acceptable salts are especially relevant.
Salts include acid addition salts and basic salts. Examples of acid
addition salts are hydrochloride salts, sodium salts, calcium
salts, potassium salts, etc. Examples of basic salts are salts
where the (remaining) counter ion is selected from alkali metals,
such as sodium and potassium, alkaline earth metals, such as
calcium, and ammonium ions (.sup.+N(R.sup.g).sub.3R.sup.h- , where
each of R.sup.g and R.sup.h independently designates optionally
substituted C.sub.1-6-alkyl, optionally substituted
C.sub.2-6-alkenyl, optionally substituted aryl, or optionally
substituted heteroaryl). Pharmaceutically acceptable salts are,
e.g., those described in Remington's Pharmaceutical Sciences, 17.
Ed. Alfonso R. Gennaro (Ed.), Mack Publishing Company, Easton, Pa.,
U.S.A., 1985 and more recent editions and in Encyclopedia of
Pharmaceutical Technology. Thus, the term "an acid addition salt or
a basic salt thereof" used herein is intended to comprise such
salts. Furthermore, the oligomers and LNAs as well as any
intermediates or starting materials therefor may also be present in
hydrate form.
[0221] Preparation of Monomers
[0222] In a preferred embodiment, nucleosides containing an
additional 2'-O,4'-C-linked ring were synthesised by the following
procedure:
[0223] Synthesis of a number of 4'-C-hydroxymethyl nucleosides have
been reported earlier (R. D. Youssefyeh, J. P. H. Verheyden and J.
G. Moffatt, J. Org. Chem., 1979, 44, 1301; G. H. Jones, M.
Taniguchi, D. Tegg and J. G. Moffatt, J. Org. Chem., 1979, 44,
1309; C. O-Yang, H. Y. Wu, E. B. Fraser-Smith and K. A. M. Walker,
Tetrahedron Lett., 1992, 33, 37; H. Thrane, J. Fensholdt, M. Regner
and J. Wengel, Tetrahedron, 1995, 51, 10389; K. D. Nielsen, F.
Kirpekar, P. Roepstorff and J. Wengel, Bioorg. Med. Chem., 1995, 3,
1493). For exemplification of synthesis of 2'-O,4'-C-linked
bicyclic nucleosides we chose a strategy starting from
4'-C-hydroxymethyl furanose derivative 31. Benzylation,
acetylation, and acetolysis followed by another acetylation
afforded furanose 33, a key intermediate for nucleoside coupling.
Stereoselective reaction with silylated thymine afforded compound
34 which was deacetylated to give nucleoside diol 35. Tosylation
followed by base-induced ring closure afforded the 2'-O,4'-C-linked
bicyclic nucleoside derivative 36. Debenzylation yielded the
unprotected bicyclic nucleoside analogue 37 which was transformed
into the 5'-O-4,4'-dimethoxytrityl protected analogue 38 and
subsequently into the phosphoramidite derivative 39 for
oligonucleotide synthesis. A similar procedure has been used for
synthesis of the corresponding uracil, adenine, cytosine and
guanine nucleosides as exemplified in the example section. This
coupling method is only one of several possible as will be apparent
for a person skilled in the art. A strategy starting from a
preformed nucleoside is also possible. Thus, for example,
conversion of uridine derivative 62 to derivative 44 was
successfully accomplished by tosylation, deisopropylidination and
base-induced ring-closure. As another example, conversion of
nucleoside 67 into nucleoside 61B has been accomplished as depicted
in FIG. 34. Conversion of the bicyclic thymine nucleoside 37 into
the corresponding 5-methyl-cytosine nucleoside 65 was accomplished
by a known reaction sequence using triazole and POCl.sub.3 followed
by benzoylation and treatment by ammonia. A similar procedure
should be applicable for the synthesis of 57C from 44. As another
example of possible strategies, coupling of precyclised furanose
derivatives already containing an additional ring with nucleobase
derivatives is possible. Such a strategy would in addition allow
preparation of the corresponding .alpha.-nucleoside analogues. When
coupling with a protected methyl furanoside of
4-C,2-O-methylene-D-ribofuranose, we obtained mainly a ring-opened
product. However, coupling of 1-O-acetyl furanose 207 or thiophenyl
furanose 212 yielded successfully LNA nucleosides with the
.alpha.-anomers as one product. Incorporation of such .alpha.-LNA
nucleosides will be possible using the standard oligomerisation
techniques (as for the LNA oligomers containing Z) yielding
.alpha.-LNA oligomers. In addition, a synthetic strategy performing
nucleoside coupling using a 4'-C-hydroxymethyl furanose already
activated for ring closure (e.g. by containing a mesyl or tosyl
group at the 4'-C-hydroxymethyl group), is possible as exemplified
by conversion of furanose 78 to nucleoside 79 followed by
deprotection and ring closure to give 36. Chemical or enzymatic
transglycosylation or anomerisation of appropriate furanose
derivatives or nucleosides are yet other possible synthetic
strategies. These and other related strategies allow for synthesis
of bicyclic nucleosides containing other nucleobases or analogues
thereof by either coupling with these nucleobases or analogues, or
starting from preformed nucleoside derivatives.
[0224] The described examples are meant to be illustrative for the
procedures and examples of this invention. The structures of the
synthesised compounds were verified using 1 D or 2D NMR techniques,
e.g. NOE experiments.
[0225] An additional embodiment of the present invention is to
provide bicyclic nucleosides containing additional rings of
different sizes and of different chemical structures. From the
methods described it is obvious for a person skilled in the art of
organic synthesis that cyclisation of other nucleosides is possible
using similar procedures, also of nucleosides containing different
C-branches. The person skilled in the art will be able to find
inspiration and guidance for the preparation of substituted
nucleoside analogue intermediates in the literature, see e.g. WO
36/14329. Regarding rings of different chemical compositions it is
clear that using similar procedures or procedures well-established
in the field of organic chemistry, synthesis of for example thio
analogues of the exemplified oxo analogues is possible as is the
synthesis of the corresponding amino analogues (using for example
nucleophilic substitution reactions or reductive alkylations).
[0226] In the example section, synthesis of the amino LNA analogues
73-74F are described. Conversion of 74 and 74D into standard
building blocks for oligomerisation was possible by 5'-O-DMT
protection and 3'-O-phosphitylation following the standard
procedures. For the amino LNA analogue, protection of the 2'-amino
functionality is needed for controlled linear oligomerisation. Such
protection can be accomplished using standard amino group
protection techniques like, e.g., Fmoc, trifluoroacetyl or BOC.
Alternatively, an N-alkyl group (e.g. benzyl, methyl, ethyl, propyl
or functionalised alkyl) can be kept on during nucleoside
transformations and oligomerisation. In FIGS. 35 and 36, strategies
using N-trifluoroacetyl and N-methyl derivatives are shown. As
outlined in FIG. 37, conversion of nucleoside 75 into the
2'-thio-LNA nucleoside analogue 76D has been successfully performed
as has the subsequent syntheses of the phosphoramidite derivative
76F. Compound 76F has the required structure for automated
synthesis of 2'-thio-LNA oligonucleotides. The N-trifluoroacetyl
2'-amino-LNA synthon 74A has the required structure for automated
synthesis of 2'-amino-LNA oligonucleotides.
[0227] Synthesis of the corresponding cytosine, guanine, and
adenine derivatives of the 2'-thio and 2'-amino LNA nucleosides can
be accomplished using strategies analogous to those shown in FIGS.
35, 36 and 37. Alternative, the stereochemistry around C-2' can be
inverted before cyclisations either by using a conveniently
configurated, e.g. an arabino-configurated, furanose synthon, or by
inverting the configuration around the C-2' carbon atom starting
from a ribo-configurated nucleoside/furanose. Subsequent activation
of the 2'-.beta.-OH, e.g. by tosylation, double nucleophilic
substitution as in the urasil/thymine example described above,
could furnish the desired bicyclic 2'-thio-LNA or 2'-amino-LNA
nucleosides. The thus obtained properly protected cytosine,
guanine, and adenine analogues can be prepared for oligomerisation
using the standard reactions (DMT-protection and phosphitylation)
as described above for other examples.
[0228] Preparation of Oligomers
[0229] Linear-, branched- (M. Grtli and B. S. Sproat, J. Chem.
Soc., Chem. Commun., 1995, 495; R. H. E. Hudson and M. J. Damha, J.
Am. Chem. Soc., 1993, 115, 2119; M. Von Buren, G. V. Petersen, K.
Rssmussen, G. Brandenburg, J. Wengel and F. Kirpekar, Tetrahedron,
1995, 51, 8491) and circular- (G. Prakash and E. T. Kool, J. Am.
Chem. Soc., 1992, 114, 3523) Oligo- and polynucleotides of the
invention may be produced using the polymerisation techniques of
nucleic acid chemistry well known to a person of ordinary skill in
the art of organic chemistry. Phosphoramidite chemistry (S. L.
Beaucage and R. P. Iyer, Tetrahedron, 1993, 49, 6123; S. L.
Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223) was used, but
e.g. H-phosphonate chemistry, phosphortriester chemistry or
enzymatic synthesis could also be used. Generally, standard
coupling conditions and the phosphoramidite approach was used, but
for some monomers of the invention longer coupling time, and/or
repeated couplings with fresh reagents, and/or use of more
concentrated coupling reagents were used. As another possibility,
activators more active than 1H-tetrazole could also be used to
increase the rate of the coupling reaction. The phosphoramidietes
39, 46, 53, 57D, 61D, and 66 all coupled with satisfactory >95%
step-wise coupling yields. An all-phosphorothioate LNA oligomer
(Table 7) was synthesised using standard procedures.
[0230] Thus, by exchanging the normal, e.g.
iodine/pyridine/H.sub.2O, oxidation used for synthesis of
phosphodiester oligomers with an oxidation using Beaucage's reagent
(commercially available), the phosphorthioate LNA oligomer was
efficiently synthesised (stepwise coupling yields >=98%). The
2'-amino-LNA and 2'methylamino-LNA oligonucleotides (Table 9) were
efficiently synthesised (step-wise coupling yields .gtoreq.98%)
using amidites 74A and 74F. The 2'-thio-LNA oligonucleotides (Table
8) were efficiently synthesised using amidite 76F using the
standard phosphoramidite procedures as described above for LNA
oligonucleotides. After synthesis of the desired sequence, work up
was done using standard conditions (cleavage from solid support and
removal of protection groups using 30% ammonia for 55.degree. C.
for 5 h). Purification of LNA oligonucleotides was done using
disposable reversed phase purification cartridges and/or reversed
phase HPLC and/or precipitation from ethanol or butanol. Capillary
gel electrophoresis, reversed phase HPLC and MALDI-MS was used to
verify the purity of the synthesised oligonucleotide analogues, and
to verify that the desired number of bicyclic nucleoside analogues
of the invention were incorporated as contemplated.
[0231] An additional aspect of the present invention is to furnish
procedures for oligonucleotide analogues containing LNA linked by
non-natural internucleoside linkages. For example, synthesis of the
corresponding phosphorothioate or phosphoramidate analogues is
possible using strategies well-established in the field of
oligonucleotide chemistry (Protocols for Oligonucleotides and
Analogs, vol 20, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa,
N.J.; S. L. Beaucage and R. P. Iyer, Tetrahedron, 1993, 49, 6123;
S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223; E.
Uhlmann and A. Peyman, Chem. Rev., 90, 543).
[0232] Thus, generally the present invention also provides the use
of an LNA as defined wherein for the preparation of an LNA modified
oligonucleotides. Is should be understood that LNA modified
oligonucleotide may comprise normal nucleosides (i.e. naturally
occurring nucleosides such as ribonucleosides and/or
dioxyribonucleosides), as well as modified nucleosides different
from those defined with the general formula II. In a particularly
interesting embodiment, incorporation of LNA modulates the ability
of the oligonucleotide to act as a substrate for nucleic acid
active enzymes.
[0233] Furthermore, solid support materials having immobilised
thereto an optionally nucleobase protected and optionally 5'-OH
protected LNA are especially interesting as material for the
synthesis of LNA modified oligonucleotides where an LNA monomer is
included in at the 3' end. In this instance, the solid support
material is preferable CPG, e.g. a readily (commercially) available
CPG material onto which a 3'-functionalised, optionally nucleobase
protected and optionally 5'-OH protected LNA is linked using the
conditions stated by the supplier for that particular material.
BioGenex Universial CPG Support (BioGenex, U.S.A.) can e.g. be
used. The 5'-OH protecting group may, e.g., be a DMT group.
3'-functional group should be selected with due regard to the
conditions applicable for the CPG material in question.
[0234] Applications
[0235] The present invention discloses the surprising finding that
various novel derivatives of bicyclic nucleoside monomers (LNAs),
when incorporated into oligonucleotides, dramatically increase the
affinity of these modified oligonucleotides for both complementary
ssDNA and ssRNA compared to the unmodified oligonucleotides. It
further discloses the surprising finding that both fully and partly
LNA modified oligonucleotides display greatly enhanced
hybridisation properties for their complementary nucleic acid
sequences. Depending on the application, the use of these LNAs thus
offers the intriguing possibility to either greatly increase the
affinity of a standard oligonucleotide without compromising
specificity (constant size of oligonucleotide) or significantly
increase the specificity without compromising affinity (reduction
in the size of the oligonucleotide). The present invention also
discloses the unexpected finding that LNA modified
oligonucleotides, in addition to greatly enhanced hybridisation
properties, display many of the useful physicochemical properties
of normal DNA and RNA oligonucleotides. Examples given herein
include excellent solubility, a response of LNA modified
oligonucleotides to salts like sodium chloride and
tetramethylammonium chloride which mimic that of the unmodified
oligonucleotides, the ability of LNA modified oligonucleotides to
act as primers for a variety of polymerases, the ability of LNA
modified nucleotides to act as primers in a target amplification
reaction using a thermostable DNA polymerase, the ability of LNA
modified oligonucleotides to act as a substrate for T4
polynucleotide kinase, the ability of biotinylated LNAs to sequence
specifically capture P(CR amplicons onto a streptavidine coated
solid surface, the ability of immobilised LNA modified
oligonucleotides to sequence specifically capture amplicons and
very importantly the ability of LNA modified oligonucleotides to
sequence specifically target double-stranded DNA by strand
invasion. Hence, it is apparent to one of ordinary skills in the
art that these novel nucleoside analogues are extremely useful
tools to improve the performance in general of oligonucleotide
based techniques in therapeutics, diagnostics and molecular
biology.
[0236] An object of the present invention is to provide monomeric
LNAs according to the invention which can be incorporated into
oligonucleotides using procedures and equipment well known to one
skilled in the art of oligonucleotide synthesis.
[0237] Another object of the present invention is to provide fully
or partly LNA modified oligonucleotides (oligomers) that are able
to hybridise in a sequence specific manner to complementary
oligonucleotides forming either duplexes or triplexes of
substantially higher affinity than the corresponding complexes
formed by unmodified oligonucleotides.
[0238] Another object of the present invention is to use LNAs to
enhance the specificity of normal oligonucleotides without
compromising affinity. This can be achieved by reducing the size
(and therefore affinity) of the normal oligonucleotide to an extent
that equals the gain in affinity resulting from the incorporation
of LNAs.
[0239] Another object of the present invention is to provide fully
or partly modified oligonucleotides containing both LNAs, normal
nucleosides and other nucleoside analogues.
[0240] A further object of the present invention is to exploit the
high affinity of LNAs to create modified oligonucleotides of
extreme affinity that are capable of binding to their target
sequences in a dsDNA molecule by way of "strand displacement".
[0241] A further object of the invention is to provide different
classes of LNAs which, when incorporated into oligonucleotides,
differ in their affinity towards their complementary nucleosides.
In accordance with the invention this can be achieved by either
substituting the normal nucleobases G, A, T, C and U with
derivatives having, for example, altered hydrogen bonding
possibilities or by using LNAs that differ in their backbone
structure. The availability of such different LNAs facilitates
exquisite tuning of the affinity of modified oligonucleotides.
[0242] Another object of the present invention is to provide LNA
modified oligonucleotides which are more resistant to nucleases
than their unmodified counterparts.
[0243] Another object of the present invention is to provide LNA
modified oligonucleotides which can recruit RNAseH.
[0244] An additional object of the present invention is to provide
LNAs that can act as substrates for DNA and RNA polymerases thereby
allowing the analogues to be either incorporated into a growing
nucleic acid chain or to act as chain terminators.
[0245] A further object of the present invention is to provide LNAs
that can act as therapeutic agents. Many examples of therapeutic
nucleoside analogues are known and similar derivatives of the
nucleoside analogues disclosed herein can be synthesised using the
procedures known from the literature (E. De Clercq, J. Med. Chem.
1995, 38, 2491; P. Herdewijn and E. De Clercq: Classical Antiviral
Agents and Design of New Antiviral Agents. In: A Textbook of Drug
Design and Development; Eds. P. Krogsgaard-Larsen, T. Liljefors and
iU. Madsen; Harwood Academic Publishers, Amsterdam, 1996, p. 425;
I. K. Larsen: Anticancer Agents, In: A Textbook of Drug Design and
Development; Eds. P. Krogsgaard-Larsen, T. Liljefors and U. Madsen;
Harwood Academic Publishers, Amsterdam, 1996, p. 460).
[0246] Double-stranded RNA has been demonstrated to posses
anti-viral activity and tumour suppressing activity (Sharp et al.,
Eur. J. Biochem. 230(1): 97-103, 1995, Lengyel-P. et al., Proc.
Nati. Acad. Sci. U.S.A., 90(13): 5893-5, 1993, and Laurent-Crawford
et al., AIDS Res. Hum. Retroviruses, 8(2): 285-90, 1992). It is
likely that double stranded LNAs may mimic the effect of
therapeutically active double stranded RNAs and accordingly such
double stranded LNAs has a potential as therapeutic drugs.
[0247] When used herein, the term "natural nucleic acid" refers to
nucleic acids in the broadest sense, like for instance nucleic
acids present in intact cells of any origin or vira or nucleic
acids released from such sources by chemical or physical means or
nucleic acids derived from such primary sources by way of
amplification. The natural nucleic acid may be single, double or
partly double stranded, and may be a relatively pure species or a
mixture of different nucleic acids. It may also be a component of a
crude biological sample containing other nucleic acids and other
cellular components. On the other hand, the term "synthetic nucleic
acids" refers to any nucleic acid produced by chemical
synthesis.
[0248] The present invention also provides the use of LNA modified
oligonucleotides in nucleic acid based therapeutic, diagnostics and
molecular biology. The LNA modified oligonucleotides can be used in
the detection, identification, capture, characterisation,
quantification and fragmentation of natural or synthetic nucleic
acids, and as blocking agents for translation and transcription in
vivo and in vitro. In many cases it will be of interest to attach
various molecules to LNA modified oligonucleotides. Such molecules
may be attached to either end of the oligonucleotide or they may be
attached at one or more internal positions. Alternatively, they may
be attached to the oligonucleotide via spacers attached to the 5'
or 3' end. Representative groups of such molecules are DNA
intercalators, photochemically active groups, thermochemically
active groups, chelating groups, reporter groups, and ligands.
Generally all methods for labelling unmodified DNA and RNA
oligonucleotides with these molecules can also be used to label LNA
modified oligonucleotides. Likewise, all methods used for detecting
labelled oligonucleotides generally apply to the corresponding
labelled, LNA modified oligonucleotides.
[0249] Therapy
[0250] The term "strand displacement" relates to a process whereby
an oligonucleotide binds to its complementary target sequence in a
double stranded DNA or RNA so as to displace the other strand from
said target strand.
[0251] In an aspect of the present invention, LNA modified
oligonucleotides capable of performing "strand displacement" are
exploited in the development of novel pharmaceutical drugs based on
the "antigene" approach. In contrast to oligonucleotides capable of
making triple helices, such "strand displacement" oligonucleotides
allow any sequence in a dsDNA to be targeted and at physiological
ionic strength and pH.
[0252] The "strand displacing" oligonucleotides can also be used
advantageously in the antisense approach in cases where the RNA
target sequence is inaccessible due to intramolecular hydrogen
bonds. Such intramolecular structures may occur in mRNAs and can
cause significant problems when attempting to "shut down" the
translation of the mRNA by the antisense approach.
[0253] Other classes of cellular RNAs, like for instance tRNAs,
rRNAs snRNAs and scRNAs, contain intramolecular structures that are
important for their function. These classes of highly structured
RNAs do not encode proteins but rather (in the form of RNA/protein
particles) participate in a range of cellular functions such as
mRNA splicing, polyadenylation, translation, editing, maintainance
of chromosome end integrity, etc. Due to their high degree of
structure, that impairs or even prevent normal oligonucleotides
from hybridising efficiently, these classes of RNAs has so far not
attracted interest as antisense targets.
[0254] The use of high affinity LNA monomers should facilitate the
construction of antisense probes of sufficient thermostability to
hybridise effectively to such target RNAs. Therefore, in a
preferred embodiment, LNA is used to confer sufficient affinity to
the oligonucleotide to allow it to hybridise to these RNA classes
thereby modulating the qualitative and/or quantitative function of
the particles in which the RNAs are found.
[0255] In some cases it may be advantageous to down-regulate the
expression of a gene whereas in other cases it may be advantageous
to activate it. As shown by Mllegaard et al. (Mllegaard, N. E.;
Buchardt, O.; Egholm, M.; Nielsen, P. E. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 3392), oligomers capable of "strand displacement"
can function as RNA transcriptional activators. In an aspect of the
present invention, the LNAs capable of "strand displacement" are
used to activate genes of therapeutic interest.
[0256] In chemotherapy of numerous viral infections and cancers,
nucleosides and nucleoside analogues have proven effective. LNA
nucleosides are potentially useful as such nucleoside based
drugs.
[0257] Various types of double-stranded RNAs inhibit the growth of
several types of cancers. Duplexes involving one or more LNA
oligonucleotide(s) are potentially useful as such double-stranded
drugs.
[0258] The invention also concerns a pharmaceutical composition
comprising a pharmaceutically active LNA modified oligonucleotide
or a pharmaceutically active LNA monomer as defined above in
combination with a pharmaceutically acceptable carrier.
[0259] Such compositions may be in a form adapted to oral,
parenteral (intravenous, intraperitoneal), intramuscular, rectal,
intranasal, dermal, vaginal, buccal, ocularly, or pulmonary
administration, preferably in a form adapted to oral
administration, and such compositions may be prepared in a manner
well-known to the person skilled in the art, e.g. as generally
described in "Remington's Pharmaceutical Sciences", 17. Ed. Alfonso
R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., U.S.A.,
1985 and more recent editions and in the monographs in the "Drugs
and the Pharmaceutical Sciences" series, Marcel Dekker.
[0260] Diagnostics
[0261] Several diagnostic and molecular biology procedures have
been developed that utilise panels of different oligonucleotides to
simultaneously analyse a target nucleic acid for the presence of a
plethora of possible mutations. Typically, the oligonucleotide
panels are immobilised in a predetermined pattern on a solid
support such that the presence of a particular mutation in the
target nucleic acid can be revealed by the position on the solid
support where it hybridises. One important prerequisite for the
successful use of panels of different oligonucleotides in the
analysis of nucleic acids is that they are all specific for their
particular target sequence under the single applied hybridisation
condition. Since the affinity and specificity of standard
oligonucleotides for their complementary target sequences depend
heavily on their sequence and size this criteria has been difficult
to fulfil so far.
[0262] In a preferred embodiment, therefore, LNAs are used as a
means to increase affinity and/or specificity of the probes and as
a means to equalise the affinity of different oligonucleotides for
their complementary sequences. As disclosed herein such affinity
modulation can be accomplished by, e.g., replacing selected
nucleosides in the oligonucleotide with an LNA carrying a similar
nucleobase. As further shown herein, different classes of LNAs
exhibit different affinities for their complementary nucleosides.
For instance, the 2-3 bridged LNA with the T-nucleobase exhibits
less affinity for the A-nucleoside than the corresponding 2-4
bridged LNA. Hence, the use of different classes of LNAs offers an
additional level for fine-tuning the affinity of a
oligonucleotide.
[0263] In another preferred embodiment the high affinity and
specificity of LNA modified oligonucleotides is exploited in the
sequence specific capture and purification of natural or synthetic
nucleic acids. In one aspect, the natural or synthetic nucleic
acids are contacted with the LNA modified oligonucleotide
immobilised on a solid surface. In this case hybridisation and
capture occurs simultaneously. The captured nucleic acids may be,
for instance, detected, characterised, quantified or amplified
directly on the surface by a variety of methods well known in the
art or it may be released from the surface, before such
characterisation or amplification occurs, by subjecting the
immobilised, modified oligonucleotide and captured nucleic acid to
dehybridising conditions, such as for example heat or by using
buffers of low ionic strength.
[0264] The solid support may be chosen from a wide range of polymer
materials such as for instance CPG (controlled pore glass),
polypropylene, polystyrene, polycarbonate or polyethylene and it
may take a variety of forms such as for instance a tube, a
micro-titer plate, a stick, a bead, a filter, etc.. The LNA
modified oligonucleotide may be immobilised to the solid support
via its 5' or 3' end (or via the terminus of linkers attached to
the 5' or 3' end) by a variety of chemical or photochemical methods
usually employed in the immobilisation of oligonucleotides or by
non-covalent coupling such as for instance via binding of a
biotinylated LNA modified oligonucleotide to immobilised
streptavidin. One preferred method for immobilising LNA modified
oligonucleotides on different solid supports is photochemical using
a photochemically active anthraquinone covalently attached to the
5' or 3' end of the modified oligonucleotide (optionally via
linkers) as described in (WO 96/31557). Thus, the present invention
also provide a surface carrying an LNA modified
oligonucleotide.
[0265] In another aspect the LNA modified oligonucleotide carries a
ligand covalently attached to either the 5' or 3' end. In this case
the LNA modified oligonucleotide is contacted with the natural or
synthetic nucleic acids in solution whereafter the hybrids formed
are captured onto a solid support carrying molecules that can
specifically bind the ligand.
[0266] In still another aspect, LNA modified oligonucleotides
capable of performing "strand displacement" are used in the capture
of natural and synthetic nucleic acids without prior denaturation.
Such modified oligonucleotides are particularly useful in cases
where the target sequence is difficult or impossible to access by
normal oligonucleotides due to the rapid formation of stable
intramolecular structures. Examples of nucleic acids containing
such structures are rRNA, tRNA, snRNA and scRNA.
[0267] In another preferred embodiment, LNA modified
oligonucleotides designed with the purpose of high specificity are
used as primers in the sequencing of nucleic acids and as primers
in any of the several well known amplification reactions, such as
the PCR reaction. As shown herein, the design of the LNA modified
oligonucleotides determines whether it will sustain a exponential
or linear target amplification. The products of the amplification
reaction can be analysed by a variety of methods applicable to the
analysis of amplification products generated with normal DNA
primers. In the particular case where the LIMA modified
oligonucleotide primers are designed to sustain a linear
amplification the resulting amplicons will carry single stranded
ends that can be targeted by complementary probes without
denaturation. Such ends could for instance be used to capture
amplicons by other complementary LNA modified oligonucleotides
attached to a solid surface.
[0268] In another aspect, LNA modified oligos capable of "strand
displacement" are used as primers in either linear or exponential
amplification reactions. The use of such oligos is expected to
enhance overall amplicon yields by effectively competing with
amplicon re-hybridisation in the later stages of the amplification
reaction. Demers, et al. (Nucl. Acid Res. 1995, Vol 23, 3050-3055)
discloses the use of high-affinity, non-extendible oligos as a
means of increasing the overall yield of a PCR reaction. It is
believed that the oligomers elicit these effect by interfering with
amplicon re-hybridisation in the later stages of the PCR reaction.
It is expected that LNA modified oligos blocked at their 3' end
will provide the same advantage. Blocking of the 3' end can be
achieved in numerous ways like for instance by exchanging the 3'
hydroxyl group with hydrogen or phosphate. Such 3' blocked LNA
modified oligos can also be used to selectively amplify closely
related nucleic acid sequences in a way similar to that described
by Yu et al. (Biotechniques, 1997, 23, 714-716).
[0269] In recent years, novel classes of probes that can be used in
for example real-time detection of amplicons generated by target
amplification reactions have been invented. One such class of
probes have been termed "Molecular Beacons". These probes are
synthesised as partly self-complementary oligonucleotides
containing a fluorophor at one end and a quencher molecule at the
other end. When free in solution the probe folds up into a hairpin
structure (guided by the self-complimentary regions) which
positions the quencher in sufficient closeness to the fluorophor to
quench its fluorescent signal. Upon hybridisation to its target
nucleic acid, the hairpin opens thereby separating the fluorophor
and quencher and giving off a fluorescent signal.
[0270] Another class of probes have been termed "Taqman probes".
These probes also contain a fluorophor and a quencher molecule.
Contrary to the Molecular Beacons, however, the quenchers ability
to quench the fluorescent signal from the fluorophor is maintained
after hybridisation of the probe to its target sequence. Instead,
the fluorescent signal is generated after hybridization by physical
detachment of either the quencher or fluorophor from the probe by
the action of the 5 ' exonuxlease activity of a polymerase which
has initiated synthesis from a primer located 5' to the binding
site of the Taqman probe.
[0271] High affinity for the target site is an important feature in
both types of probes and consequently such probes tends to be
fairly large (typically 30 to 40 mers). As a result, significant
problems are encountered in the production of high quality probes.
In a preferred embodiment, therefore, LNA is used to improve
production and subsequent performance of Taqman probes and
Molecular Beacons by reducing their size whilst retaining the
required affinity.
[0272] In a further aspect, LNAs are used to construct new affinity
pairs (either fully or partially modified oligonucleotides). The
affinity constants can easily be adjusted over a wide range and a
vast number of affinity pairs can be designed and synthesised. One
part of the affinity pair can be attached to the molecule of
interest (e.g. proteins, amplicons, enzymes, polysaccharides,
antibodies, haptens, peptides, PNA, etc.) by standard methods,
while the other part of the affinity pair can be attached to e.g. a
solid support such as beads, membranes, micro-titer plates, sticks,
tubes, etc. The solid support may be chosen from a wide range of
polymer materials such as for instance polypropylene, polystyrene,
polycarbonate or polyethylene. The affinity pairs may be used in
selective isolation, purification, capture and detection of a
diversity of the target molecules mentioned above.
[0273] The principle of capturing an LNA-tagged molecule by ways of
interaction with another complementary LNA oligonucleotide (either
fully or partially modified) can be used to create an infinite
number of novel affinity pairs.
[0274] In another preferred embodiment the high affinity and
specificity of LNA modified oligonucleotides are exploited in the
construction of probes useful in in-situ hybridisation. For
instance, LNA could be used to reduce the size of traditional DNA
probes whilst maintaining the required affinity thereby increasing
the kinetics of the probe and its ability to penetrate the sample
specimen. The ability of LNA modified oligonucleotides to "strand
invade" double stranded nucleic acid structures are also of
considerable advantage in in-situ hybridisation, because it
facilitates hybridisation without prior denaturation of the target
DNA/RNA.
[0275] In another preferred embodiment, LNA modified
oligonucleotides to be used in antisense therapeutics are designed
with the dual purpose of high affinity and ability to recruit
RNAseH. This can be achieved by, for instance, having LNA segments
flanking an unmodified central DNA segment.
[0276] The present invention also provides a kit for the isolation,
purification, amplification, detection, identification,
quantification, or capture of natural or synthetic nucleic acids,
where the kit comprises a reaction body and one or more LNA
modified oligonucleotides (oligomer) as defined herein. The LNA
modified oligonucleotides are preferably immobilised onto said
reactions body.
[0277] The present invention also provides a kit for the isolation,
purification, amplification, detection, identification,
quantification, or capture of natural or synthetic nucleic acids,
where the kit comprises a reaction body and one or more LNAs as
defined herein. The LNAs are preferably immobilised onto said
reactions body (e.g. by using the immobilising techniques described
above).
[0278] For the kits according to the invention, the reaction body
is preferably a solid support material, e.g. selected from
borosilicate glass, soda-lime glass, polystyrene, polycarbonate,
polypropylene, polyethylene, polyethyleneglycol terephthalate,
polyvinylacetate, polyvinylpyrrolidinone, polymethylmethacrylate
and polyvinylchloride, preferably polystyrene and polycarbonate.
The reaction body may be in the form of a specimen tube, a vial, a
slide, a sheet, a film, a bead, a pellet, a disc, a plate, a ring,
a rod, a net, a filter, a tray, a microtitre plate, a stick, or a
multi-bladed stick.
[0279] The kits are typically accompanied by a written instruction
sheet stating the optimal conditions for use of the kit.
[0280] The above-mentioned diagnostic and therapeutic aspects of
the present invention have been illustrated with the following
examples.
[0281] Experimental
[0282] General
[0283] All reagents were obtained from commercial suppliers and
were used without further purification. After drying any organic
phase using Na.sub.2SO.sub.4, filtration was performed. The silica
gel (0.040-0.063 mm) used for column chromatography was purchased
from Merck. NMR spectra were recorded at 300 MHz or 250 MHz for
.sup.1H NMR and 62.9 MHz for .sup.13C NMR and at 202.33 MHz for
.sup.31P NMR. .delta.-Values are in ppm relative to
tetramethylsilane as internal standard (.sup.1H NMR and .sup.13C
NMR) and relative to 85% H.sub.3PO.sub.4 as external standard
(.sup.31P NMR). Assignments of NMR peaks are given according to
standard nucleoside nomenclature. El mass spectra, FAB mass spectra
and Plasma Desorption mass spectra were recorded to gain
information on the molecular weight of synthesised compounds.
Oligonucleotide analogues were synthesised using the
phosphoramidite methodology. Purification of 5'-O-DMT-ON or
5'-O-DMT-OFF oligonucleotide analogues was accomplished using
disposable reversed phase chromatography cartridges or reversed
phase HPLC when necessary. Matrix-assisted laser desorption mass
spectra were obtained to verify the molecular weight and monomer
composition of representative oligonucleotide samples. Capillary
gel electrophoresis was performed to verify the purity of
representive oligonucleotide samples.
[0284] The specific descriptions below are accompanied by FIGS.
2-41 and Tables 1-10. Unless otherwise stated in the following
examples, "LNA" designates the 2'-4'-bridged variant illustrated
with the forumula Z in FIG. 2.
[0285] Preparation of LNA Monomers
EXAMPLE 1
3-C-Allyl-1,2-O-isopropylidene-.alpha.-D-ribofuranose (0A)
[0286] Method 1: A solution of
5-O-t-butyidimethylsilyl-1,2-O-isopropylide-
ne-.alpha.-D-ribofuran-3-ulose (Y. Yoshimura, T. Sano, A. Matsuda
and T. Ueda, Chem. Pharm. Bull., 1988, 36, 162) (17.8 g, 58.9 mmol)
in anhydrous THF (980 cm.sup.3) was stirred at 0.degree. C. and 1 M
allylmagnesium bromide in anhydrous ether (130 cm.sup.3, 130 mmol)
was added dropwise. After stirring for 2 h, a saturated aqueous
solution of ammonium chloride (800 cm.sup.3) was added and the
mixture was extracted with dichloromethane (3.times.400 cm.sup.3).
The organic phase was washed with brine (3.times.450 cm.sup.3) and
dried (Na.sub.2SO.sub.4). The solvent was removed under reduced
pressure and the residue was dissolved in anhydrous THF (700
cm.sup.3). A 1.1 M solution of tetrabutylammonium fluoride in THF
(54.4 cm.sup.3, 59.8 mmol) was added and the mixture was stirred at
room temperature for 1 h and evaporated to dryness. The residue was
dissolved in dichloromethane (1700 cm.sup.3) and was washed with a
saturated aqueous solution of sodium hydrogencarbonate (3.times.500
cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography using dichloromethane/methanol (98:2, v/v) as
eluent to give furanose 0A as a white solid material (9.42 g, 69%).
Method 2: Furanose 0A was analogously synthesised from
5-O-t-butyidiphenylsilyl-1,2-O-isopropylidene-.alpha.-D-ribofuran-3-ulose
(T. F. Tam and B. Fraser-Reid, J. Chem. Soc., Chem. Commun., 1980,
556) (9.5 g, 22.2 mmol) using: anhydrous THF (425 cm.sup.3); a 1 M
solution of allylmagnesium bromide in anhydrous ether (130
cm.sup.3, 130 mmol); a saturated aqueous solution of ammonium
chloride (490 cm.sup.3); ether for extraction (350+2.times.160
cm.sup.3); brine (2.times.160 cm.sup.3); a 1.1 M solution of
tetrabutylammonium fluoride in THF (22.3 cm.sup.3, 24.6 mmol);
anhydrous THF (400 cm.sup.3); dichloromethane (1400 cm.sup.3); a
saturated aqueous solution of sodium hydrogencarbonate (3.times.500
cm.sup.3); brine (500 cm.sup.3) and (Na.sub.2SO.sub.4).
.delta..sub.H ((CD.sub.3).sub.2SO) 5.84 (1 H, m, 2'-H), 5.65 (1 H,
d, J 3.8, 1-H), 5.12 (1H, d, J 6.1, 3'-H.sub.a), 5.06 (1H, br s,
3'-H.sub.b), 4.76 (1H, s, 3-OH), 4.64 (1H, t, J 5.4, 5-OH), 4.16 (1
H, d, J 3.8, 2-H), 3.84 (1 H, dd, J 2.2, 8.1, 4-H), 3.56 (1 H, ddd,
J 2.3, 5.6, 11.8, 5-H.sub.a), 3.42 (1 H, m, 5-H.sub.b), 2.16 (1 H,
dd, J 6.1, 14.3, 1'-H.sub.a), 1.98 (1 H, dd, J 8.2, 14.3,
1'-H.sub.b), 1.46 (3 H, s, CH.sub.3), 1.25 (3 H, s, CH.sub.3).
.delta..sub.C (CDCl.sub.3) 133.5 (C-2'), 117.9 (C-3'), 110.8
(C(CH.sub.3).sub.2), 102.9 (C-1), 82.6, 81.0, 77.7 (C-2, C-3, C-4),
59.4 (C-5), 36.4 (C-1'), 26.4, 26.3 (CH.sub.3) (Found: C, 57.4; H,
8.0; C.sub.11H.sub.18O.sub.5 requires C, 57.4; H, 7.9%).
EXAMPLE 2
3-C-Allyl-3,5-di-O-benzyl-1,2-O-isopropylideiie-.alpha.-D-ribofuranose
(0B)
[0287] A 60% suspension of sodium hydride (4.9 g, 123 mmol) in
anhydrous DMF (100 cm.sup.3) was stirred at 0.degree. C. and a
solution of furanose 0A (9.42 g, 40.9 mmol) in anhydrous DMF (65
cm.sup.3) was added dropwise over 45 min. The solution was stirred
for 1 h at 50.degree. C. and cooled to 0.degree. C. A mixture of
benzyl broimide (14.5 cm.sup.3, 121 mmol) and anhydrous DMF (14.5
cm.sup.3) was added dropwise and the mixture was stirred at room
temperature for 18 h. The reaction mixture was evaporated to
dryness and a solution of the residue in dichloromethane (700
cm.sup.3) was washed with a saturated aqueous solution of sodium
hydrogencarbonate (2.times.450 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using petroleum ether/ethylacetate (9:1, v/v) as eluent to give
compound 0B as an oil (14.5 g, 86%). .delta..sub.H ((CDCl.sub.3)
7.39-7.21 (10 H, m, Bn), 5.92 (1 H, m, 2'-H), 5.71 (1 H, d, J 3.8,
1-H), 5.17-5.09 (2 H, m, 3'-H.sub.a, 3'-H.sub.b), 4.67 (2 H, m,
Bn), 4.60 (1 H, d, J 12.2, Bn), 4.52 (1 H, d, J 12.1, Bn), 4.43 (1
H, m, 4-H), 4.42 (1 H, d, J 3.8, 2-H), 3.73 (1 H, dd, J 3.2, 10.8,
5-H.sub.a), 3.66 (1 H, dd, J 7.4, 10.8, 5-H.sub.b), 2.50 (1 H, dd,
J 7.7, 14.9, 1'-H.sub.a), 2.39 (1 H, dd, J 6.5, 14.9, 1'-H.sub.b),
1.60 (3 H, s, CH.sub.3), 1.34 (3 H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 138.7, 138.1 (Bn), 132.6 (C-2'), 128.3, 128.2, 127.7,
127.5, 127.4, 127.4 (Bn), 118.5 (C-3'), 112.6 (C(CH.sub.3).sub.2),
104.1 (C-1), 86.5, 82.1, 80.4 (C-2, C-3, C-4), 73.4, 68.6 (Brn),
67.0 (C-5), 35.8 (C-1'), 26.8, 26.6 (CH.sub.3). FAB-MS m/z 433
[M+Na]+(Found: C, 73.4; H, 7.4; C.sub.25H.sub.30O.sub.5 requires C,
73.2; H, 7.4%).
EXAMPLE 3
3-C-Allyl-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribofuranose (0C)
[0288] A solution of furanose 0B (12.42 g, 30.3 mmol) in 80%
aqueous acetic acid (150 cm.sup.3) was stirred at 90.degree. C. for
3 h. The solvent was removed under reduced pressure and the residue
was coevaporated with ethanol (3.times.75 cm.sup.3), toluene
(3.times.75 cm.sup.3) and anhydrous pyridine (2.times.75 cm.sup.3)
and redissolved in anhydrous pyridine (60 cm.sup.3). Acetic
anhydride (46 cm.sup.3) was added and the solution was stirred at
room temperature for 48 h. A mixture of ice and water (300
cm.sup.3) was added and the resulting mixture was extracted with
dichloromethane (2.times.300 cm.sup.3). The combined organic phase
was washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.200 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was evaporated and the residue was
purified using silica gel column chromatography with petroleum
ether/ethyl acetate (4:1, v/v) as eluent to give the anomeric
mixture 0C (.beta.:.alpha..about.2:1) as an oil (13.3 g, 97%).
.delta..sub.C (CDCl.sub.3) 169.7, 169.6 (C.dbd.O), 138.7, 138.4,
137.7, 137.6 (Bn), 132.4, 132.2 (C-2'), 128.4 128.4, 128.2, 128.2,
127.8, 127.7, 127.7, 127.6, 127.3, 127.3, 126.9, 126.8 (Bn), 118.5
(C-3'), 99.4, 93.5 (C-1), 84.8, 83.7, 83.2, 82.0, 79.1, 75.5 (C-2,
C-3, C-4), 73.7, 73.5, 69.3, 68.7 (Bn), 66.1 (C-5), 35.5, 34.9
(C-1), 21.1, 21.0, 20.7, 20.6 (CH.sub.3) (Found: C, 68.7; H, 6.7;
C.sub.26H.sub.30O.sub.7 requires C, 68.8; H, 6.6%).
EXAMPLE 4
1-(2-O-Acetyl-3-C-allyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)thymine
(1)
[0289] To a stirred solution of the anomeric mixture 0C
(.beta.:.alpha..about.2:1, 11.8 g, 26.0 mmol) (P. Nielsen, H. M.
Pfundheller and J. Wengel, Chem. Commur., 1997, 825; P. Nielsen, H.
M. Pfundheller, C. E. Olsen and J. Wengel, J. Chem. Soc., Perkin
Trans. 1, 1997, in the press) and thymine (6.55 g, 52.0 mmol) in
anhydrous acetonitrile (250 cm.sup.3) was added
N,O-bis(trimethylsilyl)acetamide (44.9 cm.sup.3, 182 mmol). The
reaction mixture was stirred at reflux for 1 h and cooled to
0.degree. C. Trimethylsilyl triflate (8.00 cm.sup.3, 44.0 mmol) was
added dropwise and the solution was stirred at room temperature for
12 h. An ice-cold saturated aqueous solution of sodium
hydrogencarbonate (270 cm.sup.3) was added and the mixture was
extracted with dichloromethane (3.times.125 cm.sup.3). The organic
phase was washed with saturated aqueous solutions of sodium
hydrogen-carbonate (2.times.125 cm.sup.3) and brine (2.times.125
cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography using dichloromethane/methanol (98:2, v/v) as
eluent to give nucleoside 1 as a white solid material (11.6 g,
86%). .delta..sub.H (CDCl.sub.3) 8.64 (1 H, br s, NH), 7.75 (1 H,
d, J 1.1, 6-H), 7.41-7.25 (10 H, m, Bn), 6.43 (1 H, d, J 8.2,
1'-H), 5.88 (1H, m, 2"-H), 5.66 (1 H, d, J 8.2, 2'-H), 5.12 (1 H,
s, 3"-H.sub.a), 5.07 (1 H, dd, J 1.5, 8.5, 3"-H.sub.b), 4.85 (1 H,
d, J 11.2, Bn), 4.64 (2 H, s, Bn), 4.63 (1 H, d, J 11.2, Bn), 4.33
(1 H, br s, 4'-H), 3.81 (1 H, dd, J 2.7, 11.1, 5'-H.sub.a), 3.65 (1
H, m, 5'-H.sub.b), 2.81-2.65 (2 H, m, 1"-H.sub.a, 1"-H.sub.b), 2.08
(3 H, s, COCH.sub.3), 1.52 (3 H, d, J 0.8, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 170.1 (C.dbd.O), 163.6 (C-4), 150.9 (C-2), 138.1,
136.6 (Bn), 136.0 (C-6), 131.6 (C-2"), 128.8, 128.4, 128.3, 127.6,
127.5, 127.1 (Bn), 118.5 (C-3"), 111.1 (C-5), 84.2, 83.4, 83.1,
77.4 (C-1', C-2', C-3', C-4'), 73.6, 69.2 (Bn), 65.6 (C-5'), 33.7
(C-1"), 20.8 (COCH.sub.3), 11.9 (CH.sub.3) (Found: C, 66.8; H, 6.3;
N, 5.1. C.sub.29H.sub.32O.sub.7 requires C, 66.9; H, 6.2; N,
5.4%).
EXAMPLE 5
1-(3-C-Allyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)thymine (2)
[0290] To a stirred solution of nucleoside 1 (11.6 g, 22.3 mmol) in
methanol (110 cm.sup.3) was added sodium methoxide (3.03 g, 55.5
mmol). The reaction mixture was stirred at room temperature for 16
h and neutralised with dilute hydrochloric acid. The solvent was
partly evaporated and the residue was dissolved in dichloromethane
(2.times.400 cm.sup.3). The organic phase was washed with a
saturated aqueous solution of sodium hydrogen-carbonate
(3.times.250 cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent
was removed under reduced pressure to give 2 as a white solid
material (10.1 g, 95%). .delta..sub.H (CDCl.sub.3) 8.77 (1 H, br s,
NH), 7.58 (1 H , d, J 1.2, 6-H), 7.41-7.25 (10 H, m, Bn), 6.14 (1H,
m, 2"-H), 6.12 (1 H, d, J 7.8, 1'-H), 5.23 (1 H, m, 3"-H.sub.a),
5.17 (1 H, br s, 3"-H.sub.b), 4.68 (1 H, d, J 10.8, Bn), 4.59 (2 H,
s, Bn), 4.55 (1 H, d, J 10.9 Bn), 4.39 (1 H, br s, 4'-H), 4.26 (1
H, dd J 7.8, 10.7, 2'-H), 3.84 (1 H, dd, J 3.1, 11.0, 5'-H,), 3.58
(1 H, dd, J 1.4, 11.0, 5'-H.sub.b), 3.04 (1 H, d, J 10.8, 2'-OH),
2.82-2.78 (2 H, m, 1"-H.sub.a, 1"-H.sub.b), 1.51 (3 H, d, J 1.0,
CH.sub.3). .delta..sub.C (CDCl.sub.3) 163.5 (C-4), 151.1 (C-2),
137.3, 136.7 (Bn), 136.0 (C-6), 132.1 (C-2"), 128.8, 128.5, 128.3,
127.9, 127.6 (Bn), 118.4 (C-3"), 111.1 (C-5), 87.4, 82.6, 81.1,
79.3 (C-1', C-2', C-3', C-4'), ,73.7, 69.8 (Bn), 64.7 (C-5'), 35.1
(C-1"), 11.9 (CH.sub.3). (Found: C, 67.8; H, 6.1; N, 5.5
C.sub.27H.sub.30N.sub.2O.sub.6 requires C, 67.8; H, 6.3; N,
5.9%).
EXAMPLE 6
1-(3-C-Allyl-3,5-di-O-benzyl-2-O-methanesulfonyl-.beta.-D-ribofuranosyl)th-
ymine (3)
[0291] To a stirred solution of nucleoside 2 (3.50 g, 7.31 mmol) in
anhydrous pyridine (23 cm.sup.3) at 0.degree. C was added
methanesulphonyl chloride (1.69 cm.sup.3, 21.89 mmol). The reaction
mixture was stirred for 1 h at room temperature, water (100
cm.sup.3) was added and extraction was performed using
dichloromethane (3.times.150 cm.sup.3). The organic phase was
washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.200 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue purified by silica gel column chromatography using
dichloromethane/methanol (99:1) as eluent to give 3 as a white
solid material (3.64 g, 89%). .delta..sub.H (CDCl.sub.3) 8.95 (1 H,
br s , NH), 7.71 (1 H , d, J 1.1, 6-H), 7.39-7.25 (10 H, m, Bn),
6.52 (1 H, d, J 8.0, 1'-H), 5.90 (1H, m, 2"-H), 5.34 (1 H, d, J
7.9, 2'-H), 5.20-5.09 (2 H, m, 3"-H.sub.a, 3"-H.sub.b), 4.91 (1 H,
d, J 11.2, Bn), 4.68 (1 H, d, J 11.3, Bn), 4.64 (2 H, s, Bn), 4.33
(1 H, br s, 4'-H), 3.81 (1 H, dd, J 2.5, 11.1, 5'-H.sub.a), 3.73 (1
H, dd, J 1.1, 11.1, 5'-H.sub.b), 3.08 (1 H, dd, J 5.5, 5.7,
1"-H.sub.a), 2.99 (3 H, s, CH.sub.3), 2.68 (1 H, m, 1"-H.sub.b),
1.51 (3 H, d, J 0.8, CH.sub.3). .delta..sub.C (CDCl.sub.3) 163.4
(C-4), 150.8 (C-2), 137.9, 136.3 (Bn), 135.5 (C-6), 131.0 (C-2"),
128.8, 128.3, 127.5, 127.2 (Bn), 119.3 (C-3"), 111.6 (C-5), 84.1,
83.6, 82.4, 82.2 (C-1', C-2', C-3', C-4'), 73.7, 68.9 (Bn), 66.2
(C-5'), 38.7 (CH.sub.3), 33.0 (C-1"), 11.9 (CH.sub.3) (Found: C,
60.5; H, 5.8; N, 4.9. C.sub.28H.sub.32N.sub.2O.sub.8S requires C,
60.4; H, 5.8; N, 5.0%).
EXAMPLE 7
1-(3-C-Allyl-3,5-di-O-benzyl-.beta.-D-arabinofuranosyl)thymine
(4)
[0292] A solution of nucleoside 3 (3.59 g, 6.45 mmol) in ethanol
(72 cm.sup.3), water (72 cm.sup.3) and 1 M aqueous sodium hydroxide
(20.6 cm.sup.3) was stirred under reflux for 18 h. After
neutralisation with dilute hydrochloric acid, the solvent was
removed under reduced pressure and the residue was dissolved in
dichloromethane (3.times.1 50 cm.sup.3). The organic phase was
washed with a saturated aqueous solution of sodium
hydlrogencarbonate (3.times.200 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (99:1, v/v) as eluent to give 4 as a
white solid material (2.32 g, 74%). .delta..sub.H (CDCl.sub.3) 7.60
(1 H, d, J 1.2, 6-H), 7.50-7.23 (10 H, m, Bn), 6.22 (1 H, d, J 2.9,
1'-H), 5.80 (1H, m, 2"-H), 5.15-5.08 (2 H, m, 3"-H.sub.a,
3"-H.sub.b), 4.86-4.33 (6 H, m, 2.times.Bn, 2'-H, 4'-H), 3.82-3.71
(2 H, m, 5'-H.sub.a, 5'-H.sub.b), 2.72 (1 H, m, 1"-H.sub.a), 2.52
(1 H, dd, J 7.6, 16.1, 1"-H.sub.b), 1.70 (3 H, d, J 0.9, CH.sub.3)
.delta..sub.C (CDCl.sub.3) 165.1 (C-4), 150.4 (C-2), 138.4, 136.8
(Bn), 137.7 (C-6), 132.3 (C-2"), 128.77 128.4, 128.3, 128.0, 127.9,
127.6 (Bn), 118.5, (C-3"), 107.8 (C-5), 88.0, 87.8, 83.7 (C-1',
C-3', C-4'), 73.7, 72.9, 69.4 (Bn, C-2'), 64.7 (C-5'), 31.1 (C-1"),
12.4 (CH.sub.3) (Found: C, 67.5; H, 6.3; N, 5.3.
C.sub.27H.sub.30N.sub.2O.sub.- 6,0.25H.sub.2O requires C, 67.1; H,
6.4; N, 5.8%).
EXAMPLE 8
1-(3,5-Di-O-benzyl-3-C-(2-hydroxyethyl)-.beta.-D-arabinofuranosyl)thymine
(5)
[0293] To a stirred solution of nucleoside 4 (2.26 g, 4.68 mmol) in
THF (12 cm.sup.3) and water (12 cm.sup.3) was added sodium
periodate (3.04 g, 14.2 mmol) and a 2.5% solution of osmium
tetraoxide in tert-butanol (w/w, 0.603 cm.sup.3, 40 .mu.mol). The
solution was stirred at room temperature for 45 min. Water (25
cm.sup.3) was added and the solution was extracted with
dichloromethane (2.times.50 cm.sup.3). The organic phase was washed
with a saturated aqueous solution of sodium hydrogencarbonate
(3.times.30 cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent was
removed under reduced pressure and the residue was redissolved in
THF (12 cm.sup.3) and water (12 cm.sup.3). The mixture was stirred
at room temperature and sodium boronhydride (182 mg, 4.71 mmol) was
added. After stirring for 1.5 h, water (25 cm.sup.3) was added and
the solution was extracted with dichloromethane (2.times.50
cm.sup.3). The organic phase was washed with a saturated aqueous
solution of sodium hydrogencarbonate (3.times.30 cm.sup.3) and
dried (Na.sub.2SO.sub.4). The solvent was removed under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (98:2, v/v) as eluent
to give 5 as a white solid material (1.13 g, 49%). .delta..sub.H
(CDCl.sub.3) 9.29 (1 H, br s , NH), 7.47 (1 H d, J 1.1, 6-H),
7.38-7.25 (10 H, m, Bn), 6.22 (1 H, d, J 3.4, 1'-H), 4.62 (2 H, s,
Bn), 4.60 (1 H, m, 4'-H), 4.46 (2 H, s, Bn), 4.35 (1 H, dd, J 3.4,
7.5, 2'-H), 3.83-3.73 (4 H, m, 2.times.5'-H, 2.times.2"-H), 2.67 (1
H, br s, OH), 2.07-2.01 (2 H, m, 2.times.1"-H), 1.77 (3 H, d, J
0.5, CH.sub.3). .delta..sub.C (CDCl.sub.3) 164.3 (C-4), 150.3
(C-2), 137.6, 137.4 (Bn, C-6), 136.7 (Bn), 128.6, 128.4, 128.2,
127.8, 127.6, 127.3, 127.1 (Bn), 108.4 (C-5), 88.0, 87.7, 81.6,
74.7 (C-1', C-2', C-3', C-4'), 73.7, 69.6 (Bn), 64.6 (C-5'), 57.7
(C-2"), 28.6 (C-1"), 12.4 (CH.sub.3). FAB-MS m/z 483 [M+H].sup.+,
505 [M+Na].sup.+ (Found: C, 63.6; H, 6.2; N, 5.4.
C.sub.26H.sub.30N.sub.2O.sub.7,0.5H.sub.2O requires C, 63.5; H 6.4;
N, 5.7%).
EXAMPLE 9
(1S,5R,6R,8R)-5-Hydroxy-6-(hydroxymethyl)-8-(thymin-1-yl)-2,7-dioxabicyclo-
[3.3.0]-octane (6)
[0294] A solution of nucleoside 5 (1.08 g, 2.20 mmol) in anhydrous
pyridine (5.0 cm.sup.3) was stirred at 0.degree. C. and a solution
of p-toluenesulphonyl chloride (462 mg, 2.47 mmol) in anhydrous
pyridine (2.0 cm.sup.3) was added dropwise. After stirring at room
temperature for 20 h and addition of a mixture of water and ice (70
cm.sup.3), extraction was performed with dichloromethane
(2.times.75 cm.sup.3). The organic phase was washed with a
saturated aqueous solution of sodium hydrogencarbonate (3.times.50
cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography using dichloromethane/methanol (99:1, v/v) as
eluent to give an intermediate which after evaporation was
dissolved in anhydrous DMF (4.0 cm.sup.3). The solution was added
dropwise to a stirred suspension of 60% sodium hydride (203 mg,
4.94 mmol) in anhydrous DMF (4.0 cm.sup.3) at 0.degree. C. The
mixture was stirred for 18 h and water (20 cm.sup.3) was added.
After neutralisation with hydrochloric acid, dichloromethane (75
cm.sup.3) was added. The organic phase was washed with a saturated
aqueous solution of sodium hydrogencarbonate (3.times.50 cm.sup.3)
and dried (Na.sub.2SO.sub.4). The solvent was removed under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (98:2, v/v) as eluent
to give a white solid material material (858 mg). A solution of
this white solid material (846 mg, 1.80 mmol) in ethanol (10.0
cm.sup.3) was stirred at room temperature and 20% palladium
hydroxide over carbon (400 mg) was added. The mixture was degassed
with argon and placed in a hydrogen atmosphere. After stirring for
2 h the mixture was directly purified by silica gel column
chromatography using dichloromethane/methanol (97:3, v/v) as eluent
to give 6 as a white solid material (444 mg, 82%). .delta..sub.H
((CD.sub.3).sub.2SO) 11.3 (1 H, br s, NH), 7.36 (1 H, d, J 1.1,
6-H), 5.80 (1 H, d, J 4.3, 1'-H), 5.61 (1 H, s, OH), 4.86 (1 H, m,
5'-H.sub.a), 3.89 (1 H, d, J 4.2, 2'-H), 3.85 (1 H, m, 2"-H.sub.a),
3.83-3.64 (3 H, m, 4'-H, 5'-H.sub.b, 2"-H.sub.b), 2.14 (1 H, m,
1"-H.sub.a), 1.81 (1 H, m, 1"-H.sub.b), 1.78 (3 H, d, J 1.0,
CH.sub.3). .delta..sub.C (CD.sub.3OD) 166.7 (C-4), 152.2 (C-2),
139.7 (C-6), 110.1 (C-5), 89.4, 89.1, 85.5, 85.2 (C-1', C-2', C-3',
C-4'), 71.4 (C-2"), 61.6 (C-5'), 37.0 (C-1"), 12.7 (CH.sub.3)
(Found: C, 47.4; H, 5.7; N, 9.0.
C.sub.12H.sub.16N.sub.2O.sub.6,H.sub.2O requires C, 47.7; H, 6,0;
N, 9.3%).
EXAMPLE 10
(1S,5R,6R,8R)-6-(4,4'-Dimethoxytrityloxymethyl)-5-hydroxy-8-(thymin-1yl)-2-
,7-dioxa-bicyclo[3.3.0]nonane (7)
[0295] A solution of nucleoside 6 (310 mg, 1.09 mmol) in anhydrous
pyridine (2.5 cm.sup.3) was stirred at room temperature and
4,4'-dimethoxytrityl chloride (593 mg, 1.83 mmol) was added. After
stirring for 3 h, additional 4,4'-dimethoxytrityl chloride (100 mg,
0.310 mmnol) was added. After stirring for another 2 h, methanol
(0.5 cm.sup.3) was added and the mixture was evaporated. The
residue was dissolved in dichloromethane (5 cm.sup.3) and washed
with an aqueous saturated solution of sodium hydrogencarbonate
(3.times.5 cm.sup.3). The organic phase was dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography with
dichloromethane/methanol (99:1, v/v) as eluent to give 7 as a white
solid material (618 mg, 97%). .delta..sub.H (CDCl.sub.3) 9.04 (1 H,
br s, NH), 7.47-7.16 (10 H, m, 6-H, DMT), 6.86-6.82 (4 H, m, DMT),
6.06 (1 H, d, J 4.1, 1'-H), 4.35 (1 H, d, J 4.1, 2'-H), 4.03 (1 H,
m, 4'-H), 3.89 (1 H, m, 2"-H.sub.a), 3.79 (6 H, s,
2.times.OCH.sub.3), 3.61 (1 H, m, 5'-H.sub.a), 3.32-3.26 (2H, m,
5'-H.sub.b), 1.94-1.69 (2 H, m, 1"-H.sub.a, 1"-H.sub.b,), 1.89 (3
H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3) 163.4 (C-4), 158.6
(DMT), 150.1 (C-2), 144.3 (DMT), 137.2 (C-6), 135.6, 135.3, 129.9,
129.9, 128.9, 128.1, 127.9, 126.9, 125.2, 113.2 (DMT), 109.3 (C-5),
88.7, 87.3, 86.9, 83.5, 81.0 (DMT, C-1', C-2', C-3', C-4'), 69.7
(C-2"), 62.1 (C-5'), 55.1 (OCH.sub.3), 36.5 (C-1"), 12.5
(CH.sub.3).
EXAMPLE 11
(1S,5R,6R,8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-6-(4,4'-dimet-
hoxytrityloxymethyl)-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]nonane
(8)
[0296] A solution of nucleoside 7 (436 mg, 0.743 mmol) in anhydrous
dichloromethane (2.2 cm.sup.3) and diisopropylethylamine (0.62
cm.sup.3) was stirred at room temperature and 2-cyamoethyl
N,N-diisopropylphosphora- midochloridite (0.33 cm.sup.3, 1.46 mmol)
was added. After stirring for 1.5 h, methanol (0.4 cm.sup.3) and
ethyl acetate (5 cm.sup.3) were added and the mixture was washed
with aqueous saturated solutions of sodium hydrogencarbonate
(3.times.5 cm.sup.3) and brine (3.times.5 cm.sup.3). The organic
phase was dried (Na.sub.2SO.sub.4) and evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography using dichloromethane/triethylamine (97:3, v/v) as
eluent, the solvents were evaporated to give an oil which was
dissolved in toluene (1 cm.sup.3) and precipitation from hexane at
-30.degree. C. yielded 8 as a solid white material (517 mg, 88%).
.delta..sub.P (CDCl.sub.3) 142.0, 141.9.
EXAMPLE 12
1-(3,5-Di-O-benzyl-3-C-(2-hydroxyethyl)-.beta.-D-ribofuranosyl)thymine
(9)
[0297] To a stirred solution of nucleoside 2 (1.00 g, 2.09 mmol) in
THF (5.4 cm.sup.3) and water (5.4 cm.sup.3) was added sodium
periodate (1.34 g, 6.27 mmol) and a 2.5% solution of osmium
tetraoxide in tert-butanol (w/w, 0.265 cm.sup.3, 19 .mu.mol). The
solution was stirred at room temperature for 45 min. Water (25
cm.sup.3) was added and the solution was extracted with
dichloromethane (2.times.50 cm.sup.3). The organic phase was washed
with a saturated aqueous solution of sodium hydrogencarbonate
(3.times.30 cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent was
removed under reduced pressure and the residue was redissolved in
THF (5.4 cm.sup.3) and water (5.4 cm.sup.3). The mixture was
stirred at room temperature and sodium boronhydride (79 mg, 2.08
mmol) was added. After stirring for 1.5 h, water (25 cm.sup.3) was
added and the solution was extracted with dichloromethane
(2.times.50 cm.sup.3). The organic phase was washed with a
saturated aqueous solution of sodium hydrogencarbonate (3.times.30
cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography using dichloromethane/methanol (98:2, v/v) as
eluent to give nucleoside 9 as a white solid material (488 mg,
48%). .delta..sub.H (CDCl.sub.3) 9.14 (1 H, br s, NH), 7.60 (1 H ,
d, J 1.1, 6-H), 7.40-7.22 (10 H, m, Bn), 6.25 (1 H, d, J 7.7,
1'-H), 4.59 (1 H, d, J 7.1 Bn), 4.49 (1 H, d, J 7.1 Bn), 4.39-3.30
(m, 8H, 4'-H, 2'-H, Bn, 5'-H.sub.a, 5'-H.sub.b, 2"-H.sub.a,
2"-H.sub.b), 2.23-2.00 (2 H, m, 1"-H.sub.a, 1"-H.sub.b), 1.49 (3 H,
d, J 0.7, CH.sub.3). .delta..sub.C (CDCl.sub.3) 163.5 (C-4), 151.2
(C-2), 137.1, 136.5 (Bn), 135.7 (C-6), 128.7, 128.5, 128.2, 127.8,
127.6, 127.2 (Bn), 111.3 (C-5), 87.0, 82.7, 81.1, 78.3 (C-1',C-2',
C-3', C-4'), 73.7, 69.6 (Bn), 64.4 (C-5'), 57.0 (C-2"), 32.4
(C-1"), 11.8 (CH.sub.3) (Found: C, 63.9; H, 6.3; N, 5.4.
C.sub.26H.sub.30N.sub.2O.sub.7,0.25H.sub.2O requires C, 64.1; H,
6.3; N, 7.75%).
EXAMPLE 13
1-[3-C-(2-O-t-Butyldimethylsilyloxyethyl)-3,5-di-O-benzyl-.beta.-D-ribofur-
anosyl]thymine (10)
[0298] A mixture of nucleoside 9 (1.80 g, 3.4 mmol) and
t-butyldimethylsilyl chloride (0.585 g, 3.9 mmol) was dissolved in
anhydrous pyridine (20 cm.sup.3). After 2 h at room temperature the
reaction mixture was evaporated to dryness, twice co-evaporated
with toluene (2.times.10 cm.sup.3) and re-dissolved in
dichloromethane (150 cm.sup.3). The solution was washed with a
saturated aqueous solution of sodium hydrocarbonate (2.times.50
cm.sup.3) and evaporated to give a foam. This material was purified
by preparative silica-gel HPLC using gradient elution (0-3%
methanol in dichloromethane, v/v) to give nucleoside 10 as a white
solid material (1.86 g, 92%). .delta..sub.H (CDCl.sub.3) 7.61 (1H,
d, J 1.1, 6-H), 7.35-7.20 (10H, m, Bn), 6.27 (1H, d, J 7.9, 1'-H),
4.65-4.40 (4H, m, Bn, 2'-H), 4.37 (1H, s, Bn), 4.28 (1H, t, J 7.9,
4'-H), 4.35-3.55 (4H, m, 2"-H.sub.a, 2"-H.sub.b, 5'-H.sub.a,
5'-H.sub.b), 2.30-2.05 (2H, m, 1"-H,, 1"-H.sub.b), 1.46 (3H, s,
5-CH.sub.3), 0.90 (9H, m, CH.sub.3--C--Si), 0.08 (6H, m,
CH.sub.3--Si). .delta..sub.C (CDCl.sub.3) 163.6 (C-6), 151.0 (C-2),
137.5, 136.6, 135.8 (C-5, Bn), 128.3, 128.1, 127.8, 127.2, 127.1,
126.8, 126.7 (Bn), 110.7 (C-4), 86.8, 82.5, 81.6, 78.3 (C-1', C-2',
C-3', C-4'), 73.3, 69.8 (Bn), 64.46 (C-5'), 58.2 (C-2"), 32.9
(C-1"), 25.6, 25.4, 17.9, -3.9, -5.7 (TBDMS), 11.6 (CH.sub.3).
FAB.sup.+-MS: m/z 597.19 [M+H].sup.+, 619.18 [M+Na].sup.+ (Found:
C, 64.2; H, 7.4; N, 4.2; C.sub.32H.sub.44O.sub.7N.su- b.2Si
requires C, 64.4; H, 7.4; N, 4.7%).
EXAMPLE 14
1-[3-C-(2-t-Butyldimethylsilyloxyethyl)-3,5-di-O-benzyl-.beta.-D-erythro-p-
entofuran-2-ulosyl]thymine (11)
[0299] A mixture nucleoside 10 (2.14 g, 3.59 mmol), 1.48 g (3.95
mmol) of pyridinium dichromate (1.48 g, 3.95) and activated 3A
molecular sieve powder (4 g) was suspended in anhydrous
dichloromethane (80 cm.sup.3). After cooling the mixture to
-10.degree. C., acetic anhydride (10 cm.sup.3, 98 mmol) was added
dropwise under vigorous stirring. The suspension was allowed to
warm to room temperature and stirring was continued for 1.5 h
whereupon the reaction was quenched by addition of triethylamine
(20 cm.sup.3). The mixture was diluted with dichloromethane to 300
cm.sup.3 and was washed with water (2.times.200 cm.sup.3). The
organic phase was evaporated, and the residue purified by flash
silica-gel chromatography using a gradient of 1.0, 1.2, 1.3, 1.4,
1.5% methanol in dichloromethane (v/v, total volume 250 cm.sup.3
each) to give nucleoside 11 (1.89 g, 84.4%) as a white solid
material. .delta..sub.H (CDCl.sub.3) 7.35-7.20 (11H, m, Bn, 6-H),
6.40 (1H, s, 1'-H), 4.57 (1H, 5, Bn), 4.52 (1H, s, Bn), 4.46 (1H,
d, J 11.0, Bn), 4.29 (1H, d, J 11.0, Bn), 4.07 (1H, dd, J' 0.5,
2.2, 4'-H), 3.95-3.70 (4H, m, 2"-H.sub.a, 2"-H.sub.b, 5'-H.sub.a,
5'-H.sub.b), 2.05 (1H, m, 1"-H.sub.a), 2.42 (1H, m, 1"-H.sub.b),
1.42 (3H, d, J 1.1, 5-CH.sub.3), 0.86 (9H, s, CH.sub.3--C--Si),
0.01 (6H, s, CH.sub.3--Si). .delta..sub.C (CDCl.sub.3) 202.6
(C-2'), 163.7 (C-4), 151.2 (C-2), 137.7, 136.6, 136.5 (Bn, C-6),
128.7, 128.5, 128.2, 128.1, 127.7, 126.4, 126.3 (Bn), 110.9 (C-5),
84.5, 81.3, 80.2 (C-1', C-3', C-4'), 73.6, 70.4 (Bn), 66.0 (C-5'),
57.6 (C-2"), 27.3 (C-1"), 25.9, 25.7, 18.2, -5.8, -5.9 (TBDMS),
11.7 (CH.sub.3). FAB-MS m/z 595.14 [M+H].sup.+ (Found: C, 64.1; H,
6.9; N, 4.5; C.sub.32H.sub.42O.sub.7N.sub.2Si requires C, 64.6; H,
7.1; N, 4.7%).
EXAMPLE 15
(1S,5R,6R,8R)-1-Hydroxy-5-benzyloxy-6-benzyloxymethyl-8-(thymin-1-yl)-2,7--
dioxabicyclo[3.3.0]octane (12)
[0300] Compound 11 (1.80 g, 30.3 mmol) was dissolved in 0.5% HCl in
methanol (w/w, 20 cm.sup.3) and the mixture was stirred for 30 min
at room temperature. After evaporation to dryness, the residue was
dissolved in dichloromethane (100 cm.sup.3) and washed with a
saturated aqueous solution of sodium hydrogencarbonate (2.times.40
cm.sup.3). The organic phase was evaporated and the residue was
purified by flash silica-gel chromatography eluting with 2%
methanol in dichloromethane (v/v) to yield nucleoside 12 (1.35 g,
93.5%) as a white solid material. .delta..sub.H (CDCl.sub.3)
7.37-7.27 (11H, m, Bn, 6-H), 5.87 (1H, s, 1'-H), 4.71 (2H, s, Bn),
4.64 (1H, d, J 12.0, Bn), 4.56 (1H, d, J 12.0, Bn), 4.36 (1H, t, J
5.7, 4'-H), 4.16 (1H, m, 2"-H.sub.a), 3.96 (1H, m, 2"-H.sub.b),
3.74 (2H, m, 5'-H.sub.a, 5'-H.sub.b ), 2.35-2.15 (2H, m,
1"-H.sub.a, 1"-H.sub.b), 1.88 (3H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 163.7 (C-4), 151.4 (C-2), 137.8, 137.3, 136.7 (Bn,
C-6), 128.5, 128.4, 128.0, 127.8, 127.5 (Bn), 109.9 (C-5), 108.6
(C-2'), 88.8, 87.1, 80.9 (C-1', C-3', C-4'), 73.6, 68.5, 68.1, 67.9
(C-5', C-2", Bn), 30.9 (C-1"), 12.6 (CH.sub.3). FAB-MS: m/z 481.03
[M+H].sup.+, 503.02 [M+Na].sup.+ (Found: C, 64.6; H, 5.8; N, 5.7;
C.sub.26H.sub.28O.sub.7N.sub.2 requires C, 65.0; H, 5.9; N,
5.8%).
EXAMPLE 16
(1S,5R,6R,8R)-1,5-Dihydroxy-6-hydroxymethyl-8-(thymin-1-yl)-2,7-dioxabicyc-
lo-[3.3.0]octane (13)
[0301] Compound 13 was successfully derived from compound 12 by
catalytic removal of the benzyl protecting group in the same way as
described in preparation of 6. Purification of 13 was accomplished
by column silica gel chromatography eluting with gradient
concentrations (6 to 14%) of methanol in dichloromethane.
Analytical amounts of compound 13 (up to 15 mg) were additionally
purified by reverse-phase HPLC at column (10.times.250 mm) packed
by Nucleosil C18 (10 .mu.m). Flow rate: 8 cm.sup.3/min; eluent:
0-10% acetonitrile in 60 min. Yield 82%. .delta..sub.H (CD.sub.3OD)
7.44 (1H d, J 1.2, 6-H), 5.83 (1H, s, 1'-H), 4.10-3.80 (5H, m,
5'-H.sub.a, 5'-H.sub.b, 2"-H.sub.a, 2"-H.sub.b, 4'-H), 2.39-2.25
(1H, m, 1"-H.sub.a), 2.00-1.90 (1H, m, 1"-H.sub.b), 1.87 (3H, d, J
1.2, CH.sub.3). .delta..sub.C (CD.sub.3OD) 166.3 (C-4), 152.7
(C-2), 139.8 (C-6), 110.0, 109.6 (C-2',C-5), 87.8, 85.8, 84.6
(C-1', C-3', C-4'), 68.8, 61.6 (C-5', C-2"), 35.6 (C-1"), 12.4
(CH.sub.3). FAB-MS: m/z 301.03 [M+H].sup.+ (Found: C, 46.6; H, 5.7;
N, 8.5; C.sub.12H.sub.16O.sub.7N.sub.2 requires C, 48.0; H, 5.4; N,
9.3%).
EXAMPLE 17
(1S,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-1-methoxy-8-(3-N-methylthymin--
1-yl)-2,7-dioxabicyclo[3.3.0]octane (14),
(1S,5R,6R,8R)-5-Benzyloxy-6-benz-
yloxymethyl-1-hydroxy-8-(3-N-methylthymin-1-yl)-2,7-dioxabicyclo[3.3.0]oct-
ane (15) and
(1S,5R,6R,8R)-5-Benzyloxy-6-benzyloxymethyl-1-methoxy-8-(thym-
in-1-yl)-2,7-dioxabicyclo[3.3.0]octane (16)
[0302] A mixture of compound 12 (1.04 g, 2.16 mmol) and sodium
hydride (171 mg of a 60% suspension in mineral oil, 4.30 mmol) was
dissolved in anhydrous dichloromethane (4 cm.sup.3) during 10 min
under stirring. Methyl iodide (1 cm.sup.3, 16 mmol) was added and
the reaction mixture was incubated at 36.degree. C. for 23 h. After
evaporation, the residue was purified by silica gel column
chromatography eluting with a gradient of 0.4-2.4%, methanol in
dichloromethane (v/v) to give products 14, 15 and 16 and starting
material 12 (212 mg, 20.5%). Compound 14 (47 mg, 4.3%).
.delta..sub.H (CDCl.sub.3) 7.25-7.37 (11H, m, Bn, 6-H), 6.15 (1H,
s, 1'-H), 4.74 (1H, d, J 11.5, Bn), 4.67 (1H, d, J 11.3, Bn), 4.62
(1H, d, J 12.1, Bn), 4.55 (1H, d, J 11.9, Bn), 4.34 (1H, t, J 5.6,
4'-H), 3.99, (1H, m, 2"-H.sub.a), 4.22 (1H, m, 2"-H.sub.b), 3.72
(2H, m, 5'-H.sub.a, 5'-H.sub.a), 3.41 (3H, s, CH.sub.3--O), 3.35
(3H, s, CH.sub.3--N.sup.3), 2,27 (1H, m, 1"-H.sub.a), 2.41 (1H, m,
1"-H.sub.b), 1.93 (3H, s, 5-CH.sub.3). .delta..sub.C (CDCl.sub.3)
163.3 (C-4), 151.0 (C-2), 138.2, 137.3, 135.7 (Bn, C-6), 128.3,
128.2, 127.8, 127.6, 127.4, 126.9 (Bn), 111.8 (C-5), 108.5 (C-2'),
89.1, 84.8, 79.5 (C-1', C-3', C-4'), 73.5, 68.4, 68.2, 67.3 (Bn,
C-5', C-2"), 50.8 (CH.sub.3--O), 32.6 (C-1"), 27.9 (CH.sub.3--N),
13.2 (CH.sub.3). FAB-MS: m/z 508.88 [M+H].sup.+ (Found: C, 65.7; H,
6.9; N, 4.8; C.sub.28H.sub.32O.sub.7N.sub- .2 requires C, 66.1; H,
6.3; N, 5.5%). Compound 15 (97 mg, 9.1%). .delta..sub.H
(CDCl.sub.3) 7.37-7.28 (11H, m, Bn, 6-H), 5.86 (1H, s, 1'-H), 4.72
(2H, s, Bn), 4.64 (1H, d, J 11.9, Bn), 4.58 (1H, d, J 11.9, Bn),
4.37 (1H, t, J 5.6, 4'-H), 4.13 (1H, m, 2"-H.sub.a), 3.93 (1H, m,
2"-H.sub.b), 3.75 (2H, m, 5'-H.sub.a, 5'-H.sub.b), 3.34 (1H, s,
CH.sub.3--N), 2.32-2.16 (2H, m, 1"-H.sub.a, 1"-H.sub.b), 1.93 (3H,
s, CH.sub.3). .delta..sub.C (CDCl.sub.3) 163.2 (C-4), 151.9 (C-2),
137.5, 137.1, 134.0 (Bn, C-6), 128.4, 128.3, 128.1, 127.9 127.7,
127.6, 127.3 (Bn), 108.8, 108.5 (C-2', C-5), 88.7 (C-1'), 88.0,
81.0 (C-3', C-4'), 73.5, 68.3, 67.9, 67.7 (Bn, C-5', C-2"), 30.6
(C-1"), 27.8 (CH.sub.3--N), 13.2 (CH.sub.3). FAB-MS m/z 495.28
[M+H].sup.+, 517.24 [M+Na].sup.+. Compound 16 (665 mg, 62.3%).
.delta..sub.H (CDCl.sub.3) 7.35-7.25 (11H, m, Bn, 6-H), 6.06 (1H,
s, 1'-H), 4.73 (1H, d, J 11.5, Bn), 4.66 (1H, d, J 11.3, Bn), 4.61
(1H, d, J 11.9, Bn), 4.55 (1H, d, J 12.0, Bn), 4.34 (1H, t, J 5.6,
4'-H), 4.20 (1H, m, 2"-H.sub.a), 3.98 (1H, m, 2"-H.sub.b), 3.72
(2H, m, 5'-H.sub.a, 5'-H.sub.b), 3.40 (3H, s, CH.sub.3--O),
2.45-2.35 (1H, m, 1"-H.sub.a), 2.30-2.20 (1H, m, 1"-H.sub.b), 1.90
(3H, d, J 1.1, CH.sub.3). .delta..sub.C (CDCl.sub.3) 163.2 (C-4),
150.1 (C-2), 138.2, 137.9, 137.3 (Bn, C-6), 128.4, 128.2, 127.8,
127.6 127.4, 127.1 (Bn), 110.8 (C-5), 109.3 (C-2'), 89.2, 84.2,
79.6 (C-1', C-3', C-4'), 73.6, 68.5, 68.3, 67.4 (Bn, C-5', C-2"),
50.8 (CH.sub.3--O), 32.6 (C-1"), 12.5 (CH.sub.3). FAB-MS m/z 495.22
[M+H].sup.+, 517.23 [M+Na].sup.+ (Found: C, 66.2; H, 7.2; N, 4.4;
C.sub.27H.sub.30O.sub.7N.sub.2 requires C, 65.6; H, 6.1; N,
5.7%).
EXAMPLE 18
(1S,5R,6R,8R)-5-Hydroxy-6-hydroxymethyl-1-methoxy-8-(thymin-1-yl)-2,7-diox-
abicyclo[3.3.0]octane (17)
[0303] To a solution of nucleoside 16 (1.20 g, 2.43 mmol) in
methanol (10 cm.sup.3) was added 20% palladium hydroxide over
charcoal (250 mg) and the mixture was carefully degassed under
reduced pressure. An atmosphere of hydrogen was applied and
stirring was continued for 12 h. The catalyst was removed by
filtration of the reaction mixture through a glass column
(1.times.8 cm) packed with silica gel in methanol. The column was
additionally washed with methanol (20 cm.sup.3). All fractions were
collected, evaporated to dryness and co-evaporated with petroleum
ether to yield a glass-like solid. This residue was purified by
silica gel chromatography eluting with a gradient of 5-10% methanol
in dichloromethane (v/v). The fractions containing the product were
collected, combined and evaporated to dryness. The residue was
dissolved in anhydrous methanol (5 cm.sup.3) and anhydrous benzene
(100 cm.sup.3) was added. Lyophilisation yielded nucleoside 17
(0.61 g, 79%) as a white solid material. .delta..sub.H (CD.sub.3OD)
7.45 (1H, s, 6-H), 5.93 (1H, s, 1'-H), 4.15-3.81 (5H, m,
5'-H.sub.a, 5'-H.sub.b, 2"-H.sub.a, 2"-H.sub.b, 4'-H), 3.43 (3H, s,
CH.sub.3--O), 2.47-2.40 (1H, m, 1"-H.sub.a), 2.03-1.93 (1H, m,
1"-H.sub.b), 1.92 (3H, s, CH.sub.3). .delta..sub.C (CD.sub.3OD)
164.1 (C-4), 150.1 (C-2), 138.3 (C-6), 109.6 (C-5), 108.3 (C-2'),
84.4, 84.1, 82.4 (C-1', C-3', C-4'), 68.0, 59.5 (C-5', C-2"), 49.6
(CH.sub.3--O), 34.0 (C-1"), 10.5 (CH.sub.3). FAB-MS m/z 315.13
[M+H].sup.+, 337.09 [M+Na].sup.+ (Found: C, 49.9; H, 5.7; N, 8.2;
C.sub.13H.sub.18O.sub.7N.sub.2 requires C, 49.7; H, 5.8; N,
8.9%.
EXAMPLE 19
(1S,5R,6R,8R)-6-(4,4'-Dimethoxytrityloxymethyl)-5-hydroxy-1-methoxy-8-(thy-
min-1-yl)-2,7-dioxabicyclo[3.3.0]octane (18)
[0304] A mixture of compound 17 (0.95 g, 3.03 mmol) and
4,4'-dimethoxytrityl chloride (1.54 g, 4.77 mmol) was dissolved in
anhydrous pyridine (20 cm.sup.3) and stirred for 4 h at room
temperature. The reaction mixture was evaporated to give an oily
residue which was co-evaporated with toluene (2.times.20 cm.sup.3).
Dichloromethane (50 cm.sup.3) and a saturated aqueous solution of
sodium hydrogencarbonate (50 cm.sup.3) were added, the organic
phase was separated and evaporated, and the residue purified by
silica gel HPLC (the residue was dissolved in the minimum amount of
dichloromethane containing 0.5% triethylamine (v/v) and applied to
the column equilibrated by the same solvent. The column was washed
(ethylacetate:petroleum ether:triethylamine; 15:84.5:0.5 (v/v/v,
1000 cm.sup.3) and the product was eluted in a gradient of methanol
(0-2%) in dichloromethane containing 0.5% of triethylamine (v/v/v)
to give compound 18 (1.71 g, 92.8%) as white solid material.
.delta..sub.H (CDCl.sub.3) 7.51-7.17 (10H, m, DMT, 6-H), 6.79-6.85
(4H, m, DMT), 6.04 (1H, s, 1'-H), 4.12-3.98 (3H, m, 5'-H.sub.a,
5'-H.sub.b, 4'-H), 3.77 (6H, s, CH.sub.3-DMT), 3.49 (3H, s,
CH.sub.3--O), 3.45-3.32 (2H, m, 2"-H.sub.a, 2"-H.sub.b), 2.11-2.01
(1H, m, 1"-H.sub.a), 1.94-1.87 (1H, m, 1"-H.sub.b), 1.93 (3H, s,
CH.sub.3). .delta..sub.C (CDCl.sub.3) 164.2 (C-4), 158.6, 144.7,
135.7, 130.1, 128.2, 127.9, 126.8, 113.2 (DMT), 150.7 (C-2), 137.7
(C-6), 109.8, 109.7 (C-5, C-2'), 86.5, 85.3, 85.0, 81.4 (DMT, C-1',
C-3', C-4'), 69.2, 62.4 (C-5', C-2"), 55.2 (CH.sub.3-DMT), 51.7
(CH.sub.3--O),35.5 (C-1"), 12.7 (CH.sub.3). FAB-MS m/z 617.26
[M+H].sup.+, 639.23 [M+Na].sup.+ (Found: C, 66.4; H, 6.1; N, 4.2;
C.sub.34H.sub.36O.sub.9N.sub.2 requires C, 66.2; H, 5.9; N,
4.5%).
EXAMPLE 20
(1S,5R,6R,.sup.8R)-5-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-6-(4,4'--
dimethoxytrityloxymethyl)-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0-
]octane (19)
[0305] Compound 18 (1.2 g, 1.95 mmol) was dissolved in anhydrous
dichloromethane (10 cm.sup.3). N,N-Diisopropylethylamine (1.35
cm.sup.3, 7.8 mmol) and
2-cyanoethyl-N,N-diiso-propylphosphoramidochloridite (0.92 g, 3.9
mmol) were added under stirring at room temperature. After 72 h,
the mixture was diluted to 100 cm.sup.3 by dichloromethane and
washed by a saturated aqueous solution of sodium hydrogencarbonate
(50 cm.sup.3). The organic phase was evaporated and applied to
silica gel HPLC purification using a gradient of eluent B
(petroleum ether:dichloromethane:ethyl acetate:pyridine;
45:45:10:0.5; v/v/v) in eluent A (petroleum
ether:dichloromethane:pyridine; 50:50:0.5; v/v/v). The fractions
containing the product were concentrated, co-evaporated with
toluene (10 cm.sup.3) and dried under reduced pressure. The residue
was dissolved in anhydrous benzene (20 cm.sup.3) and precipitated
by addition of this solution into anhydrous petroleum ether (400
cm.sup.3) under stirring. The resulting white solid was isolated by
filtration and dried to give compound 19 (0.96 g, 60.3%).
.delta..sub.P (CDCl.sub.3) 142.64, 142.52. FAB-MS m/z 817.26
[M+H].sup.+, 839.24 [M+Na].sup.+ (Found: C, 62.8; H, 6.4; N, 6.9;
C.sub.43H.sub.53O.sub.10N.sub.4P requires C, 63.2; H, 6.5; N,
6.9%).
EXAMPLE 21
1,2-O-Isopropylidene-3-C-vinyl-.alpha.-D-ribofuranose (20)
[0306] A solution of
5-O-t-butyldi-methylsilyl-1,2-O-isopropylidene-.alpha-
.-D-erythro-pent-3-ulofuranose (Y. Yoshimura, T. Sano, A. Matsuda,
T. Ueda, Chem. Pharm. Bull., 1988, 36, 162) (6.05 g, 0.020 mol) in
anhydrous THF (250 cm.sup.3) was stirred at 0.degree. C. and a 1 M
solution of vinylmagnesium bromide in ether (44 cm.sup.3, 44 mmol)
was added dropwise. The reaction mixture was stirred at room
temperature for 2 h, whereupon saturated aqueous ammonium chloride
(200 cm.sup.3) was added, and extraction was performed using
dichloromethane (3.times.300 cm.sup.3). The combined extract was
washed with brine (3.times.250 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed and the residue was
redissolved in anhydrous THF (225 cm.sup.3). To this mixture was
added a 1 M solution of tetrabutylammonium fluoride in THF (22
cm.sup.3, 22 mmol), stirring at room temperature was continued for
20 min whereupon the mixture was evaporated undler reduced
pressure. The residue was dissolved in dichloromethane (500
cm.sup.3) and washed with a saturated solution of sodium
hydrogencarbonate (2.times.200 cm.sup.3). The aqueous phase was
extracted using continuous extraction for 12 h and the combined
extract was dried (Na.sub.2SO.sub.4) and evaporated. The residue
was purified by silica gel columri chromatography using
dichloromethane/methanol (99:1, v/v) as eluent to give furanose 20
as a white solid material (3.24 g, 75%). .delta..sub.H (CDCl.sub.3)
5.84 (1H, d, J 3.7, 1-H), 5.74 (1H, dd, J 11.0, 17.2, 1'-H), 5.52
(1H, dd, J 1.6, 17.1, 2'-H.sub.a), 5.29 (1H, dd, J 1.3, 11.0,
2'-H.sub.b), 4.21 (1H, d, J 3.7, 2-H), 3.98 (1H, t, J 5.7, 4-H),
3.68-3.64 (2H, m, 5-H.sub.a, 5-H.sub.b), 2.88 (1H, s, 3-OH), 1.99
(1H, t, J 6.3, 5-OH), 1.60 (3H, s, CH.sub.3), 1.35 (3H, s,
CH.sub.3) .delta..sub.C (CDCl.sub.3) 133.6 (C-1'), 116.2 (C-2'),
113.0 (C(CH.sub.3).sub.2), 103.8 (C-1), 83.4, 82.4 (C-4, C-2), 79.6
(C-3), 61.3 (C-5), 26.5, 26.4 (CH.sub.3).
EXAMPLE 22
3,5-Di-O-benzyl-1,2-O-isopropylidene-3-C-vinyl-.alpha.-D-ribofuranose
(21)
[0307] A 60% suspension of sodium hydride (w/w, 1.78 g, 44.5 mmol)
in anhydrous DMF (50 cm.sup.3) was stirred at 0.degree. C. and a
solution of furanose 20 (3.20 g, 14.8 mmol) in anhydrous DMF (35
cm.sup.3) was added dropwise over 30 min. The mixture was stirred
at 50.degree. C. for 1 h and subsequently cooled to 0.degree. C. A
solution of benzyl bromide (5.3 mL, 44.5 mmol) in anhydrous DMF
(5.3 cm.sup.3) was added dropwise, and the mixture was stirred at
room temperature for 20 h. The reaction mixture was evaporated and
redissolved in dichloromethane (300 cm.sup.3), washed with
saturated aqueous sodium hydrogencarbonate (3.times.200 cm.sup.3)
and dried (Na.sub.2SO.sub.4). The solvents were removed under
reduced pressure and the residue was purified by silica gel column
chromatography using petroleum ether/ethylacetate (9:1, v/v) as
eluent to give furanose 21 as a white solid material (5.36 g, 91%).
.delta..sub.H (CDCl.sub.3) 7.40-7.26 (10H, m, Bn), 5.90 (1H, d, J
3.6, 1-H), 5.72 (1H, dd, J 11.1, 17.9, 1'-H), 5.41 (1H, dd, J 0.7,
11.1, 2'-H.sub.a), 5.30 (1H, dd, J 0.5, 17.8, 2'-H.sub.b),
4.70-4.45 (6H, m, Bn, 2-H, 4-H), 3.69 (1H, dd, J 2.6, 10.8,
5-H.sub.a), 3.50 (1H, dd, J7.9, 10.9, 5-H.sub.b), 1.64 (3H, s,
CH.sub.3), 1.40 (3H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3)
138.6, 138.3 (Bn), 134.5 (C-1'), 128.3-127.4 (Bn), 118.2 (C-2'),
112.9 (C(CH.sub.3).sub.2), 104.7 (C-1), 84.7, 81.1, 81.0 (C-2, C-3,
C-4), 73.3 (C-5), 69.4, 67.0 (Bn), 26.8, 26.6 (CH.sub.3).
EXAMPLE 23
1,2-Di-O-acetyl-3,5-di-O-benzyl-3-C-vinyl-.alpha.,.beta.-D-ribofuranose
(22)
[0308] A solution of furanose 21 (4.40 9, 11.1 mmol) in 80% aqueous
acetic acid (50 cm.sup.3) was stirred at 90.degree. C. for 8 h. The
solvents were removed and the residue was coevaporated with 99%
ethanol (3.times.25 cm.sup.3), toluene (3.times.25 cm.sup.3) and
anhydrous pyridine (2.times.25 cm.sup.3) and redissolved in
anhydrous pyridine (20 cm.sup.3). Acetic anhydride (17 cm.sup.3)
was added and the solution was stirred at room temperature for 48
h. The reaction was quenched with ice-cold water (100 cm.sup.3) and
extracted with dichloromethane (2.times.100 cm.sup.3). The combined
extract was washed with saturated aqueous sodium hydrogencarbonate
(3.times.100 cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent
was evaporated and the residue was purified by silica gel column
chromatography using petroleum ether/ethylacetate (4:1, v/v) as
eluent to give furanose 22 as an oil (4.27 g, 87%,
.alpha.:.beta..about.1:1). .delta..sub.C (CDCl.sub.3) 169.9, 169.8
(C.dbd.O), 139.0, 138.6, 138.0, 137.8 (Bn), 133.3, 132.4 (C-1'),
128.4-126.8 (Bn), 119.6, 119.5 (C-2'), 99.5, 94.0 (C-1), 85.4,
85.0, 84.3, 83.6, 77.7, 73.6, 73.5, 73.3, 70.0, 69.2, 67.5, 67.2
(C-2, C-3, C-4, C-5, Bn), 21.0, 20.9, 20.6, 20.4 (CH.sub.3).
EXAMPLE 24
1-(2-O-Acetyl-3,5-di-O-benzyl-3-C-vinyl-.beta.-D-ribofuranosyl)thymine
(23)
[0309] To a stirred solution of compound 22 (4.24 g, 9.6 mmol) and
thymine (2.43 g, 19.3 mmol) in anhydrous acetonitrile (100
cm.sup.3) was added N,O-bis(trimethylsilyl)acetamide (11.9
cm.sup.3, 48.1 mmol). The reaction mixture was stirred at reflux
for 30 min. After cooling to 0.degree. C., trimethylsilyl triflate
(3.2 cm.sup.3, 16.4 mmol) was added dropwise and the solution was
stirred for 24 h at room temperature. The reaction was quenched
with cold saturated aqueous sodium hydrogencarbonate (100 cm.sup.3)
and the resulting mixture was extracted with dichlorometharne
(3.times.50 cm.sup.3). The combined extract was washed with
saturated aqueous sodium hydrogencarbonate (2.times.50 cm.sup.3)
and brine (2.times.50 cm.sup.3) and dried (Na.sub.2SO.sub.4). The
extract was evaporated under reduced pressure and the residue was
purified by silica gel column chromatography using
dichloromethane/methanol (99:1, v/v) as eluent to give nucleoside
23 as a white foam (4.03 g, 83%). .delta..sub.H (CDCl.sub.3) 8.78
(1H, br s, NH), 7.75 (1H, s, 6-H), 7.38-7.26 (10H, m, Bn), 6.49
(1H, d, J 8.1, 1'-H), 5.99-5.88 (2H, m, 2'-H and 1"-H), 5.54-5.48
(2H, m, 2"-H.sub.a, 2"-H.sub.b), 4.91-4.50 (4H, m, Bn), 4.34 (1H,
s, 4'-H), 3.80 (1H, m, 5'-H.sub.a), 3.54 (1H, m, 5'-H.sub.b), 2.11
(3H, s, COCH.sub.3), 1.48 (3H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 170.1 (C.dbd.O) 163.8 (C-4), 151.0 (C-2), 138.9, 136.9
(Bn), 136.1 (C-6), 132.0 (C-1"), 128.7, 128.5, 128.2, 127.8, 127.7,
127.5, 127.5, 127.1 (Bn), 120.7 (C-2"), 111.3 (C-5), 85.4 (C-1'),
85.2 (C-3'), 84.3 (C-4'), 76.0 (C-2'), 73.7 (C-5'), 69.3, 67.6
(Bn), 20.6 (COCH.sub.3), 11.7 (CH.sub.3). Found: C, 66.3; H, 6.0;
N, 5.1; C.sub.28H.sub.30N.sub.2O.sub.- 7 requires C, 66.4; H, 6.0;
N, 5.5%.
EXAMPLE 25
1-(3,5-Di-O-benzyl-3-C-vinyl-.beta.-D-ribofuranosyl)thymine
(24)
[0310] To a stirred solution of nucleoside 23 (3.90 g, 7.7 mmol) in
anhydrous methanol (40 cm.sup.3) was added sodium methoxide (0.83
g, 15.4 mmol). The mixture was stirred at room temperature for 42 h
and then neutralised with dilute aqueous hydrochloric acid. The
mixture was extracted with dichloromethane (2.times.150 cm.sup.3),
and the combined extract was washed with saturated aqueous sodium
hydrogencarbonate (3.times.100 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
to give nucleoside 24 as a white foam (3.48 g, 97%). .delta..sub.H
(CDCl.sub.3) 8.89 (1H, br s, NH), 7.60 (1H, d, J 0.9, 6-H),
7.36-7.26 (10H, m, Bn), 6.23 (1H, d, J 7.8, 1'-H), 5.98 (1H, dd, J
11.2, 17.7, 1"-H), 5.66 (1H, d, J 17.7, 2"-H.sub.a), 5.55 (1H, d, J
11.5, 2"-H.sub.b), 4.75-4.37 (6H, m, 2'-H, 4'-H, Bn), 3.84 (1H, dd,
J 2.7, 10.8, 5'-H.sub.a), 3.58 (1H, d, J 11.2, 5'-H.sub.b), 3.23
(1H, d, J 10.6, 2'-OH), 1.50 (3H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 163.7 (C-4), 151 3 (C-2), 138.0, 136.9 (Bn), 136.0
(C-6), 131.2 (C-1"), 128.8, 128.6, 128.3, 127.8, 127.7, 127.3 (Bn),
120.7 (C-2"), 111.3 (C-5), 87.3 (C-1'), 84.6 (C-3'), 81.4 (C-4'),
78.0 (C-2'), 73.7 (C-5'), 70.0, 66.4 (Bn), 11.8 (CH.sub.3). Found:
C, 66.8; H, 6.2; N, 5.9; C.sub.26H.sub.28N.sub.2O.sub.6 requires C,
67.2; H, 6.1; N, 6.0%.
EXAMPLE 26
1-(3,5-Di-O-benzyl-2-O-methanesulfonyl-3-C-vinyl-.beta.-D-ribofuranosyl)th-
ymine (25)
[0311] Nucleoside 24 (2.57 g, 5.53 mmol) was dissolved in anhydrous
pyridine (18 cm.sup.3) and cooled to 0.degree. C. Methanesulfonyl
chloride (1.28 cm.sup.3, 16.6 mmol) was added dropwise and the
mixture was stirred at room temperature for 30 min. The reaction
was quenched with water (5 cm.sup.3) and the resulting mixture was
extracted with dichloromethane (3.times.80 cm.sup.3). The combined
extract was washed with saturated aqueous sodium hydrogencarbonate
(3.times.120 cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvent
was evaporated under reduced pressure and the residue was purified
by silica gel column chromatography using dichloromethane/methanol
(99:1, v/v) as eluent to give nucleoside 25 as a yellow foam (2.53
g, 84%). .delta..sub.H (CDCl.sub.3) 8.92 (1H, br s, NH), 7.71 (1H,
d, J 1.4, 6-H), 7.41-7.28 (10H, m, Bn), 6.57 (1H, d, J 7.8, 1'-H),
5.99-5.61 (4H, m, 2'-H, 1"-H and 2"-H.sub.a, 2"-H.sub.b), 4.86-4.50
(4H, m, Bn), 4.37 (1H, dd, J 1.5, 2.4, 4'-H), 8.82 (1H, dd, J 2.6,
11.0, 5'-H.sub.a), 3.55 (1H, dd, J 1.2, 11.0, 5'-H.sub.b), 3.02
(3H, s, CH.sub.3), 1.47 (3H, d, J 1.1, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 163.7 (C-4), 151.5 (C-2), 138.7 136.7 (Bn), 135.7
(C-6), 130.9 (C-1"), 128.8, 128.5, 128.4, 127.6, 127.0 (Bn), 121.8
(C-2"), 111.9 (C-5), 85.1 (C-1'), 84.5 (C-3'), 84.0 (C-4'), 80.7
(C-2'), 73.7 (C-5'), 69.2, 67.7 (Bn), 38.9 (CH.sub.3), 11.8
(CH.sub.3).
EXAMPLE 27
1-(3,5-Di-O-benzyl-3-C-vinyl-.beta.-D-arabinofuranosyl)thymine
(26)
[0312] A solution of nucleoside 25 (2.53 g, 4.66 mmol) in a mixture
of ethanol (50 cm.sup.3), water (50 cm.sup.3) and 1 M aqueous
sodium hydroxide (15 cm.sup.3) was stirred under reflux for 16 h.
The mixture was neutralised using dilute aqueous hydrochloric acid,
the solvent was evaporated under reduced pressure, and the residue
was extracted with dichloromethane (3.times.120 cm.sup.3). The
combined extract was washed with saturated aqueous sodium
hydrogencarbonate (3.times.150 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (99:1) as eluent to give 26 as a
white foam (1.61 g, 74%). .delta..sub.H (CDCl.sub.3) 9.89 (1H, br
s, NH), 7.50 (1H, d, J 1.1, 6-H), 7.41-7.26 (Bn), 6.28 (1H, d, J
2.8, 1'-H), 6.05 (1H, dd, J 11.1, 17.9, 1"-H), 5.58-5.50 (2H, m,
2"-H.sub.a, 2"-H.sub.b), 4.98 (1H, d, J 9.0, 2'-OH), 4.64-4.31 (6H,
m, 2'-H, 4'-H, Bn), 3.73 (2H, m, 5'-H.sub.a, 5'-H.sub.b), 1.73 (1H,
d, J 0.6, CH.sub.3). .delta..sub.C (CDCl.sub.3) 165.1 (C-4), 150.5
(C-2), 138.4, 138.0, 136.7 (C-6, Bn), 130.4 (C-1"), 128.8, 128.6,
128.5, 128.1, 128.0, 127.8 (Bn), 120.6 (C-2"), 108.1 (C-5), 88.6
(C-1'), 87.9 (C-3'), 87.2 (C-4'), 73.7 (C-2'), 71.8 (C-5'), 69.7,
66.3 (Bn), 12.3 (CH.sub.3). Found: C, 66.8; H, 6.2; N, 5.9;
C.sub.26H.sub.28N.sub.2O.sub.6 requires C, 67.2; H, 6.1; N,
6.0.
EXAMPLE 28
1-(3,5-Di-O-benzyl-3-C-hydroxymethyl-.beta.-D-arabinofuranosyl)thymine
(27)
[0313] To a solution of nucleoside 26 (2.00 g, 4.31 mmol) in a
mixture of THF (15 cm.sup.3) and water (15 cm.sup.3) was added
sodium periodate (2.76 g, 12.9 mmol) and a 2.5% solution of osmium
tetraoxide in t-butanol (w/w, 0.54 cm.sup.3, 43 .mu.mol). The
reaction was stirred at room temperature for 18 h, quenched with
water (50 cm.sup.3), and the mixture was extracted with
dichloromethane (2.times.100 cm.sup.3). The combined extract was
washed with saturated aqueous sodium hydrogen carbonate (3.times.75
cm.sup.3), dried (Na.sub.2SO.sub.4) and evaporated under reduced
pressure. The residue was redissolved in a mixture of THF (15
cm.sup.3) and water (15 cm.sup.3), and sodium borohydride (488 mg,
12.9 mmol) was added. The reaction mixture was stirred at room
temperature for 1 h, water (50 cm.sup.3) was added, and the mixture
was extracted with dichloromethane (2.times.100 cm.sup.3). The
combined organic phase was washed with saturated aqueous sodium
hydrogencarbonate (3.times.75 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed and the residue was
purified by silica gel column chromatography using
dichloromethane/methanol (98:2, v/v) as eluent to give nucleoside
27 as a white foam (732 mg, 36%). .delta..sub.H (CDCl.sub.3) 11.09
(1H, br s, NH), 7.41 (1H, d, J 1.0, 6-H), 7.38-7.26 (Bn), 6.16 (1H,
d, J 2.6, 1'-H), 5.12 (1H, d, J 5.4, 2'-OH), 4.66-4.29 (6H, m,
2'-H, 4'-H, Bn), 4.02-3.96 (2H, m, 1"-H.sub.a, 1"-H.sub.b), 3.90
(1H, dd, J 7.2, 9.7, 5'-H.sub.a), 3.79 (1H, dd, J 5.6, 9.7,
5'-H.sub.b), 2.49 (1H, t, J 6.4, 1"-OH), 1.68 (3H, d, J 0.6,
CH.sub.3); .delta..sub.C (CDCl.sub.3) 166.1 (C-4), 150.6 (C-2),
139.0, 137.9, 137.0 (C-6, Bn), 128.7, 128.6, 128.4, 128.3, 128.0
(Bn), 107.5 (C-5), 88.2 (C-1'), 88.1 (C-3'), 84.2 (C-4'), 73.7
(C-5'), 72.1 (C-2'), 69.3, 65.4 (Bn), 58.6 (C-1"), 12.3
(CH.sub.3).
EXAMPLE 29
(1R,2R,4R,5S)-1-Benzyloxy-2-benzyloxymethyl-4-(thymin-1-yl)-3,6-dioxabicyc-
lo-[3.2.0]heptane (28)
[0314] A solution of compound 27 (2.26 g, 4.83 mmol) in anhydrous
pyridine (20 cm.sup.3) was stirred at -40.degree. C. and a solution
of methanesulphonyl chloride (0.482 cm.sup.3, 4.83 mmol) in
anhydrous pyridine (10 cm.sup.3) was added. The reaction mixture
was stirred at room temperature for 17 h, water (50cm.sup.3) was
added, and the mixture was extracted with dichloromethane
(2.times.100 cm.sup.3). The combined organic phase was washed with
saturated aqueous sodium hydrogencarbonate (3.times.100 cm.sup.3),
dried (Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using
dichloromethane/methanol (99:1, v/v) as eluent to give an
intermediate which after evaporation of the solvents was dissolved
in anhydrous DMF (15 cm.sup.3). This solution was added dropwise to
a suspension of 60% sodium hydride (461 mg, 11.5 mmol) in anhydrous
DMF (15 cm.sup.3) at 0.degree. C. The reaction was stirred at room
temperature for 30 min, then quenched with water (60 cm.sup.3).
After neutralisation using dilute aqueous hydrochloric acid, the
mixture was dissolved in dichloromethane (150 cm.sup.3), washed
with saturated aqueous sodium hydrogencarbonate (3.times.100
cm.sup.3) and dried (Na.sub.2SO.sub.4). The solvents were
evaporated and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (99:1, v/v) as eluent
to give nucleoside 28 as a white foam (2.00 g, 93%). .delta..sub.H
(CDCl.sub.3) 9.13 (1H, br s, NH), 7.55 (1H, d, J 1.4, 6-H),
7.40-7.26 (Bn), 5.99 (1H, d, J 2.5, 1'-H), 5.30 (1H, d, J 2.7,
2'-H), 4.88-4.57 (6H, m, 1"-H.sub.a, 1"-H.sub.b, Bn), 4.22-4.19
(1H, m, 4'-H), 3.92 (1H, dd, J 6.2, 10.8, 5'-H.sub.a), 3.82 (1H,
dd, J 3.7, 10.8, 5'-H.sub.b), 1.91 (3H, d, J 1.3, CH.sub.3).
.delta..sub.C (CDCl.sub.3) 163.8 (C-4), 150.3 (C-2), 137.6 (C-6),
137.5, 137.0 (Bn), 128.7, 128.6, 128.2, 128.0, 127.8, 127.3 (Bn),
109.8 (C-5), 85.7 (C-3'), 84.1 (C-1'), 83.5 (C-4'), 79.7 (C-1"),
73.9 (C-2'), 73.6 (C-5'), 68.6, 67.8 (Bn), 12.4 (CH.sub.3). FAB m/z
451 [M+H].sup.+, 473 [M+Na].sup.+. Found: C, 66.3; H, 5.9; N, 6.1;
C.sub.25H.sub.26N.sub.2O.su- b.6 requires C, 66.7; H, 5.8; N,
6.2%.
EXAMPLE 30
(1R,2R,4R,5S)-1-Hydroxy-2-hydroxymethyl-4-(thymin-1-yl)-3,6-dioxabicyclo[3-
.2.0]-heptane (29)
[0315] To a stirred solution of nucleoside 28 (180 mg, 0.40 mmol)
in ethanol (3 cm.sup.3) was added 10% palladium hydroxide over
carbon (90 mg). The mixture was degassed several times with argon
and placed under a hydrogen atmosphere. The reaction mixture was
stirred at room temperature for 6 h, then filtered through celite.
The filtrate was evaporated under reduced pressure and the residue
was purified by silica gel column chromatography using
dichloromethane/methanol (96:4, v/v) as eluent to give nucleoside
29 as a white solid material (92 mg, 86%). .delta..sub.H
(CD.sub.3OD) 7.79 (1H, d, J 1.2, 6-H), 5.91 (1H, d, J 2.5, 1'-H),
4.96 (1H, d, J 2.5, 2'-H), 4.92 (1H, d, J 7.4, 1"-H.sub.a), 4.58
(1H, dd, J 0.9, 7.4, 1"-H.sub.b), 3.98 (1H, dd, J 7.3, 12.8,
5'-H.sub.a), 3.87-3.82 (2H, m, 4'-H, 5'-H.sub.b), 3.34 (2H, s,
3'-OH, 5'-OH), 1.87 (3H, d, J 1.3, CH.sub.3). .delta..sub.C
(CD.sub.3OD) 166.5 (C-4), 152.1 (C-2), 140.1 (C-6), 110.1 (C-5),
91.2 (C-2'), 85.1 (C-1'), 84.0 (C-4'), 79.6 (C-3'), 78.6 (C-1"),
61.1 (C-5'), 12.3 (CH.sub.3).
EXAMPLE 31
(1R,2R,4R,5S)-1-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-2-(4,4'-dimet-
hoxytrityloxymethyl)-4-(thymin-1-yl)-3,6-dioxabicyclo[3.2.0]heptane
(30)
[0316] To a solution of diol 29 (250 mg, 0.925 mmol) in anhydrous
pyridine (4 cm.sup.3) was added 4,4'-di-methoxytrityl chloride (376
mg, 1.11 mmol) and the mixture was stirred at room temperature for
18 h. The reaction was quenched with methanol (1.5 cm.sup.3) and
the mixture was evaporated under reduced pressure. A solution of
the residue in dichloromethane (30 cm.sup.3) was washed with
saturated aqueous sodium hydrogencarbonate (3.times.20 cm.sup.3),
dried (Na.sub.2SO.sub.4) and evaporated. The residue was purified
by silica gel column chromatography using dichloromethane/methanol
(98:2, v/v) as eluent to give an intermediate which was dissolved
in anhydrous dichloromethane (7.0 cm.sup.3).
N,N-Diisopropylethylamine (0.64 cm.sup.3, 3.70 mmol) followed by
2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.41 cm.sup.3,
1.85 mmol) were added and the mixture was stirred at room
temperature for 25 h. The reaction was quenched with methanol (3
cm.sup.3), and the mixture was dissolved in ethylacetate (70
cm.sup.3), washed with saturated aqueous sodium hydrogencarbonate
(3.times.50 cm.sup.3) and brine (3.times.50 cm.sup.3), dried
(Na.sub.2SO.sub.4), and was evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using
petroleum ether/dichloromethane/ethylacetate/triethy- lamine
(100:45:45:10, v/v/v/v) as eluent. The residue obtained was
dissolved in toluene (2 cm.sup.3) and precipitated under stirring
from petroleum ether at -50.degree. C. After evaporation of the
solvents, the residue was coevaporated with anhydrous acetonitrile
(4.times.5 cm.sup.3) to give 30 as a white foam (436 mg, 61%).
.sup.31P NMR (CDCl.sub.3) 146.6.
EXAMPLE 32
3,5-Di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-.alpha.-D-ribofuran-
ose (31)
[0317] To a solution of
3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene--
.alpha.-D-ribofuranose (R. D. Youssefyeh, J. P. H. Verheyden and J.
G. Moffatt, J. Org. Chem., 1979, 44, 1301) (20.1 g, 0.064 mol) in
anhydrous DMF (100 cm.sup.3) at -5.degree. C. was added a
suspension of NaH (60% in mineral oil (w/w), four portions during 1
h 30 min, total 2.85 g, 0.075 mol). Benzyl bromide (8.9 cm.sup.3,
0.075 mol) was added dropwise and stirring at room temperature was
continued for 3 h whereupon ice-cold water (50 cm.sup.3) was added.
The mixture was extracted with EtOAc (4.times.100 cm.sup.3) and the
combined organic phase was dried (Na.sub.2SO.sub.4). After
evaporation, the residue was purified by silica gel column
chromatography eluting with 5% EtOAc in petroleum ether (v/v) to
yield compound 31 (18.5 g, 71%). .delta..sub.C (CDCl.sub.3) 138.0,
137.4, 128.5, 128.3, 128.0, 127.8, 127.6 (Bn), 113.5
(C(CH.sub.3).sub.2), 104.4 (C-1), 86.5 (C-4), 78.8, 78.6 (Bn),
73.6, 72.6, 71.6 (C-2, C-3, C-5), 63.2, (C-1'), 26.7, 26.1
(CH.sub.3).
EXAMPLE 33
4-C-(Acetoxymethyl)-3,5-di-O-benzyl-1,2-O-isopropylidene-.alpha.-D-ribofur-
anose (32)
[0318] To a solution of furanose 31 (913 mg, 2.28 mmol) in
anhydrous pyridine (4.5 cm.sup.3) was dropwise added acetic
anhydride (1.08 cm.sup.3, 11.4 mmol) and the reaction mixture was
stirred at room temperature for 3 h. The reaction was quenched by
addition of ice-cold water (50 cm.sup.3) and extraction was
performed with dichloromethane (3.times.50 cm.sup.3). The combined
organic phase was washed with a saturated aqueous solution of
sodium hydrogencarbonate (2.times.50 cm.sup.3), dried
(Na.sub.2SO.sub.4) and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using
dichloromethane as eluent to give compound 32 as a clear oil (911
mg, 90%). .delta..sub.H (CDCl.sub.3) 7.34-7.25 (10H, m, Bn), 5.77
(1H, d, J 3.6, 1-H), 4.78-4.27 (8 H, m, Bn, H-5.sub.a, H-5.sub.b,
H-3, H-2), 3.58 (1 H, d, J 10.3,H-1'.sub.a), 3.48 (1 H, d, J 10.5,
H-1'.sub.b), 2.04 (3 H, s, COCH.sub.3), 1.64 (3 H, s, CH.sub.3),
1.34 (3 H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3) 171.1
(C.dbd.O), 138.2, 137.9, 128.6, 128.1, 128.0, 128.0, 127,8 (Bn),
114.0 (C(CH.sub.3).sub.2), 104.5 (C-1), 85.4 (C-4), 79.3, 78.6
(C-2, C-3), 73.7, 72.7, 71.2 (Bn, C-5), 64.9 (C-1'), 26.7, 26.3
(C(CH.sub.3).sub.2), 21.0 (COCH.sub.3). Found: C, 67.0; H, 6.5;
C.sub.25H.sub.30O.sub.7, 1/4H.sub.2O requires C, 67.2 H, 6.9%.
EXAMPLE 34
4-C-(Acetoxymethyl)-1,2-di-O-acetyl-3,5-di-O-benzyl-D-ribofuranose
(33)
[0319] A solution of furanose 32 (830 mg, 1.88 mmol) in 80% acetic
acid (10 cm.sup.3) was stirred at 90.degree. C. for 4 h. The
solvent was removed under reduced pressure and the residue was
coevaporated with ethanol (3.times.5 cm.sup.3), toluene (3.times.5
cm.sup.3) and anhydrous pyridine (3.times.5 cm.sup.3), and was
redissolved in anhydrous pyridine (3.7 cm.sup.3). Acetic anhydride
(2.85 cm.sup.3) was added and the solution was stirred for 72 h at
room temperature. The solution was poured into ice-cold water (20
cm.sup.3) and the mixture was extracted with dichloromethane
(2.times.20 cm.sup.3). The combined organic phase was washed with a
saturated aqueous solution of sodium hydrogencarbonate (2.times.20
cm.sup.3), dried (Na.sub.2SO.sub.4) and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography using dichloromethane as eluent to give 33
(.beta.:.alpha..about.1:3) as an clear oil (789 mg, 86%).
.delta..sub.C (CDCl.sub.3) 171.0, 170.3, 170.0, 169.3 (C.dbd.O),
138.1, 137.6, 136.3, 128.9, 128.6, 128.2, 128.0, 128.0, 127.9,
127.7, 124.0 (Bn), 97.8, 97.8 (C-1), 87.0, 85.0, 78.9, 74.5, 74.4,
73.8, 73.6, 72.0, 71.8, 71.0, 70.9, 64.6, 64.4 (C-2, C-3, C-4, Bn,
C-5, C-1'), 21.0, 20.8, 20.6 (COCH.sub.3). Found: C, 64.2; H, 6.3;
C.sub.26H.sub.30O.sub.9 requires C, 64.2; H, 6.2%.
EXAMPLE 35
1-(4-C-(Acetoxymethyl)-2-O-acetyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)t-
hymine (34)
[0320] To a stirred solution of the anomeric mixture 33 (736 mg,
1.51 mmol) and thymine (381 mg, 3.03 mmol) in anhydrous
acetonitrile (14.5 cm.sup.3) was added
N,O-bis(trimethyl-silyl)acetamide (2.61 cm.sup.3, 10.6 mmol). The
reaction mixture was stirred at reflux for 1 h, then cooled to
0.degree. C. Trimethylsilyl triflate (0.47 cm.sup.3, 2.56 mmol) was
added dropwise under stirring and the solution was stirred at
65.degree. C. for 2 h. The reaction was quenched with a cold
saturated aqueous solution of sodium hydrogen carbonate (15
cm.sup.3) and extraction was performed with dichloromethane
(3.times.10 cm.sup.3). The combined organic phase was washed with
saturated aqueous solutions of sodium hydrogencarbonate (2.times.10
cm.sup.3) and brine (2.times.10 cm.sup.3), and was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (98:2, v/v) as eluent to give
nucleoside 34 as a white solid material (639 mg, 76%).
.delta..sub.H (CDCl.sub.3) 8.98 (1H, br s, NH), 7.39-7.26 (11 H, m,
Bn, 6-H), 6.22 (1 H, d, J 5.3, 1'-H), 5.42 (1 H, t, J 5.4, 2'-H),
4.63-4.43 (5H, m, 3'-H, Bn), 4.41 (1 H, d, J 12.2, 5'-H.sub.a),
4.17 (1 H, d, J 12.1, 5'-H.sub.b), 3.76 (1 H, d, J 10.2,
1"-H.sub.a), 3.51 (1 H, d, J 10.4, 1"-H.sub.b), 2.09 (3 H, s,
COCH.sub.3), 2.03 (3 H, s, COCH.sub.3), 1.53 (3 H, d, J 0.9,
CH.sub.3). .delta..sub.C (CDCl.sub.3) 170.8, 170.4, (C.dbd.O),
163.9 (C-4), 150.6 (C-2), 137.4 (C-6) 137.4, 136.1, 128.9, 128.8,
128.4, 128.2, 127,9 (Bn), 111.7 (C-5), 87.2, 87.2, 86.1 (C-1',
C-3', C-4'), 77.6 (C-2'), 74.8, 73.9, 71.1, 63.8 (Bn, C-1", C-5'),
20.9, 20.8 (COCH.sub.3), 12.0 (CH.sub.3). FAB-MS m/z 553
[M+H].sup.+. Found: C, 62.7; H, 5.9; N, 4.7;
C.sub.29H.sub.32N.sub.2O.sub.9 requires C, 63.0; H, 5.8; N,
5.1%.
EXAMPLE 36
1-(3,5-Di-O-benzyl-4-C-(hydroxymethyl)-.beta.-D-ribofuranosyl)thymine
(35)
[0321] To a stirred solution of nucleoside 34 (553 mg, 1.05 mmol)
in methanol (5.5 cm.sup.3) was added sodium methoxide (287 mg, 5.25
mmol). The reaction mixture was stirred at room temperature for 10
min, then neutralised with dilute hydrochloric acid. The solvent
was partly evaporated and extraction was performed with
dichloromethane (2.times.20 cm.sup.3). The combined organic phase
was washed with saturated aqueous sodium hydrogencarbonate
(3.times.20 cm.sup.3) and was dried (Na.sub.2SO.sub.4). The solvent
was removed under reduced pressure to give 35 as a while solid
material (476 mg, 97%). .delta..sub.H (CDCl.sub.3) 7.47 (1 H, d, J
1.0 6-H), 7.36-7.22 (10 H, m, Bn), 6.07 (1 H, d, J 3.8, 1'-H), 4.87
(1 H, d, J 11.7, Bn), 4.55 (1 H, d, J 11.7, Bn), 4.50-4.32 (4 H, m,
Bn, 2'-H, 3'-H), 3.84-3.53 (4 H, m, 5'-H.sub.a, 5'-H.sub.b,
1"-H.sub.a, 1"-H.sub.b), 1.50 (3 H, d, J 1.1, CH.sub.3).
.delta..sub.C (CDCl.sub.3) 164.3 (C-4), 151.3 (C-2), 137.6 (C-6)
136.4, 136.3, 128.8, 128.6, 128.4, 128.3, 127,9 (Bn), 111.1 (C-5),
91.1, 91.0, 88.1 (C-1', C-3', C-4'), 77.4 (C-2'), 74.8, 73.8, 71.4,
63,2 (Bn, C-5', C-1"), 12.0 (CH.sub.3). FAB-MS m/z 491
[M+Na].sup.+. Found: C, 63.4; H, 6.0; N, 5.5;
C.sub.25H.sub.28N.sub.2O.sub.7, 1/4H.sub.2O requires C, 63.5; H,
6.1; N, 5.9%.
EXAMPLE 37
Intermediate 35A
[0322] A solution of nucleoside 35 (225 mg, 0.48 mmol) in anhydrous
pyridine (1.3 cm.sup.3) was stirred at 0.degree. C. and
p-toluenesulphonyl chloride (118 mg, 0.62 mmol) was added in small
portions. The solution was stirred at room temperature for 16 h and
additional p-toluenesulphonyl chloride (36 mg, 0.19 mmol) was
added. After stirring for another 4 h and addition of ice-cold
water (15 cm.sup.3), extraction was performed with dichloromethane
(2.times.15 cm.sup.3). The combined organic phase was washed with
saturated aqueous sodium hydrogencarbonate (3.times.15 cm.sup.3)
and dried (Na.sub.2SO.sub.4). The solvent was removed under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (99:1, v/v) as eluent
to give a intermediate 35A (140 mg) which was used without further
purification in the next step.
EXAMPLE 38
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-yl)-2,5-dioxabicyc-
lo-[2.2.1]heptane (36)
[0323] Intermediate 35A (159 mg) was dissolved in anhydrous DMF
(0.8 cm.sup.3). The solution was added dropwise to a stirred
suspension of 60% sodium hydride in mineral oil (w/w, 32 mg, 0.80
mmol) in anhydrous DMF (0.8 cm.sup.3) at 0.degree. C. The mixture
was stirred at room temperature for 72 h and then concentrated
under reduced pressure. The residue was dissolved in
dichloromethane (10 cm.sup.3), washed with saturated aqueous sodium
hydrogencarbonate (3.times.5 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (99:1, v/v) as eluent to give the
bicyclic nucleoside 36 as a white solid material (65.7 mg, 57%).
.delta..sub.H (CDCl.sub.3) 9.24 (1 H, br s, NH), 7.49 (1 H, s,
6-H), 7.37-7.26 (10 H, m, Bn), 5.65 (1 H, s, 1'-H), 4.70-4.71 (5 H,
m, Bn, 2'-H), 4.02-3.79 (5 H, m, 3'-H, 5'-H.sub.a, 5'-H.sub.b,
1"-H.sub.a, 1"-H.sub.b), 1.63 (3 H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 164.3 (C-4), 150.1 (C-2), 137.7, 137.1 (Bn), 135.0
(C-6), 128.8, 128.7, 128.4, 128.0, 127.9 (Bn), 110.4 (C-5), 87.5,
87.3 (C-1', C-3'), 76.7, 75.8, 73.9, 72.3, 72.1 (Bn, C-5', C-2',
C-4'), 64.5 (C-1"), 12.3 (CH.sub.3). FAB-MS m/z 451
[M+H].sup.+.
EXAMPLE 39
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2-
.2.1]-heptane (37)
[0324] A solution of nucleoside 36 (97 mg, 0.215 mmol) in ethanol
(1.5 cm.sup.3) was stirred at room temperature and 20% palladium
hydroxide over carbon (50 mg) was added. The mixture was degassed
several times with argon and placed in a hydrogen atmosphere with a
baloon. After stirring for 4 h, the mixture was purified by silica
gel column chromatography using dichloromethane-methanol (97:3,
v/v) as eluent to give nucleoside 37 as a white solid material (57
mg, 98%). .delta..sub.H ((CD.sub.3).sub.2SO) 11.33 (1H, br s, NH),
7.62 (1H, d, J 1.1 Hz, 6-H), 5.65 (1H, d, J 4.4 Hz, 3'-OH), 5.41
(1H, s, 1'-H), 5.19 (1H, t, J 5.6 Hz, 5'-OH), 4.11 (1H, s, 2'-H),
3.91 (1H, d, J 4.2 Hz, 3'-H), 3.82 (1H, d, J 7.7 Hz,1"-H.sub.a),
3.73 (1H, s, H'-5.sub.a), 3.76 (1H, s, 5'-H.sub.b), 3.63 (1H, d, J
7.7 Hz, 1"-H.sub.b), 1.78 (3H, d, J 0.7 Hz, CH.sub.3).
.delta..sub.C (CDCl.sub.3) 166.7 (C-4), 152.1 (C-2), 137.0 (C-6),
110.9 (C-5), 90,5, 88.4 (C-1', C-4'), 80.9, 72.5, 70.4 (C-2', C-3',
C-5'), 57.7 (C-1"), 12.6 (CH.sub.3). EI-MS m/z 270 [M].sup.+.
EXAMPLE 40
(1R,3R,4R,7S)-1-(4,4'-Dimethoxytrityloxymethyl)-7-hydroxy-3-thymin-1-yl)-2-
,5-dioxabicyclo[2.2.1]heptane (38)
[0325] To a solution of nucleoside 37 (1.2 g, 4.44 mmol) in
anhydrous pyridine (5 cm.sup.3) was added 4,4'-dimethoxytrityl
chloride (2.37 g, 7.0 mmol) at 0.degree. C. The solution was
stirred at room temperature for 2 h whereupon the reaction was
quenched with ice-cold water (10 cm.sup.3) and extracted with
dichloromethane (3.times.15 cm.sup.3). The combined organic phase
was washed with saturated aqueous solutions of sodium hydrogen
carbonate (3.times.10 cm.sup.3), brine (2.times.10 cm.sup.3) and
dried (Na.sub.2SO.sub.4). The solvent was removed under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (98:2, v/v) as eluent
to give nucleoside 38 as a white solid material (2.35 g, 93%).
.delta..sub.H (CDCl.sub.3) 9.89 (1H, br s, NH), 7.64 (1H, s, 6-H),
7.47-7.13 (9H, m, DMT), 6.96-6.80 (4H, m, DMT), 5.56 (1H, s, 1'-H),
4.53 (1H, br s, 2'-H), 4.31 (1H, m, 3'-H), 4.04-3.75 (9H, m,
1"-H.sub.a, 1"-H.sub.b, 3'-OH, OCH.sub.3), 3.50 (2H, br s,
5'-H.sub.a, 5'-H.sub.b), 1.65 (3H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 164.47 (C-4), 158.66 (DMT), 150.13 (C-2), 144.56,
135.46, 135.35, 134.78, 130.10, 129.14, 128.03, 127.79, 127.05
(C-6, DMT), 113.32, 113.14 (DMT), 110.36 (C-5), 89.17, 88.16, 87.05
(C-1', C-4', DMT), 79.36, 71.81, 70.25, 58.38 (C-2', C-3', C-5',
C-1"), 55.22 (OCH.sub.3), 12.57 (CH.sub.3). FAB-MS m/z 595
[M+Na].sup.+, 573 [M+H].sup.+.
EXAMPLE 41
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4'-dimet-
hoxytrityloxymethyl)-3-(thymin-1-yl)-2,5-dioxabicyclo[2.2.1]heptane
(39)
[0326] To a solution of nucleoside 38 (2.21 g, 3.86 mmol) in
anhydrous dichloromethane (6 cm.sup.3) at room temperature was
added N,N-diisopropylethylamine (4 cm.sup.3) and 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite (1 cm.sup.3, 4.48 mmol) and
stirring was continued for 1 h. MeOH (2 cm.sup.3) was added, and
the mixture was diluted with ethyl acetate (10 cm.sup.3) and washed
successively with saturated aqueous solutions of sodium
hydrogencarbonate (3.times.5 cm.sup.3) and brine (3.times.5
cm.sup.3) and was dried (Na.sub.2SO.sub.4). The solvent was
evaporated under reduced pressure, and the residue was purified by
basic alumina column chromatography with dichloromethane/methanol
(99:1, v/v) as eluent to give 39 as a white foam. This residue was
dissolved in dichloromethane (2 cm.sup.3) and the product was
precipitated from petroleum ether (100 cm.sup.3, cooled to
-30.degree. C.) under vigorous stirring. The precipitate was
collected by filtration, and was dried to give nucleoside 39 as a
white solid material (2.1 g, 70%). .delta..sub.P (CDCl.sub.3)
149.06, 148.74. FAB-MS m/z 795 [M+Na].sup.+, 773 [M+H].sup.+.
EXAMPLE 42
1-(2-O-Acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)ura-
cil (40)
[0327] To a stirred solution of the anomeric mixture 33 (3.0 g,
6.17 mmol) and uracil (1.04 g, 9.26 mmol) in anhydrous acetonitrile
(65 cm.sup.3) was added N,O-bis(trimethylsilyl)acetamide (9.16
cm.sup.3, 37.0 mmol). The reaction mixture was stirred for 1 h at
room temperature and cooled to 0.degree. C. Trimethylsilyl triflate
(1.8 cm.sup.3, 10.0 mmol) was added dropwise and the solution was
stirred at 60.degree. C. for 2 h. The reaction was quenched by
addition of a saturated aqueous solution of sodium
hydrogencarbonate (10 cm.sup.3) at 0.degree. C. and extraction was
performed with dichloromethane (3.times.20 cm.sup.3). The combined
organic phase was washed with brine (2.times.20 cm.sup.3) and was
dried (Na.sub.2SO.sub.4). The solvents were removed under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (99:1, v/v) as eluent
to give nucleoside 40 as a white solid material (2.5 g, 75%).
.delta..sub.H (CDCl.sub.3) 9.57 (1H, br s, NH), 7.63 (1H, d, J8.2,
6-H), 7.40-7.24 (10H, m, Bn), 6.18 (1H, d, J4.5, 1'-H), 5.39-5.32
(2H, m, 2'-H, 5-H), 4.61 (1H, d, J 11.6, Bn), 4.49-4.40 (5H, m,
3'-H, Bn, 1"-H.sub.a), 4.37 (1H, d, J 12.3, 1"-H.sub.b), 3.76 (1H,
d, J 10.1, 5'-H.sub.a), 3.49 (1H, d, J 10.1, 5'-H.sub.b), 2.09 (s,
3H, COCH.sub.3), 2.04 (3H, s, COCH.sub.3). .delta..sub.C
(CDCl.sub.3) 170.47, 169.94 (C.dbd.O), 163.32 (C-4), 150.30 (C-2),
140.24 (C-6), 137.15, 136.95, 128.65, 128.52, 128.32, 128.19,
128.02, 127.77 (Bn), 102.57 (C-5), 87.41, 86.14 (C-1', C-4'),
77.09, 74.84, 74.51, 73.75, 70.60, 63.73 (C-2', C-3', C-5', C-1",
Bn), 20.79, 20.68 (COCH.sub.3). FAB-MS m/z 539 [M].sup.+.
EXAMPLE 43
1-(3,5-Di-O-benzyl-4-C-hydroxymethyl-.beta.-D-ribofuranosyl)uracil
(41)
[0328] To a stirred solution of nucleoside 40 (2.0 g, 3.7 mmol) in
methanol (25 cm.sup.3) was added sodium methoxide (0.864 g, 95%,
16.0 mmol). The reaction mixture was stirred at room temperature
for 10 min and neutralised with 20% aqueous hydrochloric acid. The
solvent was partly evaporated and the residue was extracted with
ethyl acetate (3.times.50 cm.sup.3). The combined organic phase was
washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.20 cm.sup.3) and was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/dichloromethane/methanol (98.5:1.5, v/v) as
eluent to give 41 as a white solid material (1.58 g, 95%).
.delta..sub.H (CDCl.sub.3) 9.95 (1H, br s, NH), 7.69 (d, J 8.1,
6-H), 7.35-7.17 (10H, m, Bn), 6.02 (1H, d, J 2.3, 1'-H), 5.26 (1H,
d, J 8.1, 5-H), 4.80 (1H, d, J 11.7, Bn), 4.47 (1H, d, J 11.7, Bn),
4.45-4.24 (4H, m, Bn, 2'-H, 3'-H), 3.81 (1H, d, J 11.9,
1"-H.sub.a), 3.69 (2H, br s, 2'-OH, 1"-OH), 3.67 (2H, m,
5'-H.sub.a, 1"-H.sub.b), 3.48 (1H, d, J 10.3, 5'-H.sub.b).
.delta..sub.C (CDCl.sub.3) 163.78 (C-4), 150.94 (C-2), 140.61
(C-6), 137.33, 137.22, 128.59, 128.18, 128.01 (Bn), 102.16 (C-5),
91.46, 88.36 (C-1', C-4'), 76.73, 74.66, 73.71, 73.29, 70.81, 62.81
(C-2', C-3', C-5', C-1", Bn). FAB-MS m/z 455 [M+H].sup.+.
EXAMPLE 44
Intermediate 42
[0329] A solution of nucleoside 41 (1.38 g, 3.0 mmol), anhydrous
pyridine (2 cm.sup.3) and anhydrous dichloromethane (6 cm.sup.3)
was stirred at -10.degree. C. and p-toluenesulfonyl chloride (0.648
g, 3.4 mmol) was added in small portions during 1 h. The solution
was stirred at -10.degree. C. for 3 h. The reaction was quenched by
addition of ice-cold water (10 cm.sup.3) and the mixture was
extracted with dichloromethane (3.times.50 cm.sup.3). The combined
organic phase was washed with a saturated aqueous solution of
sodium hydrogencarbonate (3.times.20 cm.sup.3) and was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (99:1, v/v) as eluent to give
intermediate 42 (0.9 g) which was used without further purification
in the next step.
EXAMPLE 45
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(uracil-1-yl)-2,5-dioxabicyc-
lo[2.2.1]-heptane (43)
[0330] Compound 42 (0.7 g) was dissolved in anhydrous DMF (3
cm.sup.3) and a 60% suspension of sodium hydride (w/w, 0.096 g, 24
mmol) was added in four portions during 10 min at 0.degree. C., and
the reaction mixture was stirred at room temperature for 12 h. The
reaction was quenched with methanol (10 cm.sup.3), and the solvents
were removed under reduced pressure. The residue was dissolved in
dichloromethane (20 cm.sup.3), washed with saturated aqueous sodium
hydrogencarbonate (3.times.6 cm.sup.3) and was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/ethanol (99:1, v/v) as eluent to give
nucleoside 43 (0.30 g, 60%). .delta..sub.H (CDCl.sub.3) 9.21 (1H,
br s, NH), 7.70 (1H, d, J 8.2, 6-H), 7.37-7.24 (10H, m, Bn), 5.65
(1H, s, 1'-H), 5.52 (1H, d, J 8.2, 5-H), 4.68-4.45 (5H, m, 2'-H,
Bn), 4.02-3.55 (5H, m, 3'-H, 5'-H.sub.a, 1"-H.sub.a, 5'-H.sub.b,
1"-H.sub.b). .delta..sub.C (CDCl.sub.3) 163.33 (C-4), 149.73 (C-2),
139.18 (C-6), 137.46, 136.81, 128.58, 128.54, 128.21, 128.10,
127.79, 127.53 (Bn), 101.66 (C-5), 87.49, 87.33 (C-1', C-4'),
76.53, 75.71, 73.77, 72.33, 72.00, 64.35 (C-2', C-3', C-5', C-1",
Bn). FAB-MS m/z 459 [M+Na].sup.+.
EXAMPLE 46
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2-
.2.1]-heptane (44)
[0331] To a solution of compound 43 (0.35 g, 0.8 mmol) in absolute
ethanol (2 cm.sup.3) was added 20% palladium hydroxide over carbon
(0.37 g) and the mixture was degassed several times with hydrogen
and stirred under the atmosphere of hydrogen for 4 h. The solvent
was removed under reduced pressure and the residue was purified by
silica gel column chromatography using dichloromethane/methanol
(9:1, v/v) as eluent to give nucleoside 44 as a white solid
material (0.1 6 g, 78%). .delta..sub.H (CD.sub.3OD) 7.88 (1H, d, J
8.1, 6-H), 5.69 (1H, d, J 8.1, 5-H), 5.55 (1H, s, 1'-H), 4.28 (1H,
s, 2'-H), 4.04 (1H, s, 3'-H), 3.96 (1H, d, J 7.9, 1"-H.sub.a) 3.91
(2H, s, 5'-H), 3.76 (1H, d, J 7.9, 1"-H.sub.b). .delta..sub.C
(CD.sub.3OD) 172.95 (C-4), 151.82 (C-2), 141.17 (C-6), 101.97
(C-5), 90.52, 88.50 (C-1', C-4'), 80.88, 72.51, 70.50, 57.77 (C-2',
C-3', C-5', C-1"). FAB-MS m/z 257 [M+H].sup.+.
EXAMPLE 47
(1R,3R,4R,7S)-1-(4,4'-Dimethoxytrityloxymethyl)-7-hydroxy-3-(uracil-1-yl)--
2,5-dioxabicyclo[2.2.1]heptane (45)
[0332] To a solution of compound 44 (0.08 g, 0.31 mmol) in
anhydrous pyridine (0.5 cm.sup.3) was added 4,4'-dimethoxytrityl
chloride (0.203 g, 0.6 mmol) at 0.degree. C. and the mixture was
stirred at room temperature for 2 h. The reaction was quenched with
ice-cold water (10 cm.sup.3) and extracted with dichloromethane
(3.times.4 cm.sup.3). The combined organic phase was washed with
saturated aqueous solutions of sodium hydrogencarbonate (3.times.3
cm.sup.3) and brine (2.times.3 cm.sup.3) and was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (98:2, v/v) as eluent to give
nucleoside 45 as a white solid material (0.12 g, 69%).
.delta..sub.H (CDCl.sub.3) 9.25 (1H, br s, NH), 7.93 (1H, d, J 7.2,
6-H), 7.50-7.15 (9H, m, DMT), 6.88-6.78 (4H, m, DMT), 5.63 (1H, s,
1'-H), 5.59 (1H, d, J 8.0, 5-H), 4.48 (1H, s, 2'-H), 4.26 (1H, s,
3'-H), 3.88 (1H, d, J 8.1, 1"-H.sub.a), 3.85-3.55 (7H, m,
1"-H.sub.b, OCH.sub.3), 3.58-3.40 (2H, m, 5'-H.sub.a, 5'-H.sub.b).
.delta..sub.C (CDCl.sub.3) 164.10 (C-4), 158.60 (DMT), 150.45
(C-2), 147.53 (DMT), 144.51 (C-6), 139.72, 135.49, 135.37, 130.20,
129.28, 128.09, 127.85, 127.07 (DMT), 113.39, 113.17 (DMT), 101.79
(C-5), 88.20, 87.10, 86.87 (C-1', C-4', DMT), 79.25, 71.79, 69.70,
58.13 (C-2', C-3', C-5', C-1"), 55.33 (OCH.sub.3). FAB-MS m/z 559
[M+H].sup.+.
EXAMPLE 48
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)posphinoxy)-1-(4,4'-dimeth-
oxytrityloxymethyl)-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane
(46)
[0333] To a solution of compound 45 (0.07 g, 0.125 mmol) in
anhydrous dichloromethane (2 cm.sup.3) at room temperature was
added N,N-diisopropylethylamine (0.1 cm.sup.3) and 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite (0.07 cm.sup.3, 0.32 mmol).
After stirring for 1 h, the reaction was quenched with MeOH (2
cm.sup.3), and the resulting mixture was diluted with ethyl acetate
(5 cm.sup.3) and washed successively with saturated aqueous
solutions of sodium hydrogencarbonate (3.times.2 cm.sup.3) and
brine (3.times.2 cm.sup.3), and was dried (Na.sub.2SO.sub.4). The
solvent was evaporated under reduced pressure and the residue was
purified by silica gel column chromatography using
dichloromethane/methanol (99:1, v/v) as eluent to give a white
foam. This foam was dissolved in dichloromethane (2 cm.sup.3) and
the product was precipitated from petroleum ether (10 cm.sup.3,
cooled to -30.degree. C.) under vigorous stirring. The precipitate
was collected by filtration and was dried to give compound 46 as a
white solid material (0.055 g, 58%). .delta..sub.P (CDCl.sub.3)
149.18, 149.02.
EXAMPLE 49
9-(2-O-Acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)-2--
N-isobutyryl-guanine (47)
[0334] To a stirred suspension of the anomeric mixture 33 (1.28 g,
5.6 mmol) and 2-N-isobutyrylguanine (1.8 g, 3.7 mmol) in anhydrous
dichloroethane (60 cm.sup.3) was added
N,O-bis(trimethylsilyl)acetamide (4 cm.sup.3, 16.2 mmol). The
reaction mixture was stirred at reflux for 1 h. Trimethylsilyl
triflate (1.5 mL, 8.28 mmol) was added dropwise at 0.degree. C. and
the solution was stirred at reflux for 2 h. The reaction mixture
was allowed to cool to room temperature during 1.5 h. After
dilution to 250 cm.sup.3 by addition of dichloromethane, the
mixture was washed with a saturated aqueous solution of sodium
hydrogencarbonate (200 cm.sup.3) and water (250 cm.sup.3). The
solvent was removed under reduced pressure and the residue was
purified by silica gel column chromatography using 1.25% (200
cm.sup.3) and 1.5% (750 cm.sup.3) of methanol in dichloromethane
(v/v) as eluents to give 2.10 g (87%) of a white solid that
according to .sup.1H-NMR analysis consisted of three isomers
(ratio: 12.5:2.5:1). The main product formed in that conditions is
expected to be compound 47 (P. Garner, S. Ramakanth, J. Org. Chem.
1988, 53, 1294; H. Vorbruggen, K. Krolikiewicz, B. Bennua, Chem.
Ber. 1981, 114, 1234). The individual isomers were not isolated and
mixture was used for next step. For main product 47: .delta..sub.H
(CDCl.sub.3) 12.25 (br s, NHCO), 9.25 (br s, NH), 7.91 (s, 8-H)
7.39-7.26 (m, Bn), 6.07 (d, J 4.6, 1'-H), 5.80 (dd, J 5.8, J 4.7,
2'-H), 4.72 (d, J 5.9, 3'-H), 4.59-4.43 (m, Bn, 1"-H.sub.a), 4.16
(d, J 12.1, 1"-H.sub.b), 3.70 (d, J 10.1, 5'-H.sub.a), 3.58 (d, J
10.1, 5'-H.sub.b), 2.65 (m, CHCO), 2.05 (s, COCH.sub.3), 2.01 (s,
COCH.sub.3), 1.22 (d, J 6.7, CH.sub.3CH), 1.20 (d, J 7.0,
CH.sub.3CH). .delta..sub.C (CDCl.sub.3) 178.3 (COCH), 170.6, 179.8
(COCH.sub.3), 155.8, 148.2, 147.6 (guanine), 137.6, 137.2 (guanine,
Bn), 128.5, 128.4, 128.2, 128.1, 128.0, 127.8, 127.7 (Bn), 121.2
(guanine), 86.2, 86.0 (C-1', C-4'), 77.8 (C-3'), 74.9, 74.5, 73.7,
70.4 (Bn, C-2', C-5'), 63.5 (C-1"), 36.3 (COCH), 20.8, 20.6
(COCH.sub.3), 19.0 (CH.sub.3CH). For the mixture: FAB-MS m/z 648
[M+H].sup.+, 670 [M+Na].sup.+. Found: C, 60.8; H, 6.0; N, 10.4;
C.sub.33H.sub.36N.sub.5O.s- ub.9 requires C, 61.3; H, 5.6; N,
10.8%.
EXAMPLE 50
9-(3,5-Di-O-benzyl-4-C-hydroxymethyl-.beta.-D-ribofuranosyl)-2-N-isobutyry-
lguanine (48)
[0335] A solution of the mixture described in EXAMPLE 49 containing
compound 47 (2.10 g, 3.25 mmol) in THF/Pyridine/methanol (2:3:4,
v/v/v) (40 cm.sup.3) was cooled to -10.degree. C. and sodium
methoxide (320 mg, 5.93 mmol) was added to the stirred solution.
The reaction mixture was stirred at 10.degree. C. for 30 min and
neutralised with 2 cm.sup.3 of acetic acid. The solvent was
evaporated under reduced pressure and the residue was twice
extracted in a system of dichloromethane/water (2.times.100
cm.sup.3). The organic fractions were combined and evaporated under
reduced pressure. After co-evaporation with toluene, the residue
was purified by silica gel column chromatography in a gradient
(2-7%) of methanol in dichloromethane (v/v) to give a white solid
material (1.62 g). According to .sup.1H-NMR it consisted of three
isomers (ratio: 13.5:1.5:1). For main product 48: .delta..sub.H
(CD.sub.3OD) 8.07 (s, 8-H) 7.36-7.20 (m, Bn), 6.05 (d, J 3.9,
1'-H), 4.81 (d, J 11.5, Bn), 4.75 (m, 2'-H), 4.56 (d, J 11.5, Bn),
4.51-4.43 (m, Bn, 3'-H), 3.83 (d, J 11.7, 1"-H.sub.a), 3.65 (d, J
11.7, 1"-H.sub.b), 3.64 (d, J 10.6, 5'-H.sub.a), 3.57 (d, J 10.3,
5'-H.sub.b), 2.69 (m, CHCO), 1.20 (6 H, d, J 6.8, CH.sub.3CH).
.delta..sub.C (CD.sub.3OD) 181.6 (COCH), 157.3, 150.2, 149.5
(guanine), 139.4, 139.3, 139.0 (guanine, Bn), 129.5, 129.4, 129.3,
129.2, 129.1, 129.0, 128.9, 128.8 (Bn), 121.2 (guanine), 90.7, 89.6
(C-1', C-4'), 79.2 (C-3'), 75.8, 74.5, 74.3, 72.2 (Bn, C-2', C-5'),
63.1 (C-1"), 36.9 (COCH), 19.4 (CH.sub.3CH), 19.3 (CH.sub.3CH). For
the mixture: FAB-MS m/z 564 [M+H].sup.+.
EXAMPLE 51
Intermediate 49
[0336] A solution of the mixture described in Example 50 containing
48 (1.6 g) in anhydrous pyridine (6 cm.sup.3) was stirred at
-20.degree. C. and p-toluenesulphonyl chloride (0.81 g, 4.27 mmol)
was added. The solution was stirred for 1 h at -20.degree. C. and
for 2 h at -25.degree. C. Then the mixture was diluted to 100
cm.sup.3 by addition of dichloromethane and immediately washed with
water (2.times.100 cm.sup.3). The organic phase was separated and
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography using dichloromethane/methanol as
eluent (1-2%, v/v) to give intermediate 49 (980 mg). After elution
of compound 49 from the column, the starting mixture containing 48
(510 mg) was eluted using 8% methanol in dichloromethane (v/v) as
eluent. This material was concentrated, dried under reduced
pressure and treated in the same manner as described above to give
additionally 252 mg of the intermediate. The intermediate (1.23 g)
was purified by silica gel HPLC (PrepPak Cartridge packed by
Porasil, 15-20 .mu.m, 125A, flow rate 60 cm.sup.3/min, eluent 0-4%
of methanol in dichloromethane (v/v), 120 min). Fractions
containing intermediate 49 were pooled and concentrated to give
white solid (1.04 g). According to .sup.1H-NMR it consisted of two
main products, namely 1"-O and 2'-O monotosylated derivatives in a
ratio of 2:1. FAB-MS m/z 718 [M+H].sup.+. Found C, 60.4; H, 5.8; N,
9.3; C.sub.36H.sub.39N.sub.5O.sub.9S requires C, 60.2; H, 5.5; N,
9.8%.
EXAMPLE 52
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(2-N-isobutyrylguanin-9-yl)--
2,5-dioxabicyclo[2.2.1]heptane (50)
[0337] To a solution of intermediate 49 (940 mg) in anhydrous THF
(20 cm.sup.3) was added a 60% suspension of sodium hydride (w/w,
130 mg, 3.25 mmol) and the mixture was stirred for 1 h at room
temperature. Acetic acid (0.25 mL) was added and the mixture was
concentrated under reduced pressure. The residue was dissolved in
dichloromethane (100 cm.sup.3) and was washed with water
(2.times.100 cm.sup.3). The organic phase was separated and
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography using methanol/dichloromethane
(1-1.5%, v/v) as eluent to give nucleoside 50 as a white solid
material (451 mg, 57%). .delta..sub.H (CDCl.sub.3) 12.25 (1H, br s,
NHCO), 10.12 (1H, br s, NH), 7.84 (1H, s, 8-H), 7.31-7.15 (10H, m,
Bn), 5.72 (1H, s, 1'-H), 4.60-4.46 (5H, m, Bn, 2'-H), 4.14 (1H, s,
3'-H), 4.02 (1H, d, J 7.9, 1"-H.sub.a), 3.85 (1H, d, J 7.9,
1"-H.sub.b), 3.78 (2H, s, 5'-H), 2.81 (1H, m, CHCO), 1.24 (3H, d, J
6.8, CH.sub.3CH), 1.22 (3H, d, J 6.4, CH.sub.3CH). .delta..sub.C
(CDCl.sub.3) 179.5 (COCH), 155.6, 148.1, 14.73 (guanine), 137.3,
136.9, 136.0 (guanine, Bn), 128.4, 128.3, 127.9, 127.8, 127.5,
127.4 (Bn), 121.2 (guanine), 87.1, 86.2 (C-1', C-4'), 77.0 (C-3'),
73.6, 72.5, 72.1 (Bn, C-2', C-5'), 64.9 (C-1"), 36.1 (COCH), 19.0
(CH.sub.3CH), 18.9 (CH.sub.3CH). FAB-MS m/z 546 [M+H].sup.+. Found:
C, 63.3; H, 5.9; N, 12.5; C.sub.29H.sub.30N.sub.5O.sub.6 requires
C, 64.0; H, 5.6; N, 12.9%.
[0338] Alternative preparation of compound 50. G1AQ. To a
suspension of compound 78 (1.5 g, 2.51 mmol), N2-isobutirylguanine
(0.93 g, 4.06 mmol) in dry DCM (50 mL) was added BSA
(N,O-bistrimethylsilylacetamide; 3.33 mL, 13.5 mmol) and the
mixture was refluxed for 2 h. Trimethylsilyl triflate (1.25 mL, 6.9
mmol) was added to the mixture and refluxing was continuing for
additional 2 h. The mixture was allowed to cool to room
temperature, diluted by 200 mL of DCM and washed by saturated aq.
NaHCO.sub.3 and water. Chromatography at silica gel column (1-2.5%
of CH.sub.3OH in dichloromethane) yielded 1.05 g (55%) of the
desired isomer G1AQ and 380 mg of isomers with higher mobility
which was converted to G1AQ by repetition of the procedure
described above. Ammonium hydroxide (12 mL of 25% aq.solution) was
added to a solution of G1AQ (1.05 g in 12 mL of methanol) and the
mixture was stirred for 1 hr at room temperature. After
concentration, the product was purified by silica gel column
chromatography (1-3% CH.sub.3OH in dichloromethane) to give 700 mg
G3 as a white solid material. 700 mg of G3 in anhydroux THF (15 mL)
was treated with NaH (225 mg of 60% suspension in mineral oil). 30
min later, the reaction was quenched by addition of 1.25 mL of
acetic acid, and the mixture was concentrated under reduced
pressure. The residue was dissolved in dichloromethane, washed by
NaHCO.sub.3 and water and purified by silica gel chromatography in
gradient 0.5-3% methanol/DCM. Yield 400 mg (75%) of 50.
EXAMPLE 53
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(2-N-isobutyrylguanin-9-yl)-2,5--
dioxabicyclol[2.2.1]heptane (51)
[0339] A mixture of nucleoside 50 (717 mg, 1.31 mmol) and 10%
palladium over carbon (500 mg) was suspended in methanol (8
cm.sup.3) at room temperature. The mixture was degassed several
times under reduced pressure and placed under a hydrogen
atmosphere. After stirring for 24 h the mixture was purified by
silica gel column chromatography using methanol/dichloromethane
(8-20%, v/v) as eluent to give nucleoside 51 as a glass-like solid
(440 mg, 92%). .delta..sub.H (CD.sub.3OD) 8.12 (1H, br s, 8-H),
5.86 (1H, s, 1'-H), 4.50 (1H, s, 2'-H), 4.30 (1H, s, 3'-H), 4.05
(1H, d, J 8.0, 1"-H.sub.a), 3.95 (2H, s, 5'-H), 3.87 (1H, d, J 7.9,
1"-H.sub.b), 2.74 (1H, m, CHCO), 1.23 (6H, d, J 6.9, CH.sub.3CH).
.delta..sub.C (CD.sub.3OD, signals from the carbohydrate part)
90.2, 87.6 (C-1', C-4'), 81.1 (C-3'), 72.9, 71.3 (C-2', C-5'), 58.2
(C-1"), 37.1 (COCH), 19.5 (CH.sub.3CH). FAB-MS m/z 366
[M+H].sup.+.
EXAMPLE 54
(1R,3R,4R,7S)-1-(4,4'-Dimethoxytrityloxymethyl)-7-hydroxy-3-(2-N-isobutyry-
lguanin-9-yl)-2,5-dioxabicyclo[2.2.]heptane (52)
[0340] A mixture of compound 51 (440 mg, 1.21 mmol) and
4,4'-dimethoxytrityl chloride (573 mg, 1.69 mmol) was dissolved in
anhydrous pyridine (7 cm.sup.3) and was stirred at room temperature
for 4 h. The mixture was evaporated under reduced pressure to give
an oil. Extraction was performed in a system of
dichloromethane/water (1:1, v/v, 40 cm.sup.3). The organic phase
was separated and concentrated to give a solution in a minimal
volume of dichloromethane containing 0.5% of pyridine (v/v) which
was applied to a silica gel column equilibrated by the same
solvent. The product was eluted in gradient concentrations of
methanol (0.6-2%, v/v) in dichloromethane containing 0.5% of
pyridine (v/v) to give compound 52 as a white solid material (695
mg, 86%). .delta..sub.H (CDCl.sub.3) 12.17 (1H, br s, NHCO), 10.09
(1H, br s, NH), 7.87 (1H, s, 8-H), 7.42-6.72 (13H, m, DMT), 5.69
(1H, s, 1'-H), 4.59 (1H, s, 2'-H), 4.50 (1H, s, 3'-H), 3.98 (1H, d,
J 8.1, 1"-H.sub.a), 3.69-3.99 (9H, m, DMT, 5'-H, 1"-H.sub.b), 2.72
(1H, m, CHCO), 1.17 (6H, d, J 6.8, CH.sub.3CH). .delta..sub.C
(CDCl.sub.3) 179.8 (COCH), 158.8, 144.5, 135.6, 135.5, 130.1,
128.1, 127.7, 126.9, 113.2 (DMT), 155.8, 147.9, 147.5, 137.0, 120.8
(guanine), 87.6, 86.4, 86.1 (C-1', C-4', DMT), 79.7 (C-3'), 72.6,
71.4 (C-2', C-5'), 59.8 (C-1"), 55.2 (DMT), 36.1 (COCH), 19.1, 18.8
(CH.sub.3CH). FAB-MS m/z 668 [M+H].sup.+.
EXAMPLE 55
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4'-dimet-
hoxytrityloxymethyl)-3-(2-N-isopropyonylguanine-9-y))-2,5-dioxabicyclo[2.2-
.1]heptane (53)
[0341] Compound 52 (670 mg, 1.0 mmol) was at room temperature
dissolved in anhydrous dichloromethane (5 cm.sup.3) containing
N,N-diisopropylethylami- ne (0.38 cm.sup.3, 4 mmol). 2-Cyanoethyl
N,N-diisopropylphosphoramidochlor- idite (0.36 cm.sup.3, 2.0 mmol)
was added drop-wise with stirring. After 5 h, methanol (2 cm.sup.3)
was added and the mixture was diluted to 100 cm.sup.3 by addition
of dichloromethane and washed with a saturated aqueous solution of
sodium hydrocarbonate (50 cm.sup.3). The organic phase was
separated and the solvent was removed by evaporation under reduced
pressure. The residue was dissolved in the minimum amount of
dichloromethane/petroleum ether (1:1, v/v) containing 0.5% pyridine
(v/v) and was applied to a column packed with silica gel
equilibrated by the same solvent mixture. The column was washed by
dichloromethane/petroleum/- pyridine (75:25:0.5, v/v/v, 250
cm.sup.3) and the product was eluted using a gradient of methanol
in dichloromethane (0-1%, v/v) containing 0.5% pyridine (v/v). The
fractions containing the main product were evaporated and
co-evaporated with toluene. The residue was dissolved in anhydrous
dichloromethane (5 cm.sup.3) and precipitated from petroleum ether
(100 cm.sup.3) to give compound 53 as a white solid material (558
mg, 64%) after filtration and drying. .delta..sub.P (CDCl.sub.3)
148.17, 146.07. FAB-MS m/z 868 [M+1].sup.+.
EXAMPLE 56
1-(2-O-Acetyl-4-C-acetoxymethyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)-4--
N-benzoylcytosine (54)
[0342] To a stirred solution of the anomeric mixture 33 (4.0 g,
8.22 mmol) and 4-N-benzoylcytosine (2.79 g, 13.0 mmol) was added
N,O-bis(trimethylsilyl)acetamide (8.16 cm.sup.3, 33.0 mmol). The
reaction mixture was stirred for 1 h at room temperature and cooled
to 0.degree. C. Trimethylsilyl triflate (3.0 cm.sup.3, 16.2 mmol)
was added dropwise and the mixture was stirred at 60.degree. C. for
2 h. Saturated aqueous solutions of sodium hydrogencarbonate
(3.times.20 cm.sup.3) and brine (2.times.20 cm.sup.3) were
successively added, and the separated organic phase was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (99:1, v/v) as eluent to give
compound 54 as a white solid material (3.9 g, 74%). .delta..sub.H
(CDCl.sub.3), 8.28 (1H, d, J 7.5, 6-H), 7.94-7.90 (2H, m, Bz),
7.65-7.25 (13H, m, Bn, Bz), 7.16 (1H, d, J 7.1, 5-H), 6.22 (1H, d,
J 2.8, 1'-H), 5.51 (1H, dd, J 2.8, 5.8, 2'-H), 4.62 (1H, d, J 11.6,
Bn), 4.51 (1H, d, J 12.3, 1"-H.sub.a), 4.49-4.34 (4H, m, 3'-H, Bn),
4.21 (1H, d, J 12.3, 1"-H.sub.b), 3.85 (1H, d, J 10.3, 5'-H.sub.a),
3.47 (1H, d, J 10.3, 5'-H.sub.b), 2.13 (3H, s, COCH.sub.3), 2.06
(3H, s, COCH.sub.3). .delta..sub.C (CDCl.sub.3) 170.52, 169.61
(C.dbd.O), 166.83, 162.27 (C-4, C.dbd.O), 154.26 (C-2), 145.26
(C-6), 137.25, 136.93, 133.18, 129.0, 128.75, 128.51, 128,45,
128.18, 128.10, 127.89, 127.71 (Bn, Bz), 96.58 (C-5), 89.42, 86.52
(C-1', C-4'), 76.21, 75.10, 74.17, 73.70, 69.70, 63.97 (C-2', C-3',
Bn, C-5', C-1"), 20.82 (COCH.sub.3). FAB-MS m/z 664 [M+Na].sup.+,
642 [M+H].sup.+. Found: C, 65.0; H, 5.7, N, 6.5;
C.sub.35H.sub.35N.sub.3O.sub.9 requires C, 65.5; H, 5.5; N,
6.5%.
EXAMPLE 57
1-(3,5-Di-O-benzyl-4-C-hydroxymethyl-.beta.-D-ribofuranosyl)-4-N-benzoylcy-
tosine (55)
[0343] To a stirred solution of nucleoside 54 (3.4 g, 5.3 mmol) in
methanol (20 cm.sup.3) was added sodium methoxide (0.663 g, 11.66
mmol). The reaction mixture was stirred at room temperature for 10
min and then neutralised with 20% aqueous hydrochloric acid. The
solvent was partly evaporated and the residue was extracted with
dichloromethane (3.times.50 cm.sup.3). The combined organic phase
was washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.20 cm.sup.3) and was dried
(Na.sub.2SO.sub.4). The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography
using dichloromethane/methanol (98.5:1.5, v/v) as eluent to give
compound 55 as a white solid material (1.6 g, 54%). .delta..sub.H
(CDCl.sub.3) 9.95 (1H, br s, NH), 8.33 (1H, d, J 7.4, 6-H), 7.98
(2H, m, Bz), 7.60-7.12 (14H, m, Bn, Bz, 5-H), 6.17 (1H, d, J 1.6,
1'-H), 4.78 (1H, d, J 11.8, Bn), 4.48-4.27 (5H, m, Bn, 2'-H, 3'-H),
3.85 (1H, d, J 11.8, 5'-H.sub.a), 3.66-3.61 (2H, m, 5'-H.sub.b,
1"-H.sub.a), 3.47 (1H, d, J 10.4 1"-H.sub.b). .delta..sub.C
(CDCl.sub.3) 167.5, 162.31 (C-4, C.dbd.OD), 155.36 (C-2), 145.34
(C-6), 137.49, 137.08, 133.09, 133.01, 128.94, 128.67, 128.48,
128.30, 128.01, 127.90, 127.80 (Bn, Bz), 96.53 (C-5), 93.97, 89.35
(C-1', C-4'), 76.06, 75.28, 73.70, 72.76, 70.26, 62.44 (C-2', C-3',
Bn, C-5', C-1"). FAB-MS m/z 558 [M+H].sup.+.
EXAMPLE 58
Intermediate 56
[0344] A solution of nucleoside 55 (2.2 g, 3.94 mmol) in anhydrous
tetrahydrofuran (60 cm.sup.3) was stirred at -20.degree. C. and a
suspension of 60% sodium hydride in mineral oil (w/w, 0.252 g, 6.30
mmol) was added in seven portions during 45 min. The solution was
stirred for 15 min at -20.degree. C. followed by addition of
p-toluenesulfonyl chloride (0.901 g, 4.73 mmol) in small portions.
The solution was stirred for 4 h at -20.degree. C. Additional
sodium hydride (0.252 g, 6.30 mmol) and p-toluenesulfonyl chloride
(0.751 g, 3.93 mmol) was added. The reaction mixture was kept at
-20.degree. C. for 48 h. The reaction was quenched by addition of
ice-cold water (50 cm.sup.3) whereupon extraction was performed
with dichloromethane (3.times.60 cm.sup.3). The combined organic
phase was washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.20 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvent was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (99:1, v/v) as eluent
to give the intermediate 56 (1.80 g).
EXAMPLE 59
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(4-N-benzoylcytosin-1-yl)-2,-
5-dioxabicyclo[2.2.1]heptane (57)
[0345] Intermediate 56 (1.80 g) was dissolved in anhydrous DMF
(30.0 cm.sup.3) and a 60% suspension of sodium hydride in mineral
oil (w/w, 0.1 6 g, 3.9 mmol) was added in five portions during 30
min at 0.degree. C. The reaction mixture was stirred for 36 h at
room temperature. The reaction was quenched by adding ice-cold
water (70 cm.sup.3) and the resulting mixture was extracted with
dichloromethane (3.times.50 cm.sup.3). The combined organic phase
was washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.30 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The solvents were removed under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (99.5:0.5, v/v) as
eluent to give compound 57 as a white solid material (1.08 g, 79%).
.delta..sub.H (CDCl.sub.3) 8.95 (1H, br s, NH), 8.20 (1H, d, J 7.5,
6-H), 7.95-7.92 (2H, m, Bz), 7.66-7.22 (14H, m, Bn, Bz, 5-H), 5.78
(1H, s, 1'-H), 4.70-4.65 (3H, m, 2'-H, Bn), 4.60 (1H, d, J 11.6,
Bn), 4.47 (1H, d, J 11.6, Bn), 4.05-3.78 (5H, m, 3'-H, 5'-H.sub.a,
1"-H.sub.a, 5"-H.sub.b, 1"-H.sub.b). .delta..sub.C (CDCl.sub.3)
167.0 162.36 (C-4, C.dbd.O), 154.5 (C-2), 144.58 (C-6), 137.46,
136.93, 133.35, 132.93, 129.11, 128.67, 128.50, 128.16, 128.11,
127.68, 127.60 (Bn), 96.35 (C-5), 88.38, 87.67 (C-1', C-4'), 76.14,
75.70, 73.79, 72.27, 72.09, 64.34 (Bn, C-5', C-1", C-2', C-3').
FAB-MS m/z 540 [M+H].sup.+. Found: C, 68.0; H, 5.5, N, 7.5;
C.sub.31H.sub.29N.sub.3O.sub.6 requires C, 69.0; H, 5.4; N,
7.8%).
EXAMPLE 60
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(cytosin-1-yl)-2,5-dioxabicyclo[-
2.2.1]-heptane (57A)
[0346] To a solution of nucleoside 57 (0.3 g, 0.55 mmol) in
anhydrous methanol (22 cm.sup.3) were added 1,4-cyclohexadiene (5.0
cm.sup.3) and 10% palladium on carbon (0.314 g). The mixture was
stirred under reflux for 18 h. Additional 10% palladium on carbon
(0.380 g) and 1,4-cyclohexadiene (5.5 cm.sup.3) were added and the
mixture was refluxed for 54 h. The reaction mixture was filtered
through a pad of silica gel which was subsequently washed with
methanol (1500 cm.sup.3). The combined filtrate was evaporated
under reduced pressure and the residue was purified by silica gel
column chromatography using dichloromethane/methanol (92.5:7.5,
v/v) as eluent to give compound 57A as a white solid material
(0.051 g, 36%). .delta..sub.H ((CD.sub.3).sub.2SO) 7.73 (1H, d, J
7.7, 6-H), 7.12-7.20 (2H, br s, NH.sub.2), 5.74 (1H, d, J 7.7,
5-H), 5.61 (1H, br, s, 3'-OH), 5.39 (1H, s, 1'-H), 5.12 (1H, m,
5'-OH), 4.08 (1H, s, 2'-H), 3.80 (1H, d, J 7.7, 1"-H.sub.a), 3.81
(1H, s, 3'-H), 3.74 (2H, m, 5'-H.sub.a, 5'-H.sub.b), 3.63 (1H, d, J
7.7, 1"-H.sub.b). .delta..sub.C ((CD.sub.3).sub.2SO) 165.66 (C-4),
154.58 (C-2), 139.68 (C-6), 93.19 (C-5), 88.42, 86.73, (C-1',
C-4'), 78.87, 70.85, 68.32, 56.04 ((C-2', C-1", C-3', C-5'). FAB-MS
m/z 256 [M+H].sup.+.
EXAMPLE 61
Intermediate 57B
[0347] To nucleoside 57A (0.030 g, 0.11 mmol) suspended in
anhydrous pyridine (2.0 cm.sup.3) was added trimethylsilyl chloride
(0.14 cm.sup.3, 1.17 mmol) and stirring was continued for 1 h at
room temperature. Benzoyl chloride (0.07 cm.sup.3, 0.58 mmol) was
added at 0.degree. C. and the mixture was stirred for 2 h at room
temperature. After cooling the reaction mixture to 0.degree. C.,
water (3.0 cm.sup.3) was added. After stirring for 5 min, an
aqueous solution of ammonia (1.5 cm.sup.3, 32%, w/w) was added and
stirring was continued for 30 min at room temperature. The mixture
was evaporated under reduced pressure, and the residue was purified
by silica gel column chromatography using dichloromethane/methanol
(97.5:2.5, v/v) as eluent to give intermediate 57B as white solid
material (0.062 g).
EXAMPLE 62
(1R,3R,4R,7S)-1-(4,4'-Dimethoxytrityloxymethyl)-7-hydroxy-3-(4-N-benzoylcy-
tosine-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (57C)
[0348] To a solution of intermediate 57B (0.042 g, 0.11 mmol) in
anhydrous pyridine (1.5 cm.sup.3) was added 4,4'-dimethoxytrityl
chloride (0.06 g, 0.17 mmol). The reaction mixture was stirred at
room temperature for 3.5 h, cooled to 0.degree. C., and a saturated
aqueous solution of sodium hydrogencarbonate (20 cm.sup.3) was
added. Extraction was performed using dichloromethane (3.times.10
cm.sup.3). The combined organic phase was dried (Na.sub.2SO.sub.4)
and evaporated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography using
dichloromethane/methanol/pyridine (98.0:1 5:0.5, v/v/v) as eluent
to give nucleoside 57C as a white solid material (0.031 g, 63% from
57A). .delta..sub.H (C.sub.5D.sub.5N) 12.32 (1H, br s, NHCO),
8.75-7.06 (20H, m, DMT, Bz, H-5, H-6), 6.24 (1H, s, 1'-H), 5.11
(1-H, s, 2'-H), 4.90 (1H, s, 3'-H), 4.38 (1H, d, J 7.6,
1"-H.sub.a), 4.10 (1H, d, J 7.6, 1"-H.sub.b), 4.02 (1H, d, J 10.6,
5'-H.sub.a), 3.87 (1H, d, J 10.6, 5'-H.sub.b), 3.77, 3.76
(2.times.3H, 2.times.s, 2.times.OCH.sub.3). .delta..sub.C
(C.sub.5D.sub.5N) 169.00 (NHCO), 164.24 (C-2), 159.39 (DMT), 150.5,
145.62 (DMT), 144.31, 132.89, 130.82, 130.72, 129.09, 128.89,
128.60, 113.96 (DMT), 96.96 , 89.01, 87.18, 79.91, 72.56, 70.25
(C-5, C-1', C-4', C-2', C-1", C-3'), 59.51 (C-5'), 55.33
(OCH.sub.3). FAB-MS m/z 662 [M+H].sup.+.
EXAMPLE 63
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4'-dimet-
hoxytrityloxymethyl)-3-(4-N-benzoylcytosine-1-yl)-2,5-dioxabicyclo[2.2.1]h-
eptane (57D)
[0349] To a solution of nucleoside 57C (0.025 g, 0.03 mmol) in
anhydrous dichloromethane (1.5 cm.sup.3) was added
N,N-diisopropylethylamine (0.03 cm.sup.3, 0.17 mmol) followed by
dropwise addition of 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite (0.02 cm.sup.3, 0.09 mmol).
After stirring for 5 h at room temperature, the reaction mixture
was cooled to 0.degree. C., dichloromethane/pyridine (10.0
cm.sup.3, 99.5:0.5, v/v) was added, and washing was performed using
a saturated aqueous solution of sodium hydrogencarbonate (3.times.8
cm.sup.3). The organic phase was separated, dried
(Na.sub.2SO.sub.4) and evaporated to dryness under reduced
pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol/pyridine
(99.0:0.5:0.5, v/v/v) as eluent to give amidite 57D as a light
yellow oil (0.038 g). .delta..sub.P (CDCl.sub.3) 147.93.
EXAMPLE 64
9-(2-O-Acetyl-4-C-acetyloxymethyl-3,5-di-O-benzyl-.beta.-D-ribofuranosyl)--
6-N-benzoyl-adenine (58)
[0350] To a stirred suspension of the anomeric mixture 33 (5.0 g,
10.3 mmol) and 6-N-benzoyladenine (3.76 g, 15.7 mmol) in anhydrous
dichloromethane (200 cm.sup.3) was added
N,O-bis(trimethylsilyl)acetamide (15.54 cm.sup.3, 61.8 mmol). The
reaction mixture was stirred at reflux for 1 h and then cooled to
room temperature. Trimethylsilyl triflate (7.0 cm.sup.3, 38.7 mmol)
was added dropwise and the mixture was refluxed for 20 h. The
reaction mixture was allowed to cool to room temperature and the
volume of the mixture was reduced to 1/4 under reduced pressure.
Dichloromethane (250 cm.sup.3) was added, and the solution was
washed with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.50 cm.sup.3) and water (50 cm.sup.3).
The organic phase was dried (Na.sub.2SO.sub.4) and evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol (99.5:0.5, v/v) as
eluent to give nucleoside 58 as white solid material (3.65 g, 52%).
.delta..sub.H (CDCl.sub.3) 9.25 (1H, br s, NH), 8.71 (1H, s, 8-H),
8.24 (1H, s, 2-H), 8.0 (2H, d, J 7.5, Bz), 7.60-7.23 (13H, m, Bn,
Bz), 6.35 (1H, d, J 4.6, 1'-H), 5.99 (1H, dd, J 4.9, 5.3, 2'-H),
4.78 (1H, d, J 5.6, 3'-H), 4.64-4.42 (5H, m, Bn, 1"-H.sub.a), 4.25
(1H, d, J 12.1, 1"-H.sub.b), 3.72 (1H, d, J 10.1, 5'-H.sub.a), 3.56
(1H, d, J 10.1, 5'-H.sub.b), 2.07 (3H, s, COCH.sub.3), 2.02 (3H, s,
COCH.sub.3). .delta..sub.C (CDCl.sub.3) 170.42, 169.72
(COCH.sub.3), 164.60 (NHCO), 152.51 (C-6), 151.45 (C-2), 149.46
(C-4), 141.88 (C-8), 137.04, 137.00, 133.50, 132.60, 128.86,
128.66, 128.53, 128.41, 128.38, 128.18, 128.06, 127.91, 127.88,
127.79, 127.63, 123.26 (Bz, Bn, C-5), 86.38 (C-1'), 86.25 (C-4'),
77.74, 74.74, 74.44, 73.48 (C-2', C-3', 2.times.Bn), 70.11 (C-1"),
63.42 (C-5'), 20.70, 20.54 (COCH.sub.3). FAB-MS m/z 666
[M+H].sup.+.
EXAMPLE 65
9-(3,5-Di-O-benzyl-4-C-hydroxymethyl-.beta.-D-ribofuranosyl)-6-N-benzoylad-
enine (59)
[0351] To a stirred solution of nucleoside 58 (4.18 g, 6.28 mmol)
in methanol (50 cm.sup.3) was added sodium methoxide (0.75 g, 13.8
mmol) at 0.degree. C. The reaction mixture was stirred for 2 h, and
ice was added. The mixture was neutralised using a 20% aqueous
solution of HCl. Extraction was performed using dichloromethane
(3.times.75 cm.sup.3), the organic phase was separated, dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using
dichloromethane/methanol (98.5:1.5, v/v) as eluent to give
nucleoside 59 as a white solid material (2.68 g, 73%).
.delta..sub.H (CDCl.sub.3) 9.42 (1H, br s, NH), 8.58 (1H, s, H-8),
8.16 (1H, s, 2-H), 7.96 (2H, d, J 7.2, Bz), 7.52-7.08 (13H, m, Bn,
Bz), 6.18 (1H, d, J 2.5, 1'-H), 4.85-4.38 (4H, m, Bn, 2'-H, 3'-H),
4.33 (2H, s, Bn) 3.90 (1H, d, J 11.9, 1"-H.sub.a), 3.71 (1H, d, J
11.8, 1"-H.sub.b), 3.50-3.39 (2H, m, 5-H). .delta..sub.C
(CDCl.sub.3) 164.98 (NHCO), 152.19 (C-6), 151.00 (C-2), 149.34
(C-4), 142.28 (C-8), 137.32, 137.25, 133.46, 132.70, 128.69,
128.49, 128.40, 128.11, 128.03, 127.94, 127.83, 127.62, (Bz, Bn),
122.92 (C-5), 90.94, 88.75 (C-1', C-4'), 77.65, 74.08, 73.44,
73.20, 71.12, 62.39 (C-1", C-5', C-2', C-3', 2.times.Bn). FAB-MS
m/z 582 [M+H].sup.+. Found: C, 65.6; H, 5.5; N, 11.7;
C.sub.32H.sub.31N.sub.5O.su- b.6 requires C, 66.1; H, 5.4; N,
12.0%.
EXAMPLE 66
Intermediate 60
[0352] A solution of nucleoside 59 (2.43 g, 4.18 mmol) in anhydrous
tetrahydrofuran (25 cm.sup.3) was stirred at -20.degree. C. and a
60% suspension of sodium hydride in mineral oil (w/w, 0.28 g, 7.0
mmol) was added in four portions during 30 min. After stirring for
1 h, p-toluenesulfonyl chloride (1.34 g, 7.0 mmol) was added in
small portions. The mixture was stirred at -10.degree. C. for 15 h.
Ice-cold water (50 cm.sup.3) was added and extraction was performed
with dichloromethane (3.times.50 cm.sup.3). The combined organic
phase was washed with a saturated aqueous solution of sodium
hydrogencarbonate (2.times.25 cm.sup.3), dried (Na.sub.2SO.sub.4)
and evaporated under reduced pressure. The residue was purified by
silica gel column chromatography using dichloromethane/methanol
(99:1, v/v) as eluent to give the intermediate 60 (1.95 g).
EXAMPLE 67
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(6-N-benzoyladenin-9-yl)-2,5-
-dioxabicyclo[2.2.1]heptane (61)
[0353] Intermediate 60 (1.90 g) was dissolved in anhydrous DMF (20
cm.sup.3) and a 60% suspension of sodium hydride in mineral oil
(w/w, 0.16 g, 3.87 mmol) was added in small portions at 0.degree.
C. The mixture was stirred for 10 h at room temperature and then
concentrated under reduced pressure. The residue was dissolved in
dichloromethane (75 cm.sup.3), washed with a saturated aqueous
solution of sodium hydrogencarbonate (2.times.25 cm.sup.3), dried
(Na.sub.2SO.sub.4), and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography using
dichloromethane/methanol (99:1, v/v) as eluent to give nucleoside
61 as white solid material (1.0 g, .about.44% from 59).
.delta..sub.H (CDCl.sub.3) 8.71 (H, s, 8-H), 8.23 (1H, s, 2-H),
8.02 (2H, m, J 7.0, Bz), 7.99-7.19 (13H, m, Bn, Bz), 6.08 (1H, s,
1'-H), 4.78 (1H, s, 2'-H), 4.61-4.50 (4H, m, 2.times.Bn), 4.24 (1H,
s, 3'-H), 4.12 (1H, d, J 7.8, 1"-H.sub.a), 4.00 (1H, d, J 7.9,
1"-H.sub.b), 3.85-3.78 (2H, m, 5'-H.sub.a, 5'-H.sub.b).
.delta..sub.C (CDCl.sub.3) 164.61 (NHCO), 152.32 (C-6), 150.61
(C-2), 149.35 (C-4), 140.67 (C-8), 137.24, 136.76, 133.33, 132.66,
128.68, 128.39, 128.29, 127.94, 127.77, 127.51 (Bn, Bz), 123.43
(C-5), 87.14, 86.52 (C-1', C-4'), 77.21, 76.77, 73.56, 72.57,
72.27, 64.65 (C-2', C-3', C-1", 2.times.Bn, C-5'). FAB-MS m/z 564
[M+H].sup.+. Found: C, 66.2; H, 5.5; N, 11.4;
C.sub.32H.sub.29N.sub.5O.sub.5 requires C, 66.2; H, 5.2; N,
12.4%.
EXAMPLE 68
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(adenin-9-yl)-2,5-dioxabicyclo[2-
.2.1]-heptane (61A)
[0354] To a stirred solution of nucleoside 61 (0.80 g, 1.42 mmol)
in anhydrous dichloromethane (30 cm.sup.3) at -78.degree. C. was
dropwise during 30 min added BCl.sub.3 (1 M solution in hexane;
11.36 cm.sup.3, 11.36 mmol). The mixture was stirred for 4 h at
-78.degree. C., additional BCl.sub.3 (1 M solution in hexane, 16.0
cm.sup.3, 16.0 mmol) was added drop-wise, and the mixture was
stirred at -78.degree. C. for 3 h. Then the temperature of the
reaction mixture was raised slowly to room temperature and stirring
was continued for 30 min. Methanol (25.0 cm.sup.3) was added at
-78.degree. C., and the mixture was stirred at room temperature for
12 h. The mixture was evaporated under reduced pressure, and the
residue was purified by silica gel column chromatography using
dichloromethane/methanol (92:8, v/v) as eluent to give nucleoside
61A as a white solid material (0.332 g, 84%). .delta..sub.H
((CD.sub.3).sub.2SO) 8.22 (1H, s, 8-H), 8.15 (1H, s, 2-H), 7.33
(2H, s, NH.sub.2), 5.89 (1H, s, 1'-H), 5.83 (1H, d, J 4.2, 3'-OH),
5.14 (1H, t, J 5.9, 5'-OH), 4.14 (1H, s, 2'-H), 4.25 (1H, d, J 4.2,
3'-H), 3.92 (1H, d, J 7.8, 1"-H.sub.a), 3.81-3.41 (3H, m,
5'-H.sub.a, 5'-H.sub.b, 1"-H.sub.b). .delta..sub.C
((CD.sub.3).sub.2SO) 155.90 (C-6), 152.64 (C-2), 148.35 (C-4),
137.72 (C-8), 118.94 (C-5), 88.48, 85.17 (C-1', C-4'), 79.09,
71.34, 69.83, 56.51 (C-2', C-3', C-1", C-5'). FAB-MS m/z 280
[M+H].sup.+.
EXAMPLE 69
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(6-N-benzoyladenin-9-yl)-2,5-dio-
xabicyclo[2.2.1]heptane (61B)
[0355] To a stirred solution of nucleoside 61A (0.32 g, 1.15 mmol)
in anhydrous pyridine (1 cm.sup.3) was added trimethylsilyl
chloride (0.73 cm.sup.3, 5.73 mmol) and the mixture was stirred at
room temperature for 20 min. Benzoyl chloride (0.67 cm.sup.3, 5.73
mmol) was added at 0.degree. C., and the reaction mixture was
stirred at room temperature for 2 h. The reaction mixture was
cooled to 0.degree. C. and ice-cold water (15.0 cm3) was added.
After stirring for 5 min, a 32% (w/w) aqueous solution of ammonia
(1.5 cm.sup.3) was added and the mixture was stirred for 30 min.
The mixture was evaporated to dryness and the residue was dissolved
in water (25 cm.sup.3). After evaporation of the mixture under
reduced pressure, the residue was purified by silica gel
chromatography using dichloromethane/methanol (97:3, v/v) as eluent
to give nucleoside 61B as a white solid material (0.55 g). FAB-MS
m/z 384 [M+H].sup.+.
EXAMPLE 70
(1R,3R,4R,7S)-7-Hydroxy-1-(4,4'-dimethoxytrityloxymethyl)-3-(6-N-benzoylad-
enin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane (61C)
[0356] To a stirred solution of compound 61B (0.50 g) in anhydrous
pyridine (20 cm.sup.3) was added 4,4'-dimethoxytrityl chloride
(0.71 g, 2.09 mmol) and 4-N,N-dimethylaminopyridine (DMAP) (0.1 g).
After stirring for 2 h at room temperature and for 1 h at
50.degree. C., the reaction mixture was cooled to 0.degree. C. and
a saturated aqueous solution of sodium hydrogencarbonate (100
cm.sup.3) was added. After extraction using dichloromethane
(3.times.50 cm.sup.3), the combined organic phase was dried
(Na.sub.2SO.sub.4) and evaporated under reduced pressure. The
residue was purified by silica gel column chromatography eluting
with dichloromethane/methanol/pyridine (98.0:1.5:0.5) to give
nucleoside 61C as a white solid material (0.36 g, .about.50% from
61A). .delta..sub.H (C.sub.5D.sub.5N) 12.52 (NHCO), 9.10 (2H, d, J
7.7, Bz), 8.88 (1H, s, 8-H), 8.50-7.11 (17H, m, DMT, Bz, 2-H), 6.65
(1H, s, H-1'), 5.25 (2H, s, H-2', H-3'), 4.71 (1H, d, J 7.8,
1"-H.sub.a), 4.56 (1H, d, J 7.8, 1"-H.sub.b), 4.20 (1H, d, J 10.8,
5'-H.sub.a), 4.07 (1H, d, J 10.8, 5'-H.sub.b), 3.82, 3.81
(2.times.3H, 2.times.s, 2.times.OCH.sub.3). .delta..sub.C
(C.sub.5D.sub.5N) 167.56 (NHCO), 159.24 (C-6), 152.50, 152.08,
151.81, 145.84, 141.45, 136.52, 136.28, 132.55, 130.76, 130.70,
129.32, 128.85, 128.76, 128.46, 127.38, 126.33 (DMT, Bz, C-2, C-4,
C-8), 113.84 (C-5), 88.64, 87.20, 86.85, 80.52, 73.13, 72.16, 60.86
(C-1', C-4', DMT, C-2', C-3', C-1", C-5'), 55.24 (OCH.sub.3).
FAB-MS m/z 686 [M+H].sup.+. Found: C, 68.3; H, 5.0; N, 9.7;
C.sub.39H.sub.35N.sub.5O.sub- .7 requires C, 68.3; H, 5.1; N,
10.2%).
EXAMPLE 71
(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4'-dimet-
hoxytrityloxymethyl)-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]hep-
tane (61D)
[0357] To a solution of compound 61C (190 mg, 0.277 mmol) in
anhydrous dichloromethane (1.5 cm.sup.3) were added
N,N-diisopropylethylamine (0.16 cm.sup.3, 0.94 mmol) and
2-cyanoethyl N,N-diisopropylphosphoramidochlorid- ite (0.1
cm.sup.3, 0.42 mmol) at 0.degree. C. The mixture was allowed to
warm to room temperature and stirred for 5 h. The solution was
diluted by dichloromethane (50 cm.sup.3), washed by a saturated
aqueous solution of sodium hydrogencarbonate (2.times.30 cm.sup.3)
and evaporated under reduced pressure. The products were isolated
by silica gel HPLC (PrepPak cartridge, 25.times.100 mm, packed by
Prep Nova-Pak.RTM. HR Silica 6 .mu.m 60 .ANG.; gradient of solution
B in solution A (from 0% to 15% during 25 min and from 15% to 100%
during another 25 min, solution A:
petroleum/dichloromethane/pyridine, 60/39.6/0.4, v/v/v, solution B:
ehylacetate. The fractions containing the two main products
(retention times 30-40 min) were joined, concentrated under reduced
pressure, co-evaporated with anhydrous toluene (2.times.40
cm.sup.3) and dried overnight in vacuo. The residue was dissolved
in anhydrous dichloromethane (4 cm.sup.3) and precipitated by
adding this solution to anhydrous petroleum ether (80 cm.sup.3)
under intensive stirring. The precipitate was collected by
filtration, washed by petroleum ether (2.times.20 cm.sup.3) and
dried under reduced pressure to give compound 61D (178 mg, 73%) as
a white solid material. .delta..sub.P (CD.sub.3CN) 148.42,
147.93.
EXAMPLE 72
1-(2,3-O-isopropylidene-4-C-(4-toluenesulphonyloxymethyl)-.beta.-D-ribofur-
anosyl)uridine (62)
[0358] To a stirred solution of
1-(2,3-O-isopropylidene-4'-C-hydroxymethyl-
-.beta.-D-ribofuranosyl)uridine (2.0 g, 6.4 mmol) (R. Youssefyeh,
D. Tegg, J. P. H. Verheyden, G. H. Jones and J. G. Moffat,
Tetrahedron Lett., 1977, 5, 435; G. H. Jones, M. Taniguchi, D. Tegg
and J. G. Moffat, J. Org. Chem., 1979, 44, 1309) in anhydrous
pyridine (28 cm.sup.3) was added p-toluenesulfonyl chloride (1.46
g, 7.3 mmol) dissolved in anhydrous pyridine (10 cm.sup.3) at
-30.degree. C. After 30 min, the reaction mixture was allowed to
reach room temperature and stirring was continued at room
temperature for 12 h. The reaction was quenched with methanol (4
cm.sup.3), silica gel (2 g) was added and the solvent was removed
under reduced pressure. The residue was purified by silica gel
column chromatography using a gradient of 0-3% methanol in
dichloromethane (v/v) as eluent to give nucleoside 62 as a pale
reddish solid material (1.8 g, 60%). .delta..sub.H (CDCl.sub.3)
9.80 (1H, br s, NH), 7.80 (2H, d, J 8.3, Ts), 7.46 (1 H, d, J 8.1,
6-H), 7.35 (2H, d, J 8.01, Ts), 5.72 (1H, d, J 8.0, 5-H), 5.54 (1H,
d, J 3.5, 1'-H), 5.08 (1H, dd, J 3.5, 6.4, 2'-H), 4.94 (1H, d, J
6.4, 3'-H), 4.18 (2H, s, 1"-H), 3.82-3.7(2H, m, 5'-H), 2.45 (3H, s,
Ts), 1.46, 1.29 (6 H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3)
163.6 (C-4), 150.4 (C-2), 145.2 (C-6), 142.9, 132.5, 129.9, 128.0
(Ts), 114.7 (OCO), 102.6 (C-5), 94.9, 87.6, 83.9, 81.5 (C-4', C-1',
C-3', C-2'), 68.7, 63.5 (C-1", C-5'), 26.4, 24.7 (CH.sub.3), 21.7
(Ts). FAB-MS m/z 469 [M+H].sup.+.
EXAMPLE 73
1-(4-C-(p-Toluenesulphonyloxymethyl-.beta.-D-ribofuranosyl)uridine
(63)
[0359] Nucleoside 62 (1.33 g, 2.83 mmol) was dissolved in 80%
acetic acid (25 cm.sup.3) and stirred at 80.degree. C. for 3 h
whereupon the solvent was removed under reduced pressure. The
residue was coevaporated with ethanol (10 cm.sup.3) and purified by
silica gel column chromatography using a gradient of 0-2% methanol
in dichloromethane (v/v) as eluent to give nucleoside 63 as a white
solid material (391 mg, 33%). .delta..sub.H (CD.sub.3OD) 7.81 (1H,
d, J 8.1, 6-H), 7.77 (1H, d, J 8.4, Ts), 7.40 (2H, d, J 8.6, Ts),
5.74 (1H, d, J 6.6, 1'-H), 5.69 (1H, d, J8.1, 5-H), 4.17-4.33 (2H,
m, 2'-H, 3'-H), 3.67-3.62 (2H, m, 1"-H), 3.26-3.20 (2H, m, 5'-H),
2.43 (3H, s, Ts). .delta..sub.C (CD.sub.3OD) 166.0 (C-4), 153.0
(C-2) 146.5 (C-6), 142.5, 130.9, 129,15 (Ts), 103.1 (C-5), 89.0,
87.2 (C-1', C-4'), 75.1, 72.7, 71.3, 63.8 (C-1", C-3', C-2', C-5'),
21.6 (Ts).
EXAMPLE 74
(1S,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2-
.2.1]-heptane (44)
[0360] To a stirred solution of nucleoside 63 (64 mg, 0.14 mmol) in
anhydrous DMF (2 cm.sup.3) was slowly added sodium hydride (8.4 mg,
21 mmol, 60% suspension in mineral oil, w/w) in anhydrous DMF (2
cm.sup.3) at 0.degree. C. The reaction mixture was then heated to
120.degree. C. for 6 h. After quenching the reaction with water (2
cm.sup.3), the solvents were removed under reduced pressure and the
residue was purified by silica gel column chromatography using a
gradient of 5-7% methanol in dichloromethane (v/v) as eluent to
give nucleoside 44 as a white solid material (9 mg, 29%). NMR data
were in agreement with those reported earlier for compound 44.
EXAMPLE 75
(1S,3R,4R,7S)-7-Acetoxy-1-acetoxymethyl-3-(thymin-1-yl)-2,5-dioxabicyclo[2-
.2.1]-heptane (64)
[0361] To a stirred solution of nucleoside 37 (209.8 mg, 0.78 mmol)
in anhydrous pyridine (2.5 cm.sup.3) was added acetic anhydride
(0.3 cm.sup.3, 3.23 mmol) and a catalytic amount of DMAP (5 mg).
After stirring for 2 h, ethanol was added (4 cm.sup.3) and the
mixture was evaporated under reduced pressure. The residue was
redissolved in dichloromethane and washed with a saturated aqueous
solution of sodium hydrogencarbonate (7 cm.sup.3). The organic
phase was dried (Na.sub.2SO.sub.4), and evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol (97:3, v/v) as eluent
affording 64 as a white solid material (249 mg, 90%). .delta..sub.C
(CDCl.sub.3) 169.59, 163.20, 149.50, 133.55, 110.64, 87.05, 85.38,
77.84, 71.70, 71.02, 58.60, 20.62, 20.53, 12.78. FAB-MS m/z 355
[M+H].sup.+.
EXAMPLE 76
(1S,3R,4R,7S)-1-Hydroxymethyl-3-(5-methyl-4-N-benzoylcytosine-1-yl)-7-hydr-
oxy-2,5-dioxabicyclo[2.2.1]heptane (65)
[0362] To a solution of nucleoside 64 (232.7 mg, 0.66 mmol) in
anhydrous acetonitrile (3 cm.sup.3) was added a solution of
1,2,4-triazole (420 mg, 6.1 mmol) and POCl.sub.3 (0.12 cm.sup.3,
1.3 mmol) in anhydrous acetonitrile (5 cm.sup.3). The reaction
mixture was cooled to 0.degree. C. and anhydrous triethylamine
(0.83 cm.sup.3) was added, whereupon the mixture was kept for 90
min at room temperature. Triethyl-amine (0.54 cm.sup.3) and water
(0.14 cm.sup.3) was added, and the reaction mixture was stirred for
10 min and evaporated under reduced pressure. The residue was
dissolved in EtOAc and washed with a saturated aqueous solution of
sodium hydrogencarbonate (2.times.9 cm.sup.3) and water (9
cm.sup.3). The aqueous phase was extracted with dichloromethane
(3.times.20 cm.sup.3). The combined organic phase was evaporated
under reduced pressure and the residue was redissolved in dioxane
(4 cm.sup.3), whereupon 32% aqueous ammonia (0.7 cm.sup.3) was
added. After stirring for 3 h, the reaction mixture was evaporated
under reduced pressure and coevaporated with anhydrous pyridine.
The residue was dissolved in anhydrous pyridine (3.6 cm.sup.3) and
benzoyl chloride (0.42 cm.sup.3, 3.6 mmol) was added. After
stirring for 2 h, the reaction was quenched with water (1 cm.sup.3)
and the reaction mixture was evaporated under reduced pressure. The
residue was then redissolved in EtOAc and washed with water
(3.times.7 cm.sup.3). The organic phase was evaporated to dryness
under reduced pressure, and the residue was dissolved in
pyridine/methanol/water (13:6:1, v/v/v, 14 cm.sup.3) at 0.degree.
C., and a 2M solution of NaOH in pyridine/methanol/water (13:6:1,
v/v/v, 7 cm.sup.3) was added. After stirring for 20 min, the
reaction mixture was neutralised using a 2M solution of HCl in
dioxane, and the reaction mixture was evaporated under reduced
pressure. The residue was purified by silica column chromatography
using dichloromethane/methanol (95:5, v/v) as eluent to give
nucleoside 65 as a yellow foam (94.6 mg, 38%). .delta..sub.H
(CD.sub.3OD) 8.21 (1H, br, s), 8.02 (1H, m), 7,84-7.9 (1H, m),
7.41-7.58 (4H, m), 5.61 (1H, s), 4.36 (1H, s), 4.10 (1H, s), 3.98
(1H, d, J 8.0), 3.94 (2H, s), 3.78 (1H, d, J 7.9), 2.11 (3H, d, J
1.0). .delta..sub.C (CD.sub.3OD, selected signals) 133.66, 132.90,
130.63, 129.50, 129.28, 128.65, 90.71, 88.86, 80.57, 72.47, 70.22,
57.53, 14.01. FAB-MS m/z 374 [M+H].sup.+.
EXAMPLE 77
(1R,3R,4R,7S)-1-(4,4'-Dimethoxytrityloxymethyl)-3-(5-methyl-4-N-benzoylcyt-
osine-1-yl)-7-O-(2-cyanoethoxy(diisopropylamino)phosphinoxy)-2,5-dioxabicy-
clo[2.2.1]heptane (66)
[0363] To a stirred solution of nucleoside 65 (82 mg, 0.22 mmol) in
anhydrous pyridine (1.5 cm.sup.3) was added 4,4'-dimethoxytrityl
chloride (80 mg, 0.24 mmol) and stirring was continued at room
temperature for 12 h. Additional 4,4'-dimethoxytrityl chloride (80
mg, 0.24 mmol) was added, and stirring was continued for another 12
h. Methanol (0.5 cm.sup.3) was added and the reaction mixture was
evaporated under reduced pressure. The residue was subjected to
silica gel column chromatography using
dichloromethane/methanol/pyridine (98.5:1.0:0.5, v/v/v). The
resulting intermediate (FAB-MS m/z 676) (50.2 mg) was coevaporated
with anhydrous acetonitrile and dissolved in anhydrous
dichloromethane (0.62 cm.sup.3). N,N-Diisopropylethylamine was
added (0.1 cm.sup.3) followed by addition of 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite (0.3 cm.sup.3, 0.11 mmol).
After stirring for 3 h at room temperature, water (1 cm.sup.3) was
added and the resulting mixture was diluted with ethylacetate (10
cm.sup.3), washed with saturated aqueous solutions of sodium
hydrogencarbonate (3.times.6 cm.sup.3) and brine (3.times.6
cm.sup.3). The organic phase was dried (Na.sub.2SO.sub.4) and
evaporated under reduced pressure. The residue was purified by
silica gel column HPLC. Precipitation as described for compound 53
afforded compound 66 as a white solid material (29.5 mg, 0.03 mmol,
14%). .delta..sub.P (CH.sub.3CN) 148.46, 147.49.
EXAMPLE 78
9-(4-(Hydroxymethyl)-2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-6-N-benz-
oyladenine (67)
[0364] A mixture of oxalyl chloride (0.93 mL, 10.75 mmol) and
dichloromethane (25 mL) was cooled to -70.degree. C. Dimethyl
sulfoxide (DMSO, 1.7 mL, 22 mmol) was added drop-wise under
intensive stirring. The mixture was stirred for 10 min at
-70.degree. C. and a solution of
9-(2,3-O-isopropylidene-.beta.-D-ribofuranosyl)-6-N-benzoyladenine
(3.4 g, 8.27 mmol) in dimethylsulfoxide/dichloromethane (1:9 v/v,
20 mL) was added during 5 min. The mixture was stirred at
-60.degree. C. for 30 min. Triethylamine (7 mL, 50,3 mmol) was
added and the mixture was allowed to warm to room temperature. The
solution was diluted by dichloromethane (50 mL) and washed by water
(3.times.100 mL). Water fractions were additionally washed by 100
mL of dichloromethane. The organic phase was concentrated to an
oily mass, co-evaporated with dioxane (50 mL) and re-dissolved in
30 mL of dioxane. 37% aq. formaldehyde (2.95 mL, 33.4 mmol) and 2M
aq. NaOH (8.26 mL) were added; the mixture was stirred at room
temperature for 10 min and cooled to 0.degree. C. Sodium
borohydride (640 mg, 16.9 mmol) was added and the reaction mixture
was allowed to warm to room temperature during 15 min. The reaction
was quenched by addition of acetic acid (5 mL) and to the mixture
was added dichloromethane and a saturated aqueous solution of
sodium hydrogen carbonate (100 mL each). The organic phase was
washed with water (100 mL), concentrated in vacuo and the product
was isolated by column (2.5.times.25 cm) silica gel chromatography
by the use of 2-3.2% of methanol in dichloromethane (v/v) as
eluent. Yield 1.85 g (50.7%) of compound 67 as a white solid
material. .delta..sub.H (CDCl.sub.3) 8.72 (1H, s), 8.14 (1H, s),
8.03-8.00 (2H, m), 7.60-7.57 (1H, m), 7.56-7.46 (2H, m), 6.0 (1H,
d, J4.9), 5.35 (1H, dd, J'5.8, J"5.0), 5.13 (1H, d, J5.8),
3.87-3.78 (4H, m), 1.65 (3H, s), 1.38 (3H, s). .delta..sub.C
(CDCl.sub.3) 164.8, 152.2, 150.4, 150.2, 142.6, 133.3, 132.9,
128.8, 128.0, 124.1, 114.7, 93.3, 90.2, 8:3.8, 82.5, 65.3, 62.9,
27.3, 25.1. FAB-MS: m/z 442 [M+H].sup.+, 464 [M+Na].sup.+.
[0365] Alternative synthesis of 67. To a solution of
2',3'-O-isopropylideneadenosine (15 g) in anhydrous pyridine (250
mL) was added trimethylsilyl chloride (15.5 mL). The reaction
mixture was stirred at room temperature for 20 min and cooled to
0.degree. C. Benzoyl chloride (17 mL) was added drop-wise and the
mixture was kept at room temperature temperature for 2-3 h. Water
(50 mL) and 25% aq. ammonium hydroxide (100 mL) was added and
stirring was continued for 3 h. Then the mixture was concentrated
under reduced pressure, co-evaporated with toluene (2.times.200 mL)
and dichloromethane (DCM) and saturated sodium hydrogencarbonate
was added. The organic phase was evaporated to dryness to give a
yellow solid. Recrystallisation from ethanol resulted in 12.5 g (ca
80%) as a white solid intermediate material. Oxalyl chloride (4.68
mL) in dry DCM (120 mL) was cooled to -70.degree. C. DMSO (8.5 mL)
was added during intensive stirring. Later (10 min) a solution of
the intermediate for which the synthesis is described above (17 g)
in 100%, DMSO/DCM (100 mL) added dropwise (20 min). The temperature
was allowed to increase to -50.degree. C. over a period of 30 min
after which the reaction was quenched with triethylamine (35 mL).
To the mixture was added DCM (200 ml) which was washed with water
(3.times.200 mL). The intermediate was concentrated in vacuo ,
co-evaporated with dioxane, and redissolved in dioxane (120 mL).
Formaldehyde (37%) and 2 M aq. sodium hydroxide (40 mL) was added
and the reaction mixture was stirred for 1 h. The mixture was
neutralised with acetic acid (6 mL) and DCM (400 ml) and saturated
sodium hydrogencarbonate (400 mL) were added. The organic phase was
concentrated. The product 67 was purified by column chromatography
(silica gel, 1.5-5.0% methanol/DCM). Yield 8.5 g (46%) of 67. Data
were as stated earlier in this example.
EXAMPLE 79
9-(2,3-O-Isopropylidene-4-(p-toluenesulfonyloxymethyl)-.beta.-D-ribofurano-
syl)-6-N-benzoyladenine (68) and
9-(4-hydroxymethyl-2,3-O-isopropylidene-5-
-O-(p-toluenesulfonyl)-.beta.-D-ribofuranosyl)-6-N-benzoyladenine
[0366] A mixture of compound 67 (1.95 g, 4.42 mmol) and
p-toluenesulfonyl chloride (1.26 g, 6.63 mmol) was dissolved in 10
mL of anhydrous pyridine at 0.degree. C. The reaction mixture was
stirred for 4 h and then diluted by dichloromethane (100 mL),
washed with water (2.times.100 mL) and concentrated under reduced
pressure. The purification of the mixture by silica gel column
(2.5.times.20 cm) chromatography in a gradient (1-4%) of methanol
in dichloromethane allowed isolation of starting material 67 (360
mg, 18.5%) and two structural isomers, namely 68 (less polar
isomer; 971 mg, 36.7%) and
9-(4-hydroxymethyl-2,3-O-isopropylidene-5-O-(4'-toluen-
esulfonyl)-.beta.-D-ribofuranosyl)-N.sup.6-benzoyladenine (more
polar isomer; 352 mg, 13, 1%) as white solid materials. 68:
.delta..sub.H (CDCl.sub.3) 8.69 (1H, s), 8.11 (1H, s), 8.00 (2H,
m), 7.79 (2H, m), 7.58-7.55 (1H, m), 7.50-7.46 (2H, m), 7.34-7.32
(2H, m), 5.88 (1H, d, J4.9), 5.35 (1H, dd, J'5.8, J"5.0), 5.13 (1H,
d, J5.8), 3.87-3.78 (4H, m), 1.65 (3H, s), 1.38 (3H, s).
.delta..sub.C (CDCl.sub.3) 164.7, 152.0, 150.2, 150.1, 144.9,
142.5, 133.2, 132.7, 132.3, 129.6, 128.6, 127.9, 127.8, 123.9,
114.6, 93.1, 87.9, 83.4, 81.6, 68.3, 64.4, 27.1, 25.0, 21.5.
FAB-MAS: m/z 596 [M+H]..sup.+
EXAMPLE 80
9-(4-(p-Toluenesulfonyloxymethyl)-.beta.-D-ribofuranosyl)-6-N-benzoyladeni-
ne (69)
[0367] A solution of compound 68 (940 mg, 1.58 mmol) in 10 mL of
90% aq. trifluoroacetic acid was kept for 30 min at room
temperature and concentrated in vacuo to an oily mass. After
co-evaporation with methanol (2.times.20 mL) and toluene (20 mL)
the mixture was purified by silica column (2.times.25 cm)
chromatography in a gradient of methanol (2-5.0%) in
dichloromethane as eluent to give compound 69 (825 mg, 94%) as
white solid material. .delta..sub.H (methanol-d.sub.4) 8.67 (1H,
s), 8.53 (1H, s), 8.05 (2H, d, J7.7), 7.75 (2H, d, J8.2), 7.63 (1H,
m), 7.53 (2H, m), 7.32 (2H, d, J8.0), 5.94 (1H, d, J 7.1), 4.92
(1H, dd, J'7.1, J"5.3), 4.41 (1H, d, J5.1), 4.38 (1H, d, J10.2),
4.28 (1H, d, J10.2), 3.80 (1H, d, J12.0), 3.68 (1H, d, J12.0), 2.35
(3H, s). .delta..sub.C (methanol-d.sub.4) 168.2, 152.9, 150.8,
151.2, 146.4, 144.9, 134.7, 134.1, 134.0, 130.8, 129.7, 129.4,
129.1, 125.1, 90.0, 88.4, 75.3, 73.1, 71.1, 64.2, 21.6. FAB-MS: m/z
556 [M+H]..sup.+
EXAMPLE 81
9-(4-(p-Toluenesulfonyloxymethyl)-3,5-O-(tetraisopropyldisiloxane-1,3-diyl-
)-.beta.-D-ribofuranosyl)-6-N-benzoyladenine (70)
[0368] To a solution of compound 69 (780 mg, 1.40 mmol) in
anhydrous pyridine (7 mL) was added
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (500 .mu.L, 1.57
mmol) at 0.degree. C. After stirring for 2 h at 0.degree. C.
additional 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (50 .mu.L,
0.16 mmol) was added. The reaction mixture was allowed to warm to
room temperature, diluted by dichloromethane (100 mL) and washed by
water (2.times.100 mL). The organic phase was concentrated, and the
residue was purified by the use of preperative HPLC (PrepPak
cartridge, Porasil 15-20 .mu.m 125 .ANG.; eluent: 0-3% of methanol
in dichloromethane (v/v) in 120 min; flow rate: 60 ml/min).
Concentration in vacuo yielded 870 mg (78%) of compound 70 as a
white solid material. .delta..sub.H (CDCl3) 8.65 (1H, s), 8.03(2H,
m), 8.00 (1H, s), 7.83 (2H, d J8.4), 7.58 (1H, m), 7.49 (2H, m),
7.34 (2H, d, J8.4), 5.87 (1H, s), 5.70 (1H, d, J6.2), 4.68 (1H, d,
J6.2), 4.59 (1H, d, J10.8), 4.31 (1H, d, J11.0), 3.91 (2H, s), 2.45
(3H, s), 1.03-0.84 (28H, m). .delta..sub.C (CDCl.sub.3) 164.9,
152.2, 150.5, 150.0, 144.7, 142.9, 133.5, 132.9, 132.8, 129.7,
128.8, 128.1, 128.0, 123.6, 90.3, 85.3, 76.0, 75.0, 68.7, 65.2,
21.6, 17.5, 17.4, 17.2, 17.1, 17.0, 16.9, 13.1, 13.0, 12.5, 12.4.
FAB-MS: m/z 798 [M+H]..sup.+
EXAMPLE 82
9-(2-O,4-C-Methylene-3,5-O-(tetraisopropyldisiloxa-1,3-diyl)-.beta.-D-ribo-
furanosyl)-6-N-benzoyladenine (71)
[0369] A solution of compound 70 (540 mg, 0.68 mmol) in anhydrous
THF (20 mL) was cooled to 0.degree. C. and sodium hydride (130 mg
of 60% suspension in mineral oil, 3.25 mmol) was added under
stirring. The reaction mixture was stirred for 30 min and then
neutralised by addition of 750 .mu.L of acetic acid.
Dichloromethane (50 mL) was added, the mixture was washed by a
saturated aqueous solution of sodium hydrogen carbonate (2.times.50
mL) and concentrated under reduced pressure. The residue was
applied to a silica gel column (2.5.times.25 cm) and the product
was eluted in a gradient concentration (0.5 to 1.2%) of methanol in
dichloromethane as eluent to yield compound 71 (356 mg, 84%) as a
white foam. .delta..sub.H (CDCl.sub.3) 8.77 (1H, s), 8.28 (1H, s),
8.03(2H, m), 7.59 (1H, m), 7.50 (2H, m), 6.08 (1H, s), 4.86 (1H,
s), 4.56 (1H, s), 4.14 (1H, d, J13.2), 4.06 (1H, d, J7.7), 3.97
(1H, d, J13.2), 3.89 (1H, d, J7.7), 1.12-0.95 (28H, m).
.delta..sub.C (CDCl.sub.3) 164.8, 152.6, 150.5, 149.6, 140.7,
133.6, 132.7, 128.7, 127.9, 123.1, 89.4, 86.5, 78.9, 71.7, 71.2,
56.7, 17.3, 17.1, 17.0, 16.9, 16.8, 13.3, 13.1, 12.5, 12.2. FAB-MS:
m/z 626 [M+H]..sup.+
EXAMPLE 83
7-Hydroxy-1-hydroxymethyl-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.-
1]-heptane (61B)
[0370] Triethylamine tris-hydroflucoride (300 .mu.L, 1.84 mmol) was
added to a solution of compound 71 (420 mg, 0.067 mmol) in
anhydrous THF (7 mL). The reaction mixture was kept at room
temperature for 1 h and concentrated to an oil which was purified
by silica gel column (2.times.25 cm) chromatography eluting with
4-10% of methanol in dichloromethane (v/v). Yield 232 mg (92%) of
compound 61B as a white solid material. NMR data were identical
with those reported earlier for 61B.
EXAMPLE 84
1-(3,5-Di-O-benzyl-4-C-(p-toluenesulphonyloxymethyl)-2-O-p-toluenesulphony-
l-.beta.-D-ribofuranosyl)thymine (72)
[0371] A solution of
1-(3,5-di-O-benzyl-4-C-(hydroxymethyl)-.beta.-D-ribof-
uranosyl)thymine 35 (1.48 g, 3.16 mmol), DMAP (1.344 g, 0.011 mol)
and p-toluenesulphonyl chloride (1.45 g, 7.6 mmol) in
dichloromethane (20 ml) was stirred for 3 h at room temperature.
The reaction mixture was diluted with dichloromethane (30 ml) and
washed with saturated aqueous solutions of sodium hydrogen
carbonate (3.times.20 ml) and sodium chloride (2.times.25 ml). The
organic phase was dried (Na.sub.2SO.sub.4), filtered and evaporated
under reduced pressure. The residue was subjected to silica gel
column chromatography using methanol:dichloromethane (1:99, v/v) as
eluent to give nucleoside 72 (1.95 g, 80%) as a white solid
material. FAB-MS m/e 776. .delta..sub.C (CDCl.sub.3) 162.9, 149.8
(C-2, C-4), 145.8, 145.2 (2.times.Ts), 136.9, 136.8 (2.times.Bn),
134.3 (C-6), 132.1, 132.0, 130.0, 129.9, 129.0 128.9, 128.4, 128.3,
128.2, 128.0, 127.7 (2.times.Ts, 2.times.Bn), 111.2 (C-5), 85.3,
84.0 (C-1', C-4'), 78.9, 78.3, 75.2, 74.3, 72.7, 69.1 (C-2', C-3',
C-5', C-1", 2.times.Bn), 21.7 (2.times.CH.sub.3), 11.9 (CH.sub.3).
Anal. Calcd. for C.sub.39H.sub.40N.sub.2S.sub.2O.sub.11: C, 60.30;
H, 5.19; N, 3.61. Found: C, 59.95; H, 5.11, N 3.81.
EXAMPLE 85
1-(2-Benzylamino-2-deoxy-3,5-di-O-benzyl-2-N,4-C-methylene-.beta.-D-ribofu-
ranosyl)-thymine (73)
[0372] A solution of 72 (8.6 g, 11.1 mol) in benzyl amine (10 ml)
was stirred at 130.degree. C. for 36 h. The reaction mixture was
directly subjected to silica gel column chromatography using
methanol:dichloromethane (1:99, v/v) as eluent to give nucleoside
73 (1.79 g, 30%) as a white solid material. FAB-MS m/e 540.
.delta..sub.C (CDCl.sub.3) 163.9, 149.8 (C-2, C-4), 139.2, 137.6,
137.3 (3.times.Bn), 135.6 (C-6), 128.5, 128.4, 128.3, 128.2, 128.0,
127.7, 127.0 (3.times.Bn), 109.6 (C-5), 88.2, 86.3 (C-1', C-4'),
76.7, 73.8, 72.0, 66.0, 63.8, 57.9, 57.8, (C-2', C-3', C-5', C-1",
3.times.Bn), 12.2 (CH.sub.3). Anal. Calcd. for
C.sub.32H.sub.33N.sub.3O.sub.5.times.0.5 H.sub.2O: C, 70.06; H,
6.25; N, 7.66. Found: C, 70.00; H, 6.06; N, 7.50.
EXAMPLE 86
1-(2-Amino-2-deoxy-2-N,4-C-methylene-.beta.-D-ribofuranosyl)thymine
(74)
[0373] To a solution of nucleoside 73 (1.62 g, 0.003 mol) in
ethanol (150 ml) was added 20% palladium hydroxide on carbon (3 g)
and the suspension was stirred for 5 days under hydrogen. The
catalyst was filtered off (silica gel) and washed with methanol (20
ml). The combined filtrate was concentrated under reduced pressure
to give a white solid material which was filtered off and washed
with methanol:dichloromethane (1:4, v/v) to give a monobenzylated
intermediate (0.82 g, 76%). FAB-MS: m/e 360 (M+H).sup.+.
.sup.13C-NMR (DMSO-d.sub.6, 250 MHz): 163.7, 149.8 (C-2, C-4),
138.2 (Bn), 134.9 (C-6), 128.2, 127.5, 127.4 (Bn), 107.8 (C-5),
87.8, 87.6 (C-1', C-4'), 72.7, 68.9, 65.9, 61.7, 49.4 (C-2', C-3',
C-5', C-1", Bn), 11.9 (CH.sub.3). Anal. Calcd. for
C.sub.18H.sub.21N.sub.3O.sub- .5: C, 60.16; H, 5.89; N, 11.69.
Found: C, 59.86; H, 5.61; N, 11.56. A mixture of this intermediate
(0.1 g, 0.29 mmol), ammonium formate (0.085 g, 1.35 mmol), 10%
palladium on carbon (130 mg) in anhydrous methanol (7 ml) was
heated under reflux for 2 h. The catalyst was filtered off (silica
gel) and washed with methanol (15 ml) and the combined filtrate was
concentrated to dryness under reduced pressure. The residue was
subjected to silica gel column chromatography using
methanol:dichloromethane (1:9, v/v) as eluent to give title
compound 74 (0.053 g, 71%) as a white solid material. FAB-MS m/e
270. .delta..sub.H (DMSC)-d.sub.6) 11.29 (bs, 1H, NH), 7.73 (d, 1H,
J 1.1, 6-H), 5.31 (s, 1H, 1'-H), 5.29 (br s, 1H, 3'-OH), 5.13 (m,
1H, 5'-OH), 3.81 (s, 1H, 3'-H), 3.69 (m, 2H, 5'-H), 3.23 (s, 1H,
2'-H), 2.88 (d, 1H, J 9.8, 1"-H.sub.a), 2.55 (d, 1H, J 9.8,
1"-H.sub.b), 1.77 (d, 3H, J 0.8, CH.sub.3). .delta..sub.C
(DMSO-d.sub.6) 164.0, 150.1 (C-2, C-4), 135.6 (C-6), 107.8 (C-5),
89.5, 87.9 (C-1', C-4'), 68.7, 61.9, 57.1, 49.4, (C-2', C-3', C-5',
C-1"). Anal. Calcd. for C.sub.11H.sub.15N.sub.3O.sub.5- .times.0.5
H.sub.2O: C, 47.48; H, 5.80; N, 15.10. Found: C, 47.54; H, 5.30; N,
14.79.
[0374] Alternative method for conversion of 73 to 74. To a solution
of 73 (0.045 g, 0.0834 mmol) in methanol (6 ml) was added 10% Pd on
carbon (0.118 g) and--in three portions during 3 h--ammonium
formate (0.145 g, 0.0023 mol). The suspension was refluxed for 4.5
h. The catalyst was filtered off (silica gel) and washed with
methanol (4.times.3 ml). The combined filtrate was concentrated and
the residue was subjected to column chromatography on silica gel
using methanol:dichloromethane (1:9, v/v) as eluent to give
nucleoside 74 (0.015 g, 67%). Spectral data were in accordance with
data reported earlier in this example for 74.
EXAMPLE 87
1-(2-Amino-2-deoxy-2-N,4-C-methylene-2-N-trifluoroacetyl-.beta.-D-ribofura-
nosyl)thymine (74-COCF.sub.3)
[0375] To a suspension of nucleoside 74 (0.050 g, 0.186 mmol) in
methanol (2 mL) were added DMAP (0.013 mg, 0.106 mmol) and ethyl
trifluoroacetate (0.029 mL, 0.242 mmol) and the mixture was stirred
at room temperature for 2.5 h. The solvent was removed under
reduced pressure and the residue was subjected to column
chromatography on silica gel using methanol:dichloromethane
(2.5:97.5, v/v) as eluent to give the title nucleoside
74-COCF.sub.3 as a white solid material after evaporation of the
solvents under reduced pressure (0.055 g, 81%). FAB-MS m/z 366
[M+H].sup.+. .sup.13C NMR (CD.sub.3OD, 62.9 MHz) .delta.166.5,
157.7 (q, .sup.2J.sub.C,F 37.5 Hz, COCF.sub.3), 157.6 (q,
.sup.2J.sub.C,F 37.2 Hz, COCF.sub.3), 151.8, 136.8, 136.8, 117.6
(d, .sup.1J.sub.C,F 287.5 Hz, CF.sub.3), 117.5 (d, .sup.1J.sub.C,F
286.5 Hz, CF.sub.3), 110.8, 110.8, 90.7, 89.3, 87.7, 87.3, 70.1,
68.6, 66.2, 66.2, 64.5, 57.9, 53.3, 12.7. Anal. Calcd. for
C.sub.13H.sub.14N.sub.3O.sub.6F.sub.3: C, 42.8; H, 3.9; N, 11.5.
Found: C, 42.5; H, 4.0; N, 11.2.
EXAMPLE 88
1-(2-Amino-2-deoxy-5-O-4,4'-dimethoxytrityl-2-N,4-C-methylene-2-N-trifluor-
oacetyl-.beta.-D-ribofuranosyl)thymine (74-DMT)
[0376] To a solution of nucleoside 74-COCF.sub.3 (0.030 g, 0.082
mmol) in anhydrous pyridine (0.6 mL) at 0.degree. C. was dropwise
(during 20 min) added 4,4'-dimethoxytrityl chloride (0.054 g, 0.159
mmol) dissolved in anhydrous pyridine:dichloromethane (0.6 mL, 1:1,
v/v) and the mixture was stirred for 10 h at room temperature. A
mixture of ice and water was added (5 mL) and the resulting mixture
was extracted with dichloromethane (3.times.5 mL). The combined
organic phase was washed with a saturated aqueous solution of
sodium hydrogencarbonate (3.times.2 mL), dried (Na.sub.2SO.sub.4)
and filtered. The filtrate was evaporated to dryness under reduced
pressure and the residue was subjected to column chromatography on
silica gel using methanol:dichloromethane:pyridine (1.5:98.0:0.5,
v/v/v) as eluent to give nucleoside 74-DMT as a white solid
material after evaporation of the solvents under reduced pressure
(0.051 g, 93%). FAB-MS m/z 667 [M].sup.+, 668 [M+H].sup.+. FAB-HRMS
calcd. for C.sub.34H.sub.32N.sub.3O.sub.8F.sub.3.sup.+: 667.2142.
Found: 667.2146. .sup.13C NMR (C.sub.5D.sub.5N, 100.6 MHz)
.delta.165.1, 165.0, 159.5, 159.5, 151.4, 145.7, 136.3, 136.1,
134.8, 134.6, 130.9, 130.9, 130.9, 128.9, 128.9, 128.7, 128.7,
128.4, 127.7, 123.2, 114.1, 114.1, 114.0, 110.4, 89.4, 87.9, 87.5,
87.4, 87.2. 70.8, 69.0, 66.0, 64.4, 60.5, 60.2, 55.5, 53.6, 53.4,
49.9, 13.2, 13.1.
EXAMPLE 89
1-(2-Amino-3-O-(2-cyanoethoxy(diisopropylamino)phosphino-2-deoxy)-5-O-4,4'-
-dimethoxytrityl-2-N,4-C-methylene-2-N-trifluoroacetyl-.beta.-D-ribofurano-
syl)thymine (74A)
[0377] To a solution of nucleoside 74-DMT (0.121 g, 0.181 mmol) in
anhydrous dichloromethane (2 mL) were added
N,N-diisopropylethylamine (0.093 mL, 0.54 mmol) and 2-cyanoethyl
N,N-diisopropylphosphoramidochlori- dite (0.057 mL, 0.26 mmol) at
0.degree. C. and the mixture was stirred for 10 h at room
temperature. The mixture was diluted with dichloromethane (20 mL),
extracted with a saturated aqueous solution of sodium
hydrogencarbonate (3.times.10 mL), dried (Na.sub.2SO.sub.4) and
filtered. The filtrate was evaporated to dryness under reduced
pressure and the residue was subjected to column chromatography on
silica gel using methanol:dichloromethane:pyridine (1.5:98.0:0.5,
v/v/v) as eluent to give crude product (0.107 g) after evaporation
of the solvents under reduced pressure. The residue was dissolved
in anhydrous dichloromethane (1 mL) and by dropwise addition to
vigorously stirred petroleum ether (60-80.degree. C., 30 mL) at
-30.degree. C., nucleotide 74A precipitated to give a white solid
material after filtration (0.090 g, 57%). FAB-MS m/z 868
[M+H].sup.+, 890 [M+Na].sup.+. .sup.31P NMR (CD.sub.3CN, 121.5 MHz)
.delta.150.4, 150.2, 148.8, 149.1.
EXAMPLE 90
1-(2-Amino-2-N,4-C-methylene-3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-.be-
ta.-D-ribofuranosyl)thymine (74B)
[0378] To a solution of nucleoside 74 (0.20 g, 0.74 mmol) in
anhydrous pyridine (3 mL) at -15.degree. C. was dropwise (during 3
h) added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (0.305 mL,
0.0011 mol) and the mixture was stirred for 10 h at room
temperature. MeOH (3 mL) was added and the mixture was evaporated
to dryness under reduced pressure. The residue was subjected to
column chromatography on silica gel using methanol:dichloromethane
(1:99, v/v) to give nucleoside 74B as a white solid material after
evaporation of the solvents under reduced pressure (0.370 mg, 97%).
FAB-DAS m/z 512 [M+H].sup.+. .sup.1H NMR ((CD.sub.3).sub.2SO, 400
MHz) .delta.11.37 (bs, 1H), 7.48 (s, 1H), 5.32 (s, 1H), 4.06 (d,
1H, J 13.5 Hz), 4.00 (s, 1H), 3.84 (d, 1H, J 13.5 Hz), 3.41 (s,
1H), 2.92 (d, 1H, J 10.2 Hz), 2.64 (d, 1H, J 10.2 Hz), 1.74 (s,
3H), 1.10-0.92 (m, 28H). .sup.13C NMR ((CD).sub.3SO.sub.2, 62.9
MHz) .delta.163.8, 149.8, 134.1, 107.9, 89.5, 87.9, 70.1, 61.1,
57.9, 49.3, 17.2, 17.2, 17.0, 16.9, 16.8, 16.7, 12.6, 12.2, 11.7.
Anal. Calcd. for C.sub.23H.sub.41N.sub.3O.sub.6Si.sub.2: C, 54.0;
H, 8.1; N, 8.2. Found: C, 54.0; H, 8.3; N, 7.8.
EXAMPLE 91
1-(2-Deoxy-2-methylamino-2-N,4-C-methylene-3,5-O-(tetraisopropyldisiloxane-
-1,3-diyl)-.beta.-D-ribofuranosyl)thymine (74C)
[0379] To a solution of nucleoside 74B (0.33 g, 0.64 mmol) in
anhydrous THF:dichloromethane (4:1, v/v) at -10.degree. C. was
dropwise (during 30 min) added 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU, 0.125 mL, 0.836 mmol) and methyl iodide (0.05 mL, 0.79 mmol)
and the mixture was stirred for 48 h at 10.degree. C. Additional
DBU (0.05 mL, 0.33 mmol) and methyl iodide (0.020 mL, 0.32 mmol)
was dropwise (during 15 min) added to the reaction mixture and
stirring at 10.degree. C. was continued for 24 h. The mixture was
evaporated to dryness under reduced pressure and the residue was
subjected to column chromatography on silica gel using
methanol:dichloromethane (1:99, v/v) as eluent to give nucleoside
74C as a white solid material after evaporation of the solvents
(0.25 g, 74%). FAB-MS m/z 526 [M+H].sup.+. .sup.1H NMR (CDCl.sub.3,
400 MHz) .delta.8.19 (bs, 1H), 7.65 (d, 1H, J 1.3 Hz), 5.59 (s,
1H), 4.11 (s, 1H), 4.05 (d, 1H, J 13.2 Hz), 3.87 (d, 1H, J 13.2
Hz), 3.44 (s, 1H), 2.98 (d, 1H, J9.5 Hz), 2.71 (d, 1H, J9.5 Hz),
2.72(s, 3H), 1.91 (d, 1H, J1.1 Hz), 1.12-0.96 (m, 28 H). .sup.13C
NMR (CDCl.sub.3, 62.9 MHz) .delta.163.7, 149.6, 135.2, 109.7, 90.9,
85.7, 71.4, 67.3, 58.6, 58.2, 41.2, 17.5, 17.4, 17.3, 17.2, 17.1,
16.9, 13.3, 13.1, 13.0, 12.6, 12.1. Anal. Calcd. for
C.sub.24H.sub.44N.sub.3O.sub.6Si.sub.2, 0.25H.sub.2O: C, 54.4; H,
8.3; N, 7.9. Found: C, 54.4; H, 8.1; N, 7.7.
EXAMPLE 92
1-(2-Deoxy-2-methylamino-2-N,4-C-methylene-.beta.-D-ribofuranosyl)thymine
(74D)
[0380] To a solution of nucleoside 74C (0.40 g, 0.76 mmol) in THF
at room temperature was added a solution of tetrabutylammonium
fluoride in THF (1.0 M, 2.2 mL, 2.2 mmol) and the reaction mixture
was stirred for 20 min whereupon pyridine:water:methanol (6 mL,
3:1:1, v/v/v) was added. The mixture was added to Dowex
50.times.200 resin (2.2 g, H.sup.+(pyridinium) form, 100-200 mesh)
suspended in pyridine:water:methanol (6 mL, 3:1:1, v/v/v) and the
resulting mixture was stirred for 20 min. After filtration, the
residue was washed with pyridine:water:methanol (3.times.3 mL,
3:1:1, v/v/v) and the combined filtrate was evaporated to dryness
under reduced pressure to give an oily residue after coevaporation
with methanol (2.times.5 mL). Column chromatography on silica gel
using methanol:dichloromethane (1:49, v/v) as eluent gave
nucleoside 74D as a white solid material after evaporation of the
solvents under reduced pressure (0.17 g, 79%). FAB-MS m/z 284
[M+H].sup.+. FAB-HRMS calcd. for
C.sub.12H.sub.18N.sub.3O.sub.5.sup.+: 284.12465. Found: 284.12402.
.sup.1H NMR ((CD.sub.3).sub.2SO, 400 MHz) .delta.11.3 (bs, 1H, NH),
7.70 (d, 1H, J 1.1 Hz, 6-H), 5.50 (s, 1H, 1'-H), 5.26 (d, 1H, J 4.9
Hz, 3'-OH), 5.12 (t, 1H, J 5.7 Hz, 5'-OH), 3.87 (d, 1H, J 4.8 Hz,
3'-H), 3.67 (d, 2H, J 5.5 Hz, 5'-H), 3.12 (s, 1H, 2'-H), 2.87 (d,
1H, J 9.3 Hz, 5"H.sub.a), 2.56 (s, 3H, NCH.sub.3), 2.52-2.49 (1H,
m, 5"-H.sub.b), 1.77 (s, 3H, CH.sub.3). .sup.1H NMR (CD.sub.3OD,
400 MHz) .delta.7.80 (d, 1H, J 1.3 Hz, 6-H), 5.71 (s, 1H, 1'-H),
4.07 (s, 1H, 3'-H), 3.83 (s, 2H, 5'-H), 3.36 (s, 1H, 2'-H), 3.08
(d, 1H, J 9.9 Hz, 5"-H.sub.a), 2.68 (s, 3H, NCH.sub.3), 2.57 (d,
1H, J 9.9 Hz, 5"-H.sub.b), 1.88 (d, 3H, J 1.1 Hz, CH.sub.3).
.sup.13C NMR (CD.sub.3OD, 62.9 MHz) .delta.166.6, 151.9, 137.4,
110.4, 91.3, 85.2, 71.4, 69.1, 59.4, 58.7, 40.2, 12.2.
EXAMPLE 93
1-(2-Deoxy-5-O-4,4'-dimethoxytrityl-2-methylamino-2-N,4-C-methylene-.beta.-
-D-ribofuranosyl)thymine (74E)
[0381] To a solution of nucleoside 74D (0.135 g, 0.477 mmol) in
anhydrous pyridine (1.5 mL) at 0.degree. C. was dropwise (during 20
min) added a solution of 4,4'-dimethoxytrityl chloride (0.238 g,
0.702 mmol) in anhydrous pyridine:dichloromethane (1.0 mL, 1:1,
v/v) and the resulting mixture was stirred for 10 h at RT. A
mixture of ice and water was added (5 mL) and the mixture was
extracted with dichloromethane (3.times.10 mL). The combined
organic phase was washed with a saturated aqueous solution of
sodium hydrogencarbonate (3.times.5 mL), dried (Na.sub.2SO.sub.4)
and filtered. The filtrate was evaporated to dryness under reduced
pressure and the residue was subjected to column chromatography on
silica gel using methanol:dichloromethane:pyridine (1:98:1, v/v/v)
as eluent to give nucleoside 74E as a white solid material after
evaporation of the solvents under reduced pressure (0.20 g, 72%).
FAB-MS m/z 586 [M+H].sup.+. .sup.1H NMR (C.sub.5D.sub.5N ,400 MHz)
.delta.13.2 (bs, 1H), 7.98 (d, 1H, J 1.3 Hz), 7.98-7.00 (m, 13H),
6.12 (s, 1H), 4.78 (d, 1H, J 3.7 Hz), 3.88-3.79 (m, 4H), 3.71 (s,
3H), 3.71 (s, 3H), 3.29 (d, 1H, J 9.3 Hz), 2.84 (d, 1H, J 9.3 Hz),
2.81 (s, 3H), 1.85 (d, 3H, J 0.9 Hz). .sup.13C NMR
(C.sub.5D.sub.5N, 62.9 MHz) .delta.165.1, 159.2, 151.4, 145.9,
136.5, 136.4, 130.8, 130.7, 128.7, 128.4, 127.4, 113.8, 109.6,
89.8, 86.8, 85.1, 72.0, 68.7, 60.9, 59.4, 55.2, 40.1, 13.1. Anal.
Calcd. for C.sub.33H.sub.35N.sub.3O.sub.7,0.25H.s- ub.2O: C, 67.2;
H, 6.1; N, 7.1. Found: C, 67.2; H, 6.2; N, 6.9.
EXAMPLE 94
1-(3-O-(2-Cyanoethoxy(diisopropylamino)posphino)-5-O-4,4'-dimethoxytrityl--
2-methylmino
-2-N,4-C-methylene-2-deoxy-.beta.-D-ribofuranosyl)thymine (74F)
[0382] To a solution of nucleoside 74E (0.130 g, 0.222 mmol) in
anhydrous dichloromethane (2 mL) at 0.degree. C. were added
N,N-diisopropylethylami- ne (0.088 mL, 0.514 mmol) and 2-cyanoethyl
N,N-diisopropylphosphoramidochl- oridite (0.065 mL, 0.291 mmol) and
the mixture was stirred for 10 h at room temperature.
Dichloromethane (30 mL) was added and the mixture was extracted
with a saturated aqueous solution of sodium hydrogencarbonate
(3.times.10 mL), dried (Na.sub.2SO.sub.4) and filtered. The
filtrate was evaporated to dryness under reduced pressure and the
residue was subjected to column chromatography on silica gel using
methanol:dichloromethane:pyridine (0.5:98.5:1.0, v/v/v) as eluent
to give crude product (0.1 20 g) after evaporation of the solvents
under reduced pressure. The residue was dissolved in anhydrous
dichloromethane (1 mL) and by dropwise addition to vigorously
stirred petroleum ether (60-80.degree. C., 30 mL) at -30.degree.
C., nucleotide 74F precipitated to give a white solid material
after filtration (0.090 g, 52%). .sup.31P NMR (CD.sub.3CN, 121.5
MHz) .delta.147.7.
EXAMPLE 95
1-(3,5-Di-O-benzyl-4-C-(p-toluenesulphonyloxymethyl)-2-O-p-toluenesulphony-
l-.beta.-D-ribofuranosyl)uracil (75)
[0383] To a stirred solution of
1-(3,5-di-O-benzyl-4-C-hydroxymethyl-.beta-
.-D-ribofuranosyl)uracil 41 (3.55 g, 7.81 mmol) in dichloromethane
(50 cm.sup.3) were added DMAP (3.82 g) and p-toluenesulphonyl
chloride (4.47 g, 23.5 mmol) at room temperature. Stirring was
continued for 2 h, and dichloromethane (100 cm.sup.3) was added.
The reaction mixture was washed with a saturated aqueous solution
of sodium hydrogen carbonate (2.times.75 cm.sup.3) and dried
(Na.sub.2SO.sub.4). The organic phase was evaporated under reduced
pressure and the residue was purified by silica gel column
chromatography using dichloromethane/methanol (99.5:0.5, v/v) as
eluent to give nucleoside 75 (4.65 g, 78%) as a white solid
material. .delta..sub.H (CDCl.sub.3) 8.49 (1H, br s, NH), 7.67 (1H,
d, J 8.3, 6-H), 7.51-7.03 (18H, m, Bn, Ts), 6.0 (1H, d, J 7.6,
1'-H), 5.05 (1H, m, 2'-H), 4.91 (2H, m, 5-H, Bn), 4.513 (2H, m,
Bn), 4.42 (1H, d, J 10.4, Bn), 4.31 (1H, d, J 4.9, 3'-H), 4.05 (2H,
m, 1"-H), 3.75-3.64 (2H, m, 5'-H), 2.41 (3H, s, CH.sub.3), 2.34
(3H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3) 162.2 (C-4), 149.5
(C-2), 146.0, 145.3 (Ts), 139.0 (C-6), 136.7, 131.9, 130.0, 129.9,
128.9, 128.7, 128.5, 128.4, 128.3, 128.2, 128.0, 127.6 (Bn, Ts)
102.7 (C-5), 85.5 (1'-C), 84.4 (4'-C), 79.2, 78.3, 75.1, 74.3,
72.4, 69.1 (Bn, 3'-C, 2'-C, 5'-C, 1"-C ), 21.7, 21.6 (Ts). FAB-MS
m/z 763. Found: C, 61.2 ;H, 4.4; N, 3.3;
C.sub.38H.sub.38N.sub.2O.sub.11S.sub.2 requires C, 59.8; H,5.0; N,
3.6.
EXAMPLE 96
1-(2-Deoxy-3,5-di-O-benzyl-2-S,4-C-methylene-2-mercapto-.beta.-D-ribofuran-
osyl)thymine (76)
[0384] To a stirred solution of nucleoside 75 (3.70 g, 4.86 mmol)
in DMF (40 cm.sup.3) was added potassium thioacetate (0.83 g, 7.28
mmol). The mixture was stirred and heated at 110.degree. C. for 80
h. After evaporation under reduced pressure, H.sub.2O (100
cm.sup.3) was added. Extraction was performed with dichloromethane
(4.times.50 cm.sup.3) and the combined organic phase was dried
(Na.sub.2SO.sub.4), filtered and evaporated under reduced pressure.
The residue was purified by silica gel chromatography using
dichloromethane/methanol (99.6:0.4, v/v) as eluent: to give
nucleoside 76 (1.65 g, 75%) as a white solid material.
.delta..sub.H (CDCl.sub.3) 9.08 (1H, br s, NH), 7.98 (1H, d, J 8.1,
6-H), 7.39-7.20 (10H, m, Bn), 5.85 (1H, s, 1'-H), 5.26 (1H, d, J
8.1, 5-H), 4.61 (1H, d J 11.4, 5'-H), 4.56 (2H, s, Bn), 4.45 (1H,
d, J 11.4, Bn), 4.14 (1H, d, J 1.7, 3'-H), 3.82 (2H, m, Bn), 3.72
(1H, d, J 1.9, 2'-H), 3.02 (1H, d, J 9.9, 1"H.sub.a), 2.78 (1H, d,
J 9.9, 1"-H.sub.b). .delta..sub.C (CDCl.sub.3) 1 63.4 (C-4), 150.0
(C-2), 139.9 (C-6), 137.2, 136.8, 128.6, 128.5, 128.2, 127.9, 127.7
(Bn), 100.8 (C-5), 90.8, 88.8 (C-1', C-4'), 76.5, 73.8, 72.0, 70.0
(2.times.Bn, C-3', C-5'), 49.52 (C-2'), 35.63 (C-1"). FAB-MS m/z
453. Found: C, 63.4; H, 5.1;N, 5.9; C.sub.24H.sub.24N.sub.2O.sub.5S
requires C, 63.7; H, 5.3; N, 6.1.
EXAMPLE 97
1-(2-O-p-Toluenesulfonyl-4-C-(p-toluenesulfonyloxymethyl)-.beta.-D-ribofur-
anosyl)uracil (76A)
[0385] To a solution of compound 75 (0.80 g, 1.0 mmol) in absolute
ethanol (2 cm.sup.3) was added 20% palladium hydroxide over carbon
(0.80 g) and the mixture was degassed several times with hydrogen
and stirring was continued under hydrogen for 48 h. The catalyst
was filtered off and the filtrate was evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol (99:1, v/v) as eluent
to give nucleoside 76A (0.30 g, 49%) as a white solid material.
.delta..sub.H (CD.sub.3OD) 7.67 (4H, m), 7.45 (1H, d, J 8.2 Hz),
7.34 (4H, m), 5.86 (1H, d, J8.0 Hz), 5.40 (1H, d, J 8.1 Hz), 4.95
(1H, m), 4.35 (1H, d, J5.0 Hz), 4.17 (2H, m), 3.61 (2H, s), 2.40
(6H, s). .delta..sub.C (CD.sub.3OD) 165.4, 151.6, 147.5, 146.6,
141.3, 134.0, 133.8, 131.4, 130.9, 129.2, 128.9, 103.7, 88.0, 85.4,
80.7, 72.4, 71.0, 64.3, 21.7, 21.6. FAB-MS m/z 583 [M+H].sup.+.
EXAMPLE 98
1-(3,5-O-(Tetraisopropyldisiloxa-1,3-diyl)-2-O-p-toluenesulfonyl-4-C-(p-to-
luenesulfonyloxymethyl)-.beta.-D-ribofuranosyl)uracil (76B)
[0386] To a stirred solution of nucleoside 76A (0.27 g, 0.46 mmol)
in anhydrous pyridine 4 cm.sup.3) was added
1,3-dichloro-1,1,3,3-tetraisopro- pyldisiloxane (0.22 cm.sup.3,
0.70 mmol). After stirring for 48 h, the mixture was cooled to
0.degree. C. and a saturated aqueous solution of sodium hydrogen
carbonate (15 cm.sup.3) was added. The mixture was extracted with
dichloromethane (3.times.10 cm.sup.3) and the combined organic
phase was dried (Na.sub.2SO.sub.4) and filtered. The solvent was
evaporated under reduced pressure and the residue was purified by
silica gel chromatography using dichloromethane/methanol (99.5:0.5,
v/v) as eluent to give nucleoside 76B (0.37 g, 97%) as a white
solid material. .delta..sub.H (CDCl.sub.3) 8.70 (1H, br s), 7.80
(4H, m), 7.36 (4H, m), 6.98 (1H, d, J 8.1 Hz), 5.64 (1H, d, J 8.0
Hz), 5.18 (2H, m), 4.98 (1H, d, J 7.0 Hz), 4.39-4.32 (2H, m),
3.92-3.76 (2H, s), 2.45 (6H, s), 1.27-0.66 (28H, m). .delta..sub.C
(CDCl.sub.3) 162.9, 149.3, 145.6, 144.8, 143.9, 132.9, 130.1,
129.9, 128.2, 128.1, 102.2, 94.6, 84.7, 80.4, 72.8, 67.8, 64.6,
21.7, 17.3, 17.2, 17.1, 16.9, 16.8, 13.1, 12.8, 12.3. FAB-MS m/z
825 [M+H].sup.+.
EXAMPLE 99
1-(2-Deoxy-2-mercapto-2-S,4-C-methylene-3,5-O-(tetraisopropyldisiloxa-1,3--
diyl)-.beta.-D-ribofuranosyl)uracil (76C)
[0387] To a stirred solution of nucleoside 76B (0.26 g, 0.32 mmol)
in DMF (5 cm.sup.3) was added potassium thioacetate (0.054 g, 0.47
mmol). The reaction mixture was stirred at 110.degree. C. for 20 h.
After evaporation of the mixture under reduced pressure, H.sub.2O
(20 cm.sup.3) was added. Extraction was performed with
dichloromethane (3.times.10 cm.sup.3) and the combined organic
phase was dried (Na.sub.2SO.sub.4), filtered and evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol (99.25:0.75, v/v) as
eluent to give nucleoside 76C (0.125 g, 77%) as a white solid
material. .delta..sub.H (CDCl3) 8.55 (1H, br s), 8.02 (1H, d, J 8.1
Hz), 5.82 (1H, s, 1'-H), 5.65 (1H, d, J 8.1 Hz), 4.37 (1Hz), 4.37
(1H, d, J 2.1 Hz), 4.10 (1H, d, J 13.2 Hz), 3.90 (1H, d, J 13.1
Hz), 3.53 (1H, d, J 2.1 Hz), 2.92 (1H, d, J 10.1 Hz), 2.74 (1H, d,
J 10.0 Hz), 1.30-0.80 (28H, m). .delta..sub.C (CDCl.sub.3) 163.2,
149.8, 139.6, 100.9, 91.4, 90.7, 71.5, 59.8, 51.5, 34.4, 17.5,
17.3, 17.1, 16.9, 15.5, 13.6, 13.3, 13.1, 12.9, 12.3. FAB-MSm/z 515
[M+H].sup.+.
EXAMPLE 100
1-(2-Deoxy-2-mercapto-2-S,4-C-methylene-.beta.-D-ribofuranosyl)uracil
(76D)
[0388] To a stirred solution of nucleoside 76C (25 mg, 0.049 rnmol)
in THF (1.0 cm.sup.3) was added a solution of tetrabutylammonium
flouride (0.20 cm.sup.3of a 1M solution in THF, 0.20 mmol) at
0.degree. C. After stirring the mixture at 0.degree. C. for 1 h,
H.sub.2O (5 cm.sup.3) was added and the mixture was evaporated. The
residue was purified by silica gel column chromatography using
dichloromethane/mel:hanol (97:3, v/v) as eluent to give nucleoside
76D (9.0 mg, 69%) as a white solid material. .delta..sub.H
(CD.sub.3OD) 8.19 (1H, d, J 8.1 Hz, 6-H), 5.77 (1H, s, 1'-H), 5.65
(1H, d, J 8.1 Hz, 5-H), 4.31 (1H, d J 2.1 Hz, 3'-H), 3.86 (2H, s,
5'-H), 3.53 (1H, d, J 2.2 Hz, 2'-H), 2.93 (1H, d, J 10.3 Hz,
1"-H.sub.a), 2.73 (1H, d, J 10.3 Hz, 1"-H.sub.b). .delta..sub.C
(CD.sub.3OD) 166.5, 152.0, 141.7, 101.2, 92.1, 92.0, 71.4, 59.9,
53.6, 35.4. FAB-MS m/z 273 [M+H].sup.+.
EXAMPLE 101
1-(2-Deoxy-5-O-(4,4'-dimethoxytrityl)-2-mercapto-2-S,4-C-methylene-.beta.--
D-ribofuranosyl)uracil (76E)
[0389] To a solution of 76D (0.2 g, 0.37 mmol) in anhydrous
pyridine (5 cm.sup.3) was added 4,4'-dimethoxytrityl chloride
(0.186 g, 0.55 mmol) at room temperature. The solution was stirred
for 5 h whereupon the reaction mixture was cooled to 0.degree. C. A
saturated aqueous solution of sodium hydrogen carbonate (30
cm.sup.3) was added and the resulting mixture was extracted with
dichloromethane (3.times.50 cm.sup.3). The combined organic phase
was separated and dried (Na.sub.2SO.sub.4). The solvent was removed
under reduced pressure and the residue was purified by silica gel
column chromatography with dichloromethane/methanol/pyridine
(98.5:1.0:0.5 v/v) as eluent to give nucleoside 76E as a white
brownish solid material (0.175 g, 83%). .delta..sub.C (CDCl.sub.3)
164.5, 159.4, 151.6, 145.7, 139.9, 136.4, 136.0, 135.6, 130.9,
130.8, 128.8, 128.5, 128.4, 127.5, 127.4, 122.7, 113.9, 101.5,
91.7, 90.2, 87.6, 71.8, 61.9, 55.3, 53.7, 36.2, 30.6. FAB-MS m/z
574 [M].sup.+, 575 [M+H].sup.+(Found: C, 65.2; H, 5.4; N, 5.0;
C.sub.31, H.sub.30N.sub.2O.sub.7S requires C, 64.8; H, 5.3; N,
4.9%).
EXAMPLE 102
1-(3-O-(2-Cyanoethoxy(diisopropylamino)phosphino)-(2-deoxy-5-O-(4,4'-dimet-
hoxytrityl)-2-mercapto-2-S,4-C-methylene-.beta.-D-ribofuranosyl)uracil
(76F)
[0390] To a solution of 76E (0.160 g, 0.28 mmol) in anhydrous
dichloromethane (2 cm.sup.3) at 0.degree. C. were added
N,N-diisopropylethylamine (0.27 cm.sup.3) and 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite (97 mg, 0.42 mmol). Stirring
was continued at room temperature for 5 h. The reaction mixture was
cooled to 0.degree. C. and a saturated aqueous solutions of sodium
hydrogen carbonate (30 cm.sup.3) was added. Extraction was
performed using dichloromethane (3.times.20 cm.sup.3) and the
combined organic phase was dried (Na.sub.2SO.sub.4) and evaporated
to dryness under reduced pressure. The residue was purified by
silica gel column chromatography using
dichloromethane/methanol/pyridine (99:0.5:0:5 v/v) as eluent to
give a white foam. This residue was dissolved in dichloromethane (2
cm.sup.3) and the product was precipitated from light petroleum
(100 cm.sup.3, cooled to -40.degree. C.) under vigorous stirring.
The precipitate was collected by filtration, and was finally dried
to give nucleoside 76F as a white solid material (95 mg, 44%).
.delta..sub.P (CDCl.sub.3) 148.9, 149.0.
EXAMPLE 103
3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-(p-toulenesulfonyloxymethyl)-.bet-
a.-D-ribofuranose (77)
[0391] A solution of
3,5-di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropyliden-
e-.alpha.-D-ribofuranose 31 (15.38 g, 38.4 mmol), anhydrous
pyridine (20 cm.sup.3) and anhydrous dichloromethane (80 ml) was
stirred at -5.degree. C. p-Toulenesulphonyl chloride (8.75 g, 46.0
mmol) dissolved in anhydrous dichloromethane (8 cm.sup.3 ) was
added during 15 min. The solution was stirred at room temperature
for 17 h. The reaction was quenched with ice-cold H.sub.2O (200
cm.sup.3). Extraction was performed with dichloromethane
(5.times.150 cm.sup.3) and the combined organic phase was washed
with saturated aqueous solutions of sodium hydrogen carbonate
(3.times.100 cm.sup.3) and brine (3.times.100 cm.sup.3), dried
(Na.sub.2SO.sub.4), filtered and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography using
dicloromethane:methanol (98.5:1.5, v/v) as eluent to give 77 as a
clear oil (17.4 g, 82%). .delta..sub.H (CDCl.sub.3) 7.79-7.19 (14H,
m, Bn), 5.66 (1H, d, J 3.6, 1-H), 4.69-4.20 (8H, m, Bn, 5-H.sub.a,
5-H.sub.b, 3-H, 2-H), 3.53 (1H, d, J 10.3, 1'-H.sub.a), 3.46 (1H,
d, J 10.3, 1'-H.sub.b), 2.40 (3H, s, CH.sub.3), 1.29 (3H, s,
CH.sub.3), 1.26 (3H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3)
144.6, 137.9, 137.3, 133.0, 129.8, 128.4, 128.3, 128.1, 128.0,
127.9, 127.7, 127.6 (aromatic), 113.6 (C(CH.sub.3).sub.2), 104.2,
(C-1), 84.7 (C-4), 79.0, 78.7, 73.7, 72.7, 70.7, 70.2, (Bn, C-2,
C-3, C-5, C-1'), 26.3, 26.0 (C(CH.sub.3).sub.2), 21.6 (CH.sub.3).
FAB-MS m/z 555 [M+H].sup.+. (Found: C, 64.8; H, 6.2;
C.sub.30H.sub.34O.sub.8S requires C, 64.9; H, 6.1%).
EXAMPLE 104
1,2-Di-O-acetyl-3,5-di-O-benzyl-4-C-(p-toluenesulfonyloxymethyl)-.alpha.,.-
beta.-D-ribofuranose (78)
[0392] A solution of furanose 77 (17.4 g, 31.4 mmol) in 80% acetic
acid (250 cm.sup.3) was stirred at 60.degree. C. for 20 h. The
solvent was removed in vacuo and the residue was coevaporated with
toluene (3.times.20 cm.sup.3). The residue was redissolved in
anhydrous pyridine (100 cm.sup.3). Acetic anhydride (14.2 cm.sup.3)
was added and the solution was stirred for 15 h at room
temperature. The reaction was quenched by addition of ice-cold
H.sub.2O (200 cm.sup.3), and the mixture was extracted with
dichloromethane (4.times.150 cm.sup.3). The combined organic phase
was washed with saturated aqueous solutions of sodium hydrogen
carbonate (2.times.125 cm.sup.3) and brine (3.times.150 cm.sup.3),
dried (Na.sub.2SO.sub.4), filtered and evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography using dicloromethane:methanol (98.5:1.5, v/v) as
eluent to give 78 (.alpha.,.beta..about.1:1) as a clear oil (13.5
g, 72%). .delta..sub.C (CDCl.sub.3) 169.8, 169.6, 69.4, 168.8
(C.dbd.O), 144.7, 137.7, 137.5, 132.8, 129.7, 129.6, 128.5, 128.4,
128.3, 128.2, 128.0, 127.8, 127.7, 127.6 (Bn), 97.4,94.2 (C-1),
86.4, 84.2 (C-4), 78.9, 77.5, 74.5, 74.1, 73.7, 73.5, 71.8, 70.6,
70.5, 69.6, 69.5 (Bn, C-2, C-3, C-1'), 21.6, 21.0, 20.8, 20.6, 20.4
(COCH.sub.3, C(CH.sub.3).sub.2). FAB-MS m/z 599 [M+H).sup.+.
[0393] Alternative Procedure for the Preparation of Compound
78.
3-O-Benzyl-1,2:5,6-di-O-isopropylidene-.alpha.-D-glucofuranose
(30B)
[0394] To a solution of
1,2:5,6-Di-O-isopropylidene-.alpha.-D-allofuranose (30A) (obtained
from Pfanstiehl Laboratories Inc.) (40 g) in dimethylformarrmide at
0.degree. C. was added sodium hydride in smaller portions. The
reaction mixture was stirred for 1 h, benzyl bromide was added drop
wise over a period of 1 h. The reaction mixture was stirred at room
temperature for 16 h. Methanol was added to quench the reaction and
dimethylformamide was removed under pressure. The syrup was
extracted with ethyl acetate and washed with brine. Evaporation of
the ethyl acetate layer yielded a semisolid (93%). Homogeneous by
TLC.
3-O-Benzyl-1,2-O-isopropylidene-.alpha.-D-glucofuranose (30C)
[0395] Partial hydrolysis of 30B (50 g) was achieved in 75% acetic
acid in a period of 20 h. Concentration to a smaller volume and
extraction with ethyl acetate yielded 30C, 40 g, (90%). Homogeneous
by TLC.
3-O-Benzyl-1,2-O-isopropylidene-.alpha.-D-ribo-pentodialdofuranose
(30D)
[0396] A solution of 30C (40 g) in water/methanol (1:1) was slowly
added with stirring to a solution of sodium periodate in water at
0.degree. C.The reaction was stirred for 2 h, ethylene glycol was
added and the mixture was extracted with ethyl acetate. The dried
extract was evaporated to yield 30D, 32 g, (89%). Homogeneous by
TLC. In this step addition of methanol is essential for the
completion of the reaction.
3-O-Benzyl-4-(hydroxymethyl)-1,2-O-isopropylidene-.alpha.-D-erythro-pentof-
uranose (30E)
[0397] Aqueous 37% formaldehyde and 1N sodium hydroxide were added
at 0.degree. C. to a stirred solution of 30D (32 g) in water and
tetrahydrofuran (1:1), the reaction was continued for 16 h,
extracted in ethyl acetate and washed with brine. Evaporation of
the organic layer afforded a syrup which crystallised from
ether/petroleum ether as white solid, 23 g, the filtrate was an oil
which solidified as a low melting solid, 10 g, total yield of 30E,
92%. [23 g (white solid was 99% pure by TLC), 10 g of low melting
solid (had faster moving impurities by TLC, approximately 75%
pure)]. In this step addition of tetrahydrofuran is very important
for the time and reaction completion.
3,5-Di-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-.alpha.-D-ribofuran-
ose (31)
[0398] Benzylation of 30E (20 g) with NaH 60% and BnBr at
-10.degree. C. yielded a mixture of two isomers. Flash column
chromatography afforded 31 as the major isomer, 14 g (54%).
Homogeneous by TLC.
3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-tosyl-.alpha.-D-ribofuranose
(77)
[0399] A solution of 31 (12.5 g) in pyridine at 0.degree. C. was
treated with p-toluenesulphonyl chloride and the reaction was
continued at room temperature for 14-16 h. Removal of pyridine,
extraction with methylene chloride and saturated bicarbonate
solution afforded 77, 14 g, (80%). Homogeneous by TLC.
1,2-di-O-acetyl-3,5-di-O-benzyl-4-C-tosyl-D-ribofuranose (78)
[0400] Hydrolysis of 77 (14 g) was done in 75% acetic acid at
65.degree. C. for 18 h. The solvent was removed under pressure and
the residue was treated with ethanol (3.times.100), toluene
(3.times.50) and anhydrous pyridine (2.times.50). (This compound 78
crystallised from petroleum ether as fine white solid.) The residue
was taken in dry pyridine and treated with acetic anhydride at room
temperature for 8 h. Extraction with ethyl acetate and saturated
bicarbonate followed by washing with brine afforded 78 as a mixture
of .alpha. and .beta. anomers, 12 g, (83%). A direct comparison
with an authentic sample of 78 (TLC, HPLC, NMR) confirmed its
identity and purity.
EXAMPLE 105
1-(2-O-Acetyl-3,5-di-O-benzyl-4-C-(p-toulenesulfonyloxymethyl)-.beta.-D-ri-
bofuranosyl)-thymine (79)
[0401] To a stirred solution of the anomeric mixture 78 (12.8 g,
21.4 mmol) and thymine (5.38 9, 42.7 mmol) in anhydrous
acetonitrile (182 cm.sup.3) was added
N,O-bis(trimethylsilyl)acetamide (31.68 ml, 128.23 mmol). The
reaction mixture was stirred for 1 h at room temperature, and
stirring was continued at 60.degree. C. for 1.5 h. After cooling to
0.degree. C., trimethylsilyl triflate (6.57 ml, 30.33 mmol) was
added dropwise, and the mixture was stirred at 60.degree. C. for 10
h. The reaction mixture was neutralised with an ice-cold saturated
aqueous solution of sodium hydrogen carbonate (90 mL). The reaction
mixture was filtered, and the filtrate was concentrated under
reduced pressure to half volume. Extraction was performed using
dichloromethane (4.times.200 cm.sup.3). The combined organic phase
was washed with saturated aqueous solutions of sodium hydrogen
carbonate (3.times.150 cm.sup.3) and brine (3.times.150 ml), dried
(Na.sub.2SO.sub.4), filtered and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography using
dicloromethane:methanol (99:1 to 98:2, v/v) as eluent to give
nucleoside 79 as a white solid material (13.1 g, 92%).
.delta..sub.H (CDCl.sub.3) 9.04 (s, 1H, NH), 7.73-7.19 (15H, m,
6-H, aromatic), 5.94 (1H, d, J 5.5, 1'-H), 5.37 (1H, d, J5.6,
2'-H), 4.57-4.40 (5H, m, 3'-H, 5'-H.sub.a, 5'-H.sub.b, Bn), 4.14
(2H, s, Bn), 3.75 (1H, d, J 10.2, 1"-H.sub.a), 3.57 (1H, d, J 10.2,
1"-H.sub.b), 2.41 (3H, s, CH.sub.3C.sub.6H.sub.5), 2.02 (3H, s,
COCH.sub.3), 1.54 (3H, s, CH.sub.3). .delta..sub.C (CDCl.sub.3)
169.8 (C=O), 163.5 (C-4), 150.2 (C-2), 145.0, 136.8, 135.6, 132.1,
129.7, 128.5, 128.0, 127.9, 127.8, 127.5 (aromatic), 113.5 (C-5),
86.8, 85.3, 77.6, 74.6, 74.3, 73.6, 70.8, 68.8 (Bn, C-1', C-3',
C-2', C-4'), 21.3 (CH.sub.3), 20.5 (COCH.sub.3), 11.8 (CH.sub.3).
FAB-MS m/z 665 [M+H].sup.+(Found C, 61.2; H, 5.3; N, 4.1; S, 4.7,
C.sub.34H.sub.36O.sub.10N.sub.2S requires C, 61.4; H, 5.4; N, 4.2;
S, 4.8).
EXAMPLE 106
1-(3,5-Di-O-benzyl-4-C(p-toulenesulfonyloxymethyl)-.beta.-D-ribofuranosyl)-
thymine (80)
[0402] Nucleoside 79 (13.1 g, 19.7 mmol) was dissolved in a
solution of ammonia in methanol (200 cm.sup.3, prepared by diluting
saturated methanolic ammonia with an equal volume of methanol) and
stirred at room temperature for 4 h. The reaction mixture was
subsequently evaporated, and the residue was dissolved in
dichloromethane (400 cm.sup.3). The organic phase was washed with
brine (3.times.150 cm.sup.3), dried (Na.sub.2SO.sub.4), filtered
and evaporated under reduced pressure. The residue was purified by
silica gel column chromatography using dicloromethane:methanol
(99.5:0.5, v/v) as eluent to give nucleoside 80 as a white solid
material (10.7 g, 87%). .delta..sub.H (CDCl.sub.3) 9.66 (s, 1H,
NH), 7.71-7.21 (15H, m, 6-H, aromatic), 5.72 (1H, d, J 5.1, 1'-H),
4.75, 4.55 (2H, each d, J 11.5, Bn), 4.51 (2H, s, Bn), 4.37 (1H, t,
J 5.4, 2'-H), 4.30-4.12 (3H, m, 3'-H, Bn), 3.76 (1H, d, J 10.2,
1"-H.sub.a), 3.59 (1H, d, J 10.2, 1"-H.sub.b), 2.39 (3H, s,
CH.sub.3C.sub.6H.sub.5), 1.48 (3H, s, CH.sub.3). .delta..sub.C
(CDCl.sub.3) 163.8 (C-4), 150.9 (C-2), 145.0, 137.0, 136.9, 135.9,
132.3, 129.8, 128.7, 128.6, 128.2, 128.1, 128.0, 127.6 (aromatic),
111.0 (C-5), 89.6, 85.3, 78.4, 74.5, 73.8, 71.1, 69.7, (Bn, C-1',
C-3', C-2', C-4', C-1"), 21.6 (CH.sub.3), 12.0 (CH.sub.3). FAB-MS
m/z 623 [M+H].sup.+(Found C, 61.5; H, 5.2; N, 4.4; S, 5.2,
C.sub.32H.sub.34O.sub.9N.sub.2S requires C, 61.7; H, 5.4; N, 4.5;
S, 5.1).
EXAMPLE 107
(1S,3R,4R,7S)-7-Benzyloxy-1-benzoyloxymiethyl-3-(thymin-1-yl)-2,5-dioxabic-
yclo-[2.2.1]heptane (36)
[0403] To a stirred solution of nucleoside 80 (10.65 g, 17.1 mmol)
in anhydrous DMF (150 cm.sup.3) was added a 60% suspension of
sodium hydride in mineral oil (0.9 g, 22.2 mmol) in small portions
at 0.degree. C. The mixture was stirred at 0.degree. C. for 15 h
whereupon additional 60% sodium hydridle (0.205 g, 5.12 mmol) was
added, and the reaction mixture was stirred for additional 22 h at
0.degree. C. Methanol (20 cm.sup.3) was added and the reaction
mixture was subsequently concentrated under reduced pressure to
half volume. Ice-cold H.sub.2O (300 cm.sup.3) was added and
extraction was performed with dichloromethane (5.times.150
cm.sup.3). The combined organic phase was washed with saturated
aqueous solutions of sodium hydrogen carbonate (3.times.40
cm.sup.3) and brine (3.times.40 cm.sup.3), dried
(Na.sub.2SO.sub.4), filtered and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography using
dichloromethane:methanol (99.5:0.5, v/v) as eluent to give
nucleoside 36 as a white solid material (7.1 g, 92%). Spectral data
were in accordance with data given earlier for 36 (Found C, 66.2;
H, 5.8; N, 6.1; C.sub.25H.sub.26N.sub.2O.sub.6 requires C, 66.6; H,
5.8; N, 6.2).
EXAMPLE 108
3,5-Di-O-benzyl-1,2-O-isopropylidene-4-C-methanesulfonyloxymethyl-.alpha.--
D-ribofuranose (200)
[0404] To a stirred solution of furanose 31 (2.16 g, 5.39 mmol) in
anhydrous pyridine (3 mL) at 0.degree. C. was added dropwise
methanesulfonyl chloride (0.61 mL, 16.0 mmol). The reaction mixture
was stirred for 20 min at room temperature, quenched with ice-cold
water (300 mL) and extracted with dichloromethane (2.times.300 mL).
The combined extracts were washed with saturated aqueous sodium
hydrogen carbonate (300 mL) and then dried (MgSO.sub.4). The
solvent was removed by distillation under reduced pressure and the
residue was purified by chromatography over silica gel with
dichloromethane as eluent to give the product 200 as a clear oil
(2.55 g, 99%); .sup.1H NMR (CDCl.sub.3): .delta.7.37-7.24 (10 H, m,
Bn), 5.78 (1 H, d, J 3.8 Hz, H-1), 4.85 (1 H, d, J 11.7 Hz, Bn),
4.73 (1 H, d, J 11.9 Hz, Bn), 4.64 (1 H, dd, J 4.0, 5.3 Hz, H-2),
4.54 (1 H, d, J 11.9 Hz, H-5'), 4.52 (1 H, d, J 11.9 Hz, Bn), 4.46
(1 H, d, J 11.9 Hz, H-5'), 4.41 (1 H, d, J 11.8 Hz, Bn), 3.60 (1 H,
d, J 10.4 Hz, H-5), 3.50 (1 H, d, J 10.5 Hz, H-5), 3.06 (3 H, s,
SO.sub.2CH.sub.3), 1.68 (3 H, s, CH.sub.3), 1.34 (3 H, s,
CH.sub.3); .sup.13C NMR (CDCl.sub.3): .delta.137.79, 137.31,
128.54, 128.48, 128.16, 128.01, 127.87, 127.79 (Bn), 113.66
(C(CH.sub.3).sub.2), 104.46 (C-1), 84.88 (C-4), 78.48, 78.41 (C-2,
C-3), 73.65, 72.63, 70.78, 70.16 (Bn, C-5, C-5'), 37.84
(SO.sub.2CH.sub.3), 26.20 (CH.sub.3), 25.69 (CH.sub.3); MS FAB: 501
(M+Na, 100%). Found: C, 60.37; H, 6.29; S, 6.53;
C.sub.24H.sub.30O.sub.8S requires C, 60.24; H, 6.32; S, 6.70%.
EXAMPLE 109
Methyl
3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-.alpha.-D-ribofuranosi-
de (201)
[0405] A solution of furanose 200 (1.133 g, 2.37 mmol) in
methanolic hydrochloric acid (20% w/w, 31.7 mL) and water (4.4 mL)
was stirred at room temperature for 2 h. After neutralisation with
sodium hydrogen carbonate (s), the solution was extracted with
dichloromethane (2.times.150 mL). The combined extracts were washed
with water (150 mL) and then dried (MgSO.sub.4). The solvent was
removed by distillation under reduced pressure and the residue
purified by chromatography over silica gel with
dichloromethane:methanol (99:1) as eluent to give the product 201
(.beta.:.alpha..about.2:1) as a clear oil (1.018 g, 95%); .sup.1H
NMR (CDCl.sub.3): .delta.7.39-7.22 (m, Bn), 4.86 (br s, Bn),
4.69-3.99 (m, Bn, H-5', H-1, H-2, H-3), 3.68 (d, J 8.9 Hz, H-5
.beta.), 3.51 (d, J 9.8 Hz, H-5 .alpha.), 3.46 (s,
OCH.sub.3.alpha.), 3.34 (d, J 9.1 Hz, H-5 .beta.), 3.32 (d, J 9.7
Hz, H-5 .alpha.),3.28 (s, OCH.sub.3 .beta.), 2.97 3 H, s,
SO.sub.2CH.sub.3 .beta.), 2.93 (3 H, s, SO.sub.2CH.sub.3 .alpha.);
.sup.13C NMR (CDCl.sub.3): .delta.137.74, 136.98, 128.70, 128.64,
128.58, 128.56, 128.37, 128.21, 128.15, 128.09, 127.98, 127.86,
127.83 (Bn), 107.54 (C-1 .beta.), 103.39 (C-1 .alpha.), 84.65,
83.18, 81.90, 78,87 (C-4, C-3), 75.04, 74.07, 73.73, 73.70, 73.38,
72.56, 72.11, 70.85, 70.55, 70.20 (C-2, Bn, C-5, C-5'), 55.90
(OCH.sub.3 .alpha.), 54.96 (OCH.sub.3 .beta.), 37.18
(SO.sub.2CH.sub.3 .beta.), 37.07 (SO.sub.2CH.sub.3 .alpha.); MS
FAB: 475 (M+Na, 25%). Found: C, 58.40; H, 6.33;
C.sub.24H.sub.30O.sub.8S requires C, 58.39; H, 6.24%.
EXAMPLE 110
(3R)- and
(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-diox-
abicyclo[2.2.1]heptane (202 and 203)
[0406] A solution of 201 (3.32 g, 7.34 mmol) in anhydrous DMF (25
mL) was stirred at 0.degree. C. and a 60% oil dispersion of sodium
hydride (700 mg, 16.9 mmol) was added. The mixture was stirred at
room temperature for 90 min, quenched with waler (300 mL) and
extracted with diethyl ether (2.times.300 mL). The combined extract
was washed with water (200 mL) and dried (MgSO.sub.4). The solvent
was removed under reduced pressure and the residue was purified by
chromatography over silica gel with dichloromethane as eluent to
give the two products 202 and 203 as clear oils (1.571 g, 60% and
0.777 g, 30% respectively).
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo[2.-
2.1]heptane (202). .sup.1H NMR (CDCl.sub.3): .delta.7.36-7.26 (10
H, m, Bn), 4.81 (1 H, s, H-1), 4.65 (1 H, d, J 11.9 Hz, Bn), 4.61
(2 H, s, Bn), 4.56 (1 H, d, J 11.9 Hz, Bn), 4.11 (1 H, s, H-2),
4.09 (1 H, s, H-3), 4.01 (1 H, d, J 7.5 Hz, H-5'), 3.80-3.77 (3 H,
m, H-5', H-5), 3.39 (3 H, s, OCH.sub.3); .sup.13C NMR (CDCl.sub.3):
.delta.138.05, 137.36, 128.47, 128.44, 127.88, 127.73, 127.63 (Bn),
104.97 (C-1), 85.13 (C-4), 79.16 (C-3), 77.18 (C-2), 73.64 (Bn),
72.26, 72.10 (Bn, C-5'), 66.50 (C-5), 55.34 (OCH.sub.3); MS FAB:
379 (M+Na, 28%). Found: C, 70.55; H, 6.97; C.sub.21H.sub.24O.sub.5
requires C, 70.77; H, 6.79%.
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-methoxy-2,5-dioxabicyclo-[2-
.2.1]heptane (203). .sup.1H NMR (CDCl.sub.3): .delta.7.36-7.26 (10
H, m, Bn), 5.00 (1 H, s, H-1), 4.67-4.54 (4 H, m, Bn), 4.18 (1 H,
s, H-2), 3.99 (1H, s, H-3), 3.99-3.90 (2 H, m, H-5'), 3.75-3.68 (2
H, m, H-5), 3.49 (3 H, s, OCH.sub.3); .sup.13C NMR (CDCl.sub.3):
.delta.137.83, 137.53, 128.51, 128.48, 127.96, 127.82, 127.71,
127.62 (Bn), 104.05 (C-1), 88.44 (C-4), 79.54 (C-3), 77.16 (C-2),
73.68 (Bn), 72.61 (C-5'), 72.24 (Bn), 65.73 (C-5), 56.20
(OCH.sub.3); MS FAB: 379 (M+Na, 100%).
EXAMPLE 111
(1R,2S,3S)-2-Benzyloxy-3-benzyloxymethyl-1-(methoxy(thymin-1-yl)methyl)-3--
trimethylsilyloxytetrahydrofuran (204)
[0407] A solution of 202 (216 mg, 0.606 mmol) and thymine (153 mg,
1.22 mmol) in anhydrous acetonitrile (9.3 mL) was added BSA
(N,O-bis(trimethylsilyl)acetamide, 0.90 mL, 3.6 mmol) and stirred
under reflux for 15 min. The solution was cooled to 0.degree. C.
and trimethylsilyl triflate (0.153 mL, 0.777 mmol) was added
dropwise. After stirring at room temperature for 18 h and at
60.degree. C. for 24 h, the reaction was quenched with a saturated
aqueous solution of sodium hydrogen carbonate (20 mL), and
extraction was performed using dichloromethane (2.times.50 mL). The
combined extract was washed with a saturated aqueous solution of
sodium hydrogen carbonate (50 mL) and dried (MgSO.sub.4). The
solvent was removed under reduced pressure and the residue was
purified by chromatography over silica gel with
dichloromethane:methanol (98:2) as eluent to give the product 204
(mixture of diastereomers.about.1.7:1) as a solid (196 mg, 67%).
.sup.1H NMR (CDCl.sub.3): .delta.7.36-7.14 (m, Bn, H-6), 5.77 (1 H,
d, J 7.9 Hz, H-1'), 5.57 (1 H, d, J 5.8 Hz, H-1'), 4.68-4.43 (m,
Bn, H-2'), 4.12-3.68 (m, H-5', H-5', H-3'), 3.32 (s, OCH.sub.3),
3.24 (s, OCH.sub.3), 1.93 (d, J 0.9 Hz, CH.sub.3), 1.86 (d, J 1.1
Hz, CH.sub.3), 0.14 (s, Si(CH.sub.3).sub.3), 0.12 (s,
Si(CH.sub.3).sub.3); .sup.13C NMR (CDCl.sub.3): .delta.163.68,
163.55 (C-4), 151.58, 151.07 (C-2), 137.84, 137.74, 137.32 (Bn),
135.93, 135.10 (C-6), 128.57, 128.,12, 128.41, 128.10, 127.95,
127.85, 127.77, 127.74 (Bn), 111.38, 111.01 (C-5), 86.89, 85.61,
85.40, 84.72, 83.40, 83.31, 82.10 (C-1', C-2', C-3', C-4'), 75.20,
73.98, 73.62, 73.59, 72.55, 72.13, 71.04, 70.74 (Bn, C-5', C-5"),
56.82, 56.54 (OCH.sub.3), 12.47, 12.38 (CH.sub.3), 1.72, 1.69
(Si(CH.sub.3).sub.3); MS FAB: 555 (M+H, 65%), 577 (M+Na, 70%).
Found: C, 62.76; H, 6.88; N, 4.94; C.sub.29H.sub.38N.sub.2O.sub.7Si
requires C, 62.79; H, 6.90; N, 5.05%.
EXAMPLE 112
(1R,2S,3S)-2-Benzyloxy-3-benzyloxymethyl-1-(methoxy(6-N-benzoyladenin-9-yl-
)-methyl)-3-trimethylsilyloxytetrahydrofuran (205)
[0408] A solution of 202 (240 mg, 0.673 mmol) and
6-N-benzoyladenine (301 mg, 1.26 mmol) in anhydrous acetonitrile
(8.2 mL) was added BSA (0.67 mL, 2.7 mmol) and stirred at room
temperature for 1 h. The solution was cooled to 0.degree. C. and
trimethylsilyl triflate (0.25 mL, 1.33 mmol) was added dropwise.
After stirring at 65.degree. C. for 18 h, the reaction was quenched
with a saturated aqueous solution of sodium hydrogen carbonate (50
mL), extracted with dichloromethane (2.times.50 mL). The combined
extract was dried (MgSO.sub.4). The solvent was removed under
reduced pressure and the residue was purified by chromatography
over silica gel with dichloromethane:methanol (98:2) as eluent to
give the product 205 (mixture of diastereomers.about.1.8:1) as a
solid (185 mg, 41%). .sup.1H NMR (CDCl.sub.3): .delta.8.78 (s,
H-8), 8.21 (s, H-2), 8.17 (s, H-2), 8.03-8.00 (m, Bz), 7.61-7.49
(m, Bz), 7.36-7.23 (m, Bn), 7.07-7.04 (m, Bz), 5.85 (1 H, d, J 7.9
Hz, H-1'), 5.76 (1 H, d, J 6.0 Hz, H-1'), 4.74-4.40 (m, Bn, H-2'),
4.22-3.62 (m, H-5', H-5', H-3'), 3.33 (s, OCH.sub.33.24 (s,
OCH.sub.3), 0.15 (s, Si(CH.sub.3).sub.3), 0.14 (s,
Si(CH.sub.3).sub.3); .sup.13C NMR (CDCl.sub.3): .delta.164.68
(HNC.dbd.O), 153.17, 152.99 (C-6), 149.47 (C-2), 141.82, 141.66
(C-8), 137.74, 137.71, 137.65 (Bn), 133.87, 132.87, 132.78 (Bz),
128.97, 128.93, 128.45, 128.42, 128.38, 128.14, 127.97, 127.88,
127.82, 127.78 (Bn, Bz), 123.66, 122.85 (C-5), 86.41, 86.23, 85.70,
85.24, 84.78, 83.73, 83.58, 82.79 (C-1', C-2', C-3', C-4'), 75.32,
74.55, 73.61, 72.18, 711.98, 70.85, 70.59 (Bn, C-5', C-5"), 57.23,
57.04 (OCH.sub.3), 1.78 (Si(CH.sub.3).sub.3); MS FAB: 668 (M+H,
50%), 690 (M+Na, 100%). Found: C, 64.07; H, 6.01; N, 9.94;
C.sub.29H.sub.38N.sub.2O.sub.7Si,0.5H.sub.2O requires C, 63.88; H,
6.25; N, 10.34%.
EXAMPLE 113
(1R,2R,3R)-2-Benzyloxy-3-benzyloxymethyl-3-hydroxytetrahydrofurfural
(206)
[0409] A solution of 202/203 (252 mg, 0.707 mmol) in 80% acetic
acid (3.8 mL) was stirred at 90.degree. C. for 2 h whereupon the
solvent was removed by distillation under reduced pressure. The
residue was coevaporated in toluene (3.times.10 mL) to give the
product 206 as an oil (242 mg, 100%). .sup.1H NMR (CDCl.sub.3):
.delta.9.66 (1 H, d, J 0.8 Hz, H-1), 7.36-7.25 (10 H, m, Bn), 4.68
(1 H, d, J 11.9 Hz, Bn), 4.60-4.39 (5 H, m, Bn, H-2, H-3),
3.98-3.92 (2 H, m, H-5), 3.85 (1 H, d, J 9.3 Hz, H-5'), 3.52 (1 H,
d, J 9.2 Hz, H-5'); .sup.13C NMR (CDCl.sub.3): .delta.203.64 (C-1),
137.39, 137.19, 128.61, 128.54, 128.29, 128.12, 127.87, 127.83
(Bn), 87.17, 87.05 (C-4, C-2), 80.98 (C-3), 75.00, 73.70, 71.86
(Bn, C-5'), 67.84 (C-5); MS FAB: 707 (2.times.M+Na, 100%).
EXAMPLE 114
(1S,3S,4R,7S)-3-Acetoxy-7-benzyloxy-1-benzyloxymethyl-2,5-dioxabicyclol2.2-
.1]-heptane (207)
[0410] To a stirred solution of 206 (230 mg, 0.672 mmol) in
anhydrous pyridine (2.0 mL) was added acetic anhydride (0.18 mL,
1.91 mmol). The reaction mixture was stirred for 23 h at room
temperature, water (0.13 mL) was added, and the solvent was removed
by distillation under reduced pressure. The residue was
coevaporated in toluene (3.times.10 mL) and purified by
chromatography over silica gel with dichloromethane:methanol (99:1)
as eluent to give the product 207 as an clear oil (56.7 mg, 23%);
.sup.1H NMR (CDCl.sub.3): .delta.7.38-7.26 (10 H, m, Bn), 6.00 (1
H, s, H-1), 4.68 (1 H, d, J 12.0 Hz, Bn), 4.62 (1 H, d, J 12.2 Hz,
Bn), 4.60 (1 H, d, J 12.4 Hz, Bn), 4.56 (1 H, d, J 12.2 Hz, Bn),
4.17 (1 H, s, H-2), 4.14 (1 H, s, H-3), 4.01 (1 H,d, J 7.7 Hz,
H-5'), 3.81-3.78 (3 H, m, H-5', H-5), 20.06 (3 H, s, COCH.sub.3);
.sup.13C NMR (CDCl.sub.3): .delta.169.18 (C=O), 137.92, 137.48,
128.52, 128.45, 128.03, 127.77, 127.73, 127.68 (Bn), 95.95 (C-1),
86.49 (C-4), 78.27, 76.58 (C-3, C-2), 73.65 (Bn), 72.26, 71.96 (Bn,
C-5'), 65.49 (C-5), 20.98 (COCH.sub.3); MS FAB: 407 (M+Na, 55%).
Found: C, 68.80; H, 6.11; C.sub.22H.sub.24O.sub.6 requires C,
68.74; H, 6.29%.
EXAMPLE 115
(1S,3S,4R,7S)-3-(6-N-Benzoyladenin-9-yl)-7-benzyloxy-1-benzyloxymethyl-2,5-
-dioxabcyclo[2.2.1]heptane (208)
[0411] A solution of furanose 207 (167 mg, 0.434 mmol) and
6-N-benzoyladenine (194 mg, 0.813 mmol) in anhydrous acetonitrile
(5.3 mL) was added BSA (0.43 mL, 1.76 mmol) and stirred at room
temperature for 1 h. The solution was cooled to .sup.0.degree. C.
and trimethylsilyl triflate (0.16 mL, 0.86 mmol) was added
dropwise. After stirring at 65.degree. C. for 2 h, the reaction was
quenched with a saturated aqueous solution of sodium hydrogen
carbonate (40 mL) and the mixture was extracted with
dichloromethane (2.times.50 mL). The combined extract was dried
(MgSO.sub.4). The solvent was removed under reduced pressure and
the residue was purified by chromatography over silica gel with
dichloromethane:methanol (98:2) as eluent to give the product 208
as a solid (111 mg, 45%); .sup.1H NMR (CDCl.sub.3): .delta.8.82 (1
H, s, H-8), 8.14 (1 H, s, H-2), 7.59-7.26 (15 H, m, Bz, Bn), 6.74(1
H, s, H-1'), 4.92 (1 H, s, H-2'), 4.74-4.39 (4 H, m, Bn), 4.42 (1
H, s, H-3'), 4.19-4.10 (2 H,m H-5"), 3.92 (1 H, d, J 11.8 Hz,
H-5'), 3.88 (1 H, d, J 11.5 Hz, H-5'); MS FAB: 564 (M+H, 100%).
EXAMPLE 116
Methyl
2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-D-ribofuran-
oside (209)
[0412] To a stirred solution of 201 (687 mg, 1.52 mmol) in
anhydrous pyridine (4 mL) at 0.degree. C. was added dropwise acetic
anhydride (0.43 mL, 4.56 mmol). The reaction mixture was stirred
for 2 days at room temperature, quenched with saturated aqueous
sodium hydrogen carbonate (75 mL) and extracted with
dichloromethane (150+75 mL). The combined extract was dried
(MgSO.sub.4), the solvent was removed by distillation under reduced
pressure and the residue was purified by chromatography over silica
gel with dichloromethane as eluent to give the product 209 as a
clear oil (.beta.:.alpha..about.3:1, 750 mg, 100%); MS FAB: 463
(M-OCH.sub.3, 100%), 517 (M+Na, 28%); Found: C, 58.53; H, 6.16;
C.sub.24H.sub.30O.sub.9S requires C, 58.29; H, 6.11%. Methyl
2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-.beta.-D-ribofura-
noside (209.beta.). .sup.1H NMR (CDCl.sub.3): .delta.7.36-7.18 (10
H, m, Bn), 5.27 (1 H, d, J4.9 Hz, H-2), 4.88 (1 H, s, H-1),
4.55-4.44 (6 H, m, H-5', Bn), 4.35 (1 H, d, J 5.0 Hz, H-3), 3.73 (1
H, d, J 9.2 Hz, H-5), 3.38 (1 H, d, J 9.3 Hz, H-5), 3.30 (3 H, s,
OCH.sub.3), 2.95 (3 H, s, SO.sub.2CH.sub.3), 2.11 (3 H, s,
OCCH.sub.3); .sup.13C NMR (CDCl.sub.3): .delta.169.91 (C=O),
137.83, 137.28, 128.49, 128.44, 127.99, 127.87, 127.77 (Bn), 105.40
(C-1), 82.65, 81.05, ,74.55, 73.62, 73.56, 71.86, 70.22 (C-2, C-3,
C-4, C-5, C-5', Bn), 55.03 (OCH.sub.3), 37.14 (SO.sub.2CH.sub.3),
20.73 (OCCH.sub.3). Methyl 2-O-acetyl-3,5-di-O-benzyl-
-4-C-methanesulfonyloxymethyl-.alpha.-D-ribofuranoside
(209.alpha.). .sup.1H NMR (CDCl.sub.3): .delta.7.36-7.18 (10 H, m,
Bn), 5.09 (1 H, d, J 4.5 Hz, H-1), 4.95 (1 H, dd J 4.5, 6.8 Hz,
H-2), 4.65-4.44 (6 H, m, H-5', Bn), 4.27 (1 H, d, J 6.6 Hz, H-3),
3.49 (1 H, d, J 9.9 Hz, H-5), 3.46 (3 H, s, OCH.sub.3), 3.36 (1 H,
d, J 9.9 Hz, H-5), 2.92 (3 H, s, SO.sub.2CH.sub.3), 2.14 (3 H, s,
OCCH.sub.3); .sup.13C NMR (CDCl.sub.3): .delta.170.41 (C=O),
137.59, 137.28, 128.56, 128.51, 128.49, 128.44, 127.98, 127.88
(Bn), 102.35 (C-1), 84.25, 77.53, 74.66, 73.67, 72.12, 70.39, 70.28
(C-2, C-3, C-4, C-5, C-5', Bn), 56.07 (OCH.sub.3), 36.94
(SO.sub.2CH.sub.3), 20.63 (OCCH.sub.3).
EXAMPLE 117
Phenyl
2-O-acetyl-3,5-di-O-benzyl-4-C-methanesulfonyloxymethyl-1-thio-.bet-
a.-D-ribofuranoside (210)
[0413] Method a. A stirred solution of 209 (738 mg, 1.49 mmol) in
anhydrous dichloromethane (6.4 mL) was added
phenylthiotrimethylsilane (2.42 mL, 12.8 mmol) and cooled to
0.degree. C. Trimethylsilyl triflate (0.67 mL, 3.67 mmol) was added
dropwise and the solution was stirred at room temperature for 4 h.
The reaction was quenched with a saturated aqueous solution of
sodium hydrogen carbonate (100 mL) and extracted with
dichloromethane (2.times.200 mL). The combined extract was dried
(MgSO.sub.4) and the solvent removed by distillation under reduced
pressure. The residue was purified by chromatography over silica
gel with dichloromethane as eluent to give the product 210 as a
clear oil (564 mg, 66%) and unreacted starting material (191 mg,
26%); Method b. A stirred solution of 211(86 mg, 0.165 mmol) in
anhydrous dichloromethane (0.49 mL) was added
phenylthiotrimethylsilane (0.16 mL, 0.825 mmol) and cooled to
0.degree. C. Trimethylsilyl trifiate (0.037 mL, 0.206 mmol) was
added and the solution was stirred at room temperature for 2 h. The
reaction was quenched with a saturated aqueous solution of sodium
hydrogen carbonate (15 mL) and the resulting mixture was extracted
with dichloromethane (2.times.25 mL). The combined extract was
dried (MgSO.sub.4) and the solvent removed by distillation under
reduced pressure. The residue was purified by chromatography over
silica gel with dichloromethane as eluent to give the product 210
as a clear oil (75 mg, 79%); .sup.1H NMR (CDCl.sub.3):
.delta.7.47-7.19 (15 H, m, Bn, SPh), 5.48 (1 H, d, J 3.6 Hz, H-2),
5.34 (1 H, dd, J 3.7, 5.2 Hz, H-1), 4.54-4.36 (7 H, m, H-3, H-5',
Bn), 3.66 (1 H, d, J 9.7 H-5), 3.48 (1 H, d, J 9.5 Hz, H-5), 2.89
(3 H, S, SO.sub.2CH.sub.3), 2.09 (3 H, s, OCCH.sub.3); .sup.13C NMR
(CDCl.sub.3): .delta.169.93 (C=O), 137.69, 137.08, 132.65, 132.45,
129.15, 128.53, 128.52, 128.18, 128.14, 128.08, 127.91, 127.85 (Bn,
SPh), 87.99, 84.35, 80.34, 75.33, 74.20, 73.67, 70.83, 69.34 (C-1,
C-2, C-3, C-4, C-5, C-5', Bn), 37.27 (SO.sub.2CH.sub.3), 20.68
(OCCH.sub.3); MS FAB: 463 (M-SPh, 100%), 595 (M+Na, 24%); Found: C,
61.17; H, 5.55; C.sub.29H.sub.32O.sub.8S.sub.2 requires C, 60.82;
H, 5.63%.
EXAMPLE 118
1,2-Di-O-acetyl-3,5-di-O-benzyl-4-C-methanesulphonyloxymethyl-D-ribofurano-
se (211)
[0414] A solution of 201 (150 mg; 0.313 mmol) in 80% aqueous acetic
acid (1.5 mL) was stirred at 90.degree. C. for 3 h. The solvent was
removed by distillation under reduced pressure and the residue was
coevaporated in ethanol (3.times.5 mL), toluene (3.times.5 mL) and
pyridine (2.times.5 mL). The residue was redissolved in anhydrous
pyridine (0.62 mL) and added acetic anhydride (0.47 mL) and the
solution was stirred at room temperature for 16 h. The reaction was
quenched with water (50 mL) and the resulting mixture extracted
with dichloromethane (2.times.50 mL). The combined extract was
washed with an aqueous saturated solution of sodium hydrogen
carbonate (50 mL) and dried (MgSO.sub.4). The solvent was
evaporated and the residue purified on column chromatography over
silica gel with dichloromethane as eluent to give the product 211
as an oil (99 mg, 60%); .sup.1H NMR (CDCl.sub.3): .delta.7.39-7.21
(m, Bn), 6.38 (d, J 4.6 Hz, H-1 .beta.), 6.15 (s, H-1 .alpha.),
5.35 (d, J 4.9 Hz, H-2 .alpha.), 5.17 (dd, J 6.3, 4.9 Hz, H-2
.alpha.4.69-4.23 (m, H-3, Bn), 3.64 (d, J 9.7 Hz, H-5 .alpha.),
3.52 (d, J 10.1 Hz, H-2 .beta.), 3.45 (d, J 9.7 Hz, H-5 .alpha.),
3.39 (d, J 9.9 Hz, H-2 .beta.), 2.99 (s, SO.sub.2CH.sub.3 .alpha.),
2.96 (s, SO.sub.2CH.sub.3 .beta.), 2.14, 2.13, 2.06, 1.90
(4.times.s, COCH.sub.3); .sup.13C NMR (CDCl.sub.3): .delta.169.68,
169.00 (C.dbd.O), 137.68, 137.05, 128.60, 128.55, 128.50, 128.21,
128.12, 128.04, 127.94, 127.82, 127.79 (Bn), 99.35 (C-1 .alpha.),
94.24 (C-1 .alpha.), 86.36 (C-4 .beta.), 84.28 (C-4 .alpha.),
79.15, 77.47, 74.58, 74.06, 73.73, 73.56, 71.67, 70.57, 70.19,
69.84 (Bn, C-2, C-3, C-5, C-5'), 37.61 (SO.sub.2CH.sub.3 .beta.),
37.48 (SO.sub.2CH.sub.3 .alpha.), 21.07, 20.74, 20.63, 20.39
(COCH.sub.3); MS FAB: 545 (M+Na, 13%). Found: C, 57.70; H, 5.56;
C.sub.25H.sub.30O.sub.10S requires C, 57.46; H, 5.79%.
EXAMPLE 119
(3R)- and
(3S)-(1S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-d-
ioxabicyclo[2.2.1]heptane (212)
[0415] A solution of 210 (553 mg, 0.966 mmol) in methanol saturated
with ammonia (35 mL) was stirred at room temperature for 2 h
whereupon the solvent removed by distillation under reduced
pressure. The residue was redissolved in anhydrous DMF (3.5 mL) and
the solution stirred at 0.degree. C. A 60% suspension of sodium
hydride (118 mg, 2.88 mmol) was added and the mixture stirred at
room temperature for 12 h. The reaction was quenched with a
saturated aqueous solution of sodium hydrogen carbonate (100 mL)
and the resulting mixture was extracted with dichloromethane
(2.times.100 mL). The combined extract was dried (MgSO.sub.4) and
the solvent was removed by distillation under reduced pressure. The
residue was purified by chromatography over silica gel with
dichloromethane as eluent to give the product 212 as a clear oil
(404 mg, 96%). MS FAB: 435 (M+H, 35%), 457 (M+Na, 16%); Found: C,
71.76; H, 6.18; C.sub.26H.sub.26O.sub.4S requires C, 71.86; H,
6.03%.
(1S,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-phenylthio-2,5-dioxabicyclo-
[2.2.1]-heptane (212.beta.). .sup.1H NMR (CDCl.sub.3):
.delta.7.46-7.26 (15 H, m, Bn, SPh), 5.35 (1 H, s, H-1), 4.68-4.56
(4 H, m, Bn), 4.31 (1 H, s, H-2), 4.10 (1 H, s, H-3), 4.09 (1 H, d,
J 7.3 Hz, H-5'), 3.93 (1 H, d, J 7.8 Hz, H-5'), 3.79 (2 H, m, H-5);
.sup.13C NMR (CDCl.sub.3): .delta.138.03, 137.45, 133.42, 132.36,
129.19, 128.55, 128.46, 128.05, 127.84, 127.83, 127.76 (Bn, SPh),
89.96 (C-1), 87.18 (C-4), 79.71 (C-2), 79.40 (C-3), 73.64 (Bn),
73.23 (C-5'), 72.30 (Bn), 66.31 (C-5).
(1S,3S,4R,7S)-7-Benzyloxy-1-benzyloxymethyl
-3-phenylthio-2,5-dioxabicycl- o[2.2.1]heptane (212.alpha.).
.sup.1H NMR (CDCl.sub.3): .delta.7.52-7.19 (15 H, m, Bn, SPh), 5.52
(1 H, s, H-1), 4.70-4.50 (4 H, m, Bn), 4.41 (1 H, s, H-2), 4.18 (1
H, d, J 7.8 Hz, H-5'), 4.08 (1 H, d, J 8.4 Hz, H-5'), 4.07 (1 H, s,
H-3), 3.78 (1 H, d, J 11.3 Hz, H-5), 3.72 (1 H, d, J 11.5 Hz, H-5);
.sup.13C NMR (CDCl.sub.3): .delta.137.89, 137.46, 135.29, 130.93,
129.13, 128.99, 128.57, 128.48, 127.81, 127.76, 127.58, 126.95 (Bn,
SPh), 91.87 (C-1), 88.59 (C-4), 80.07, 79.14 (C-2, C-3), 73.65,
73.40, 72.04 (Bn, C-5'), 65.62 (C-5).
EXAMPLE 120
(3R)- and (3S)-(1S,4R,7S)-7-Benzyloxy-l -be
nzyloxymethyl-3-(thymin-1-yl)-- 2,5-dioxabicyclo[2.2.1]heptane
(36+213)
[0416] Thymine (175 mg, 1.38 mmol) was stirred in
hexamethyldisilazane (6.8 mL) at reflux and ammonium sulphate (5
mg) was added. After stirring for 16 h, the clear solution was
cooled to 40.degree. C. and the solvent was removed by distillation
under reduced pressure. To the residue was added a solution of 212
(201 mg, 0.463 mmol) in anhydrous dichloromethane (4.6 mL) and 4
.ANG. molecular sieves (180 mg). After stirring at room temperature
for 10 min, NBS (107 mg, 0.602 mmol) was added and the mixture
stirred for another 30 min. The reaction was quenched with a
saturated aqueous solution of sodium thiosulphate (25 mL) and the
resulting mixture was extracted with dichloromethane (2.times.50
mL). The combined extract was dried (MgSO.sub.4) and evaporated,
and the residue was purified on column chromatography over silica
gel with dichloromethane:methanol (97:3) as eluent to give the
product 36+213 and as an anomeric mixture
(.beta.:.alpha..about.1:2) (127 mg, 61%); .sup.1H NMR (CDCl.sub.3):
.delta.7.49 (d, J 0.9 Hz, H-6 .beta.), 7.46 (d, J 1.0 Hz, H-6
.alpha.), 7.39-7.25 (m, Bn), 5.94 (s, H-1' .alpha.), 5.64 (s,
H-1'.beta.), 4.71-4.50 (m, Bn, H-2'), 4.23 (s, H-3' .alpha.), 4.16
(d, J 8.6 Hz, H-5".alpha.), 4.09-3.78 (m, H-5', H-5", H-3' .beta.),
1.94 (d, J 0.9 Hz, CH.sub.3 .alpha.), 1.62 (d, J 1.2 Hz, CH.sub.3
.beta.); MS FAB: 551 (M+H, 96%).
EXAMPLE 121
(3R)- and
(3S)-(1S,4R,7S)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2,5-di-
oxabicyclo[2.2.1]heptane (37+214)
[0417] A solution of 36+213 (175 mg, 0.39 mmol) in ethanol (2.7 mL)
was stirred at room temperature and 20% palladium hydroxide over
carbon (50 mg) was added. The mixture was degassed several times
with argon and placed under a hydrogen atmosphere. After stirring
for 18 h, the mixture was purified on column chromatography over
silica gel with dichloromethane:methanol (95:5) as eluent to give a
mixture of 37 and 214 (1:1.2) (26 mg, 25%); .sup.1H NMR
(CD.sub.3OD): .delta.7.78 (d, J 1.3 Hz, H-6 .alpha.), 7.73 (d, J
1.2 Hz, H-6 .beta.), 5.88 (s, H-1 .alpha.), 5.53 (s, H-1' .beta.),
4.38 (s, H-2' .alpha.), 4.34 (s, H-3' .alpha.), 4.26 (s,
H-2'.beta.), 4.08-3.69 (m, H-5', H-5", H-3'.beta.), 1.92 (d, J 1.2
Hz, CH.sub.3 .alpha.), 1.88 (d, J 1.1 Hz, CH.sub.3 .beta.);
.sup.13C NMR (CD.sub.3OD): .delta.138.00 (C-6 .alpha.) 136.96 (C-6
.beta.), 110.80 (C-5 .beta.), 110.08 (C-5 .alpha.), 92.49, 89.01
(C-4', C-1' .alpha.), 90.46, 88.37 (C-4', C-1' .beta.), 80.89,
74.27, 73.34 (C-2', C-3', C-5' .alpha.), 80.59, 72.47, 70.39 (C-2',
C-3', C-5' .beta.), 59.29 (C-5" .alpha.), 57.61 (C-5" .beta.),
12.52 (CH.sub.3 .alpha.), 12.39 (CH.sub.3 .beta.); MS EI: 270 (M+,
100%).
[0418] Preparation of LNA Phosphoramidites
EXAMPLE 122
4-N-Benzoyl-LNA-C
[(1R,3R,4R,7S)-3-(4-N-benzoylcytosine-1-yl)-1-(hydroxyme-
thyl)-7-hydroxy-2,5-dioxabicyclo{2.2.1}heptane]
[0419] LNA-C (formula Z) was taken in absolute ethanol and heated
at reflux. To the refluxing solution, benzoic anhydride (2
equivalents) was added and the reaction was followed by HPLC
(Eluant: 20% acetonitrile in 0.1M TEAA, pH 7.0, flow rate: 1
ml/min., Novapak C-18 analytical column). Additional anhydride was
added at 0.5-2 h intervals till no more increase in product was
observed by HPLC. Reaction mixture was concentrated on rotavap.
Residue was repeatedly washed with ether, filtered and dried to
give an off white solid. Yield: 45%.
[0420] General Method for Dimethoxytritylation of Base Protected
LNA Nucleosides (LNA-C.sup.B.sup..sub.z, LNA-T, LNA-G.sup.iBu,
LNA-A.sup.Bz).
[0421] Base protected LNA-nucleoside was coevaporated with pyridine
(2.times.) and was stirred with dimethoxytrityl chloride (1.5
equivalents) in pyridine (.about.10 ml/g of nucleoside). The
reaction was followed by HPLC (50% acetonitrile in 0.1M TEAA, pH
7.0, for 5 min., 50-100% acetonitrile in 10 min. and 100%
acetonitrile for 5 min., flow rate: 1 ml/min., Novapak C-1 8
column). When >95% of the starting material had reacted,
reaction mixture was cooled in ice. Reaction was quenched by
addition of cold saturated NaHCO.sub.3 (.about.5 ml.times.vol. of
pyridine). The mixture was extracted with dichloromethan(3
(3.times.half the vol. of sodium bicarbonate). Organic extractions
were combined, dried over anhydrous sodium sulfate, filtered and
concentrated on rotavap. Residue was dried in vacuo and purified by
silica gel chromatography using 0.5% pyridine and 0-2% methanol in
dichloromethane as eluant. Fractions containing pure products were
combined and concentrated on rotavap. Residue was coevaporated with
anhydrous acetonitrile (3.times.) and dried in vacuo. General
method for phosphitylation of protected LNA nucleosides. Base
protected dimethoxytrityl-LNA nucleoside was coevaporated with
anhydrous dichloromethane (2.times.) and was taken in anhydrous
dichloromethane (10 ml/g of nucleoside for A,G & T and
.about.30 ml/g for C). To this
bis(diisopropylamino)(2-cyanoethyl)phosphite (1.05-1.10
equivalent), followed by tetrazole (0.95 equivalent) were added.
Mixture was stirred at room temperature and reaction was followed
by HPLC (70% acetonitrile in 0.1M TEAA, pH 7, 2 min., 70-100%
acetonitrile in 8 min., and 100% acetonitrile in 5 min., flow rate:
1 ml/min., Novapak C-18 column). Once the reaction had proceeded to
>90% and no more increase in amidite formation was observed upon
further stirring, the mixture was cooled in ice. It was diluted
with dichloromethane (.about.15-20 times the original volume) and
washed with cold saturated sodium bicarbonate (2.times.) followed
by cold brine (1.times.). Organic layer was dried over anhydrous
sodium sulfate, filtered and concentrated on rotavap. Residue was
coevaporated with anhydrous acetonitrile (3.times.) and dried in
vacuo overnight. HPLC purity ranged from 93-98%.
[0422] Preparation of LNA Nucleoside 5'-triphosphates
EXAMPLE 123
[0423] Synthesis of LNA Nucleoside 5'-triphosphatles.
[0424] (Tetrahedron Letters 1988, 29 4525). In a 13.times.100 mm
polypropylene tube, nucleosides 37, 44, 51, 4-N-benzoylated 57A or
61B (93.8 .mu.mol) was suspended in 1 mL pyridine (dried by
CaH.sub.2). The solution was evaporated in a speedvac, under high
vacuum, to dryness. The residue was twice resuspended in
acetonitrile (dried by CaH.sub.2) and evaporated to dryness. The
nucleoside was suspended in 313 .mu.L trimethyl phosphate (dried by
4 .ANG. molecular sieves), to which 30.1 mg Proton Sponge.TM. (1.5
equivalents) were added. The mixture was sealed, vortexed, and
cooled to 0.degree. C. POCl.sub.3 (9.8 .mu.L, 1.1 equivalent) was
added with vortexing. The reaction was allowed to proceed at
0.degree. C. for 2.5 hours. During this interval, 469 .mu.mols
sodium pyrophosphate (5 equivalents) were dissolved in 5 mL water
and passed through 5 mL Dow 50 H.sup.+ ion exchange resin. When the
effluent turned acidic, it was collected in 220.mu.L tributylamine
and evaporated to a syrup. The TBA pyrophosphate was coevaporated
three times with dry acetonitrile. Finally, the dried pyrophosphate
was dissolved in 1.3 mL DMF (4 .ANG. sieves). After 2.5 hours
reaction time, the TBA pyrophosphate and 130 .mu.L tributylamine
were added to the nucleoside solution with vigorous vortexing.
After 1 minute, the reaction was quenched by adding 3 mL 0.1 M
triethylammnonium acetate, pH 7.5. Assay by Mono Q chromatography
showed 49% nucleoside 5'-triphosphate. The reaction mixture was
diluted to 100 mL with water and adsorbed onto a Q Sepharose ion
exchange column, washed with water, and eluted with a linear
gradient of 0 to 700 mM NaCl in 5 mM sodium phosphate, pH 7.5.
Fractions containing triphosphate were assayed by Mono Q ion
exchange chromatography. Fractions containing triphosphate were
pooled and concentrated to the point of NaCl saturation. The
product was desalted on a C.sub.18 cartridge. The triphospate was
quantitated by UV spectroscopy and adjusted to 10 mM solution.
Yields were 17-44%. LNA nucleosides prepared by this method were,
U, T, A, G, and C.
[0425] Preparation of LNA Modified Oligonucleotides
EXAMPLE 124
[0426] Synthesis of Oligonucleotides Containing LNAs of Formula V,
X, Y and Z.sup.T, Z.sup.U, Z.sup.G, Z.sup.C, Z.sup.A,
Z.sup.MeC.
[0427] The bicyclic nucleoside 3'-O-phospohoramidite analogues 8,
19, 30, 39, 46, 53, 57D, 61D, and 66 as well as commercial
3'-O-phosphoramidites were used to synthesise example LNA
oligonucleotides of the invention (0.2 to 5 .mu.mol scale)
containing one or more of the LNAs of types V, X, Y and Z.sup.T,
Z.sup.U, Z.sup.G, Z.sup.C, Z.sup.A, and Z.sup.MeC. The purity and
composition of the synthesised LNA oligonucleotides was verified by
capillary gel electrophoresis, and/or HPLC and/or MALDI-MS. In
general, satisfactory coupling efficiencies were obtained for all
the monomers. The best coupling efficiencies (.about.95-100%) were
obtained for LNAs 39, 46, 53, 57D, 61D, and 66 (leading to LNA
monomers of formula Z) giving very satisfactory results when
synthesising fully modified LNA oligonucleotides or when
incorporating LNAs in otherwise unmodified DNA or RNA stands or
LNAs into an all-phosphorothioate oligonucleotide. LNA
oligonucleotides were dissolved in pure water and the concentration
determined as OD.sub.260. Solubiliities in all cases were
excellent. For plain DNA/RNA synthesis and partially modified LNA
oligomers, a standard CPG support or a polystyrene support, was
used. For the synthesis of fully modified LNA oligomers (e.g.
5'-d(GTGATATGC)-3'), a BioGenex Universial CPG Support (BioGenex,
U.S.A.) was used, or LNA derivatised supports were used.)
EXAMPLE 125
[0428] Synthesis of Phosphorothioate LNA Oligonucleotides.
[0429] The all-phosphorothioate LNA (Table 7) was synthesised on an
automated DNA synthesiser using similar conditions as those
described earlier (Example 124). Beaucages' reagent was used as
sulphurising agent. The stepwise coupling yields were >98%.
After completion of the syntheses, deprotection and cleavage from
the solid support was effected using concentrated ammonia
(55.degree. C., 14 h).
EXAMPLE 126
[0430] Synthesis of 2'-Thio-LNA Oligonucleotides.
[0431] The 2'-thio-LNA oligonucleotides (containing monomer U.sup.s
(formula Z (thio-variant) of FIG. 2), FIG. 37, Table 8) were
synthesised on an automated DNA synthesiser using standard
conditions (Example 124). The step-wise coupling yield for amidite
76F was approximately 85% (12 min couplings; improved purity of
amidite 76F is expected to result in increased coupling yield).
After completion of the syntheses, deprotection and cleavage from
the solid support was effected using concentrated ammonia
(55.degree. C., 8 h).
EXAMPLE 127
[0432] Synthesis of 2'-Amino-LNA Oligonucleotides.
[0433] By procedures similar to those described in Example 126,
2'-Amino-LNA oligonucleotides (containing monomer T.sup.NH and
monomer T.sup.NMe (formula Z (amino variants) of FIG. 2), FIGS. 35
and 36) was efficiently obtained on an automated DNA synthesiser
using amidites 74A and 74F (.gtoreq.98% stepwise coupling
yields).
EXAMPLE 128
[0434] Fluorescein-Labeling of LNA Oligomers.
[0435] LNA oligomers (formula Z of FIG. 2) AL16
(5'-d(TGTGTGAAATTGTTAT)-3'- ; LNA nucleotides in bold) and AL17
(5'-d(ATAAAGTGTAAAG)-3'; LNA nucleotides in bold) were succesfully
labeled with fluorescein using the FluoroAmp T4 Kinase Green
Oligonucleotide Labeling System as described by the manufacturer
(Promega). EBriefly, 16 nmol of either LNA-oligomer AL16 or AL17
was 5'-thiophosphate labelled in a 50 .mu.l reaction buffer
containing T4 kinase and .gamma.-S-ATP. The reactions were
incubated for 2 h at 37.degree. C. The thiophosphorylated LNA
oligos were precipitated by the addition of 5 .mu.l of
oligonucleotide precipitant (Promega) and 165 .mu.l of ice cold
(-20.degree. C.) 95% ethanol. After centrifugation the pellets were
washed once with 500 .mu.l of ice cold (-20.degree. C) 70% ethanol
and redissolved in 25 .mu.l of PBSE buffer. Freshly prepared
5-maleimide-fluorescein solution (50 .mu.g in 5 .mu.l DMSO) were
added to the thiophosphorylated LNA oligos and the reaction
mixtures incubated at 68.degree. C. for 30 min. Additional
5-maleimide-fluorescein (50 .mu.g in 5 .mu.l DMSO) were added to
each LNA oligo and the reaction mixtures incubated for an
additional 60 min. After incubation 10 .mu.l of oligonucleotide
precipitant was added to each reaction mixture followed by 180
.mu.l ice-cold (-20.degree. C.) and 100 .mu.l
N,N-dimethylformamide. The fluorescein labeled LNA oligos were
isolated by centrifugation followed by aspiration of the
supernatant. The fluorescein labelled LNA-oligomers were purified
by reversed-phase HPLC as follows: column Delta-Pack C-18, 300A,
0.4.times.30 cm; eluent 0-50% acetonitrile in 0.04 M
triethylammonium buffer (pH 7.0); flow rate 1.5 ml/min. The
fractions containing LNA-oligos were pooled and evaporated under
reduced pressure (oil pump and speed-vac system) during 12 h.
[0436] Hybridisation Data
EXAMPLE 129
[0437] Thermostability of Oligonucleotides Containing Monomers of
Formula V, X, Y and Z.sup.T z.sup.U, z.sup.G, z.sup.C, z.sup.A,
Z.sup.MeC.
[0438] The thermostability of the LNA modified oligonucleotides
were determined spectrophotometrically using a spectrophotometer
equipped with a thermoregulated Peltier element. Hybridisation
mixtures of 1 ml were prepared containing either of 3 different
buffers (10 mM Na.sub.2HPO.sub.4, pH 7.0, 100 mM NaCl, 0.1 mM EDTA;
10 mM Na.sub.2HPO.sub.4 pH 7.0, 0.1 mM EDTA; 3M
tetrametylammoniumchlorid (TMAC), 10 mM Na.sub.2HPO.sub.4, pH 7.0,
0.1 mM E DTA) and equimolar (1 .mu.M or 1.5 .mu.M) amounts of the
different LNA modified oligonucleotides and their complementary or
mismatched DNA or RNA oligonucleotides. Identical hybridisation
mixtures using the unmodified oligonucleotides were prepared as
references. The T.sub.m's were obtained as the first derivative of
the melting curves. Tables 1-4 summarise the results (LNAs are
marked with bold). FIG. 2 illustrates the monomeric LNAs used. The
nomenclature V, X, Y and Z.sup.T, Z.sup.U, Z.sup.G, Z.sup.C,
Z.sup.A, Z.sup.MeC refer to structures V, X, Y and Z of FIG. 2. In
the tables, the nucleobases of the LNA monomers are indicated.
Furthermore, for the thio and amino variants of the LNA structure Z
of the last two tables, the nomenclature used is, e.g., Z.sup.TS
and Z.sup.TNH, respectively. LNAs containing structure Z were
particularly thoroughly examined (see Table 1). When three Z.sup.T
residues were incorporated into an oligonucleotide of mixed
sequence the T.sub.m's obtained in NaCl buffer with both
complementary DNA (10) and RNA (16) oligonucleotides were
substantially higher (RNA: roughly 7.degree. C. and DNA: roughly
5.degree. C. per modification) than the T.sub.m of the
corresponding duplexes with unmodified oligonucleotides (1 and 8).
Similar results were obtained with LNAs containing two Z.sup.T
residues and either one Z.sup.G (21 and 24B) or Z.sup.U (25),
Z.sup.C (69), Z.sup.MeC (65), and Z.sup.A (58) residues. When
mismatches were introduced into the target RNA or DNA
oligonucleotides the T.sub.m of the LNA modified oligonucleotides
in all cases dropped significantly (11-15A and 17; 18-20 and
22-24A; 26-31; 57 and 59-60; 63-64 and 66, and 67), unambiguously
demonstrating that the LNA modified oligonucleotides hybridise to
their target sequences obeying the Watson-Crick hydrogen bonding
rules. In all cases the drop in T.sub.m of the LNA modified
oligonucleotides upon introduction of mismatches was equal to or
greater than that of the corresponding unmodified oligonucleotides
(2-7 and 9; 33-38), showing that the LNA modified oligonucleotide
are at least as specific as their natural counterparts. A lowering
of the ionic strength of the hybridisation buffer (from 10 mM
Na.sub.2HPO.sub.4, pH 7.0, 100 mM NaCl, 0.1 mM EDTA to 10 mM
Na.sub.2HPO.sub.4 pH 7.0, 0.1 mM EDTA) lowers the T.sub.m of the
LNA modified oligonucleotides for their complementary DNA oligos
(40, 41) or RNA oligonucleotides (40A, 41A). A similar effect is
observed with the unmodified oligonucleotides and its complementary
DNA oligo (39) or RNA oligo (39A).
[0439] Addition of 3M tetrametylammoniumchlorid (TMAC) to the
hybridisation buffer significantly increases the T.sub.m of the LNA
modified oligonucleotide for their complementary DNA oligos (10,
21, 25). Moreover, TMAC levels out the diffeneces in the T.sub.m's
of the different oligonucleotides which is observed in the NaCl
buffer (lowest T.sub.m in the NaCl buffer 44.degree. C. and highest
49.degree. C. as opposed to 56.degree. C. and 57.degree. C. in
TMAC). Introduction of mismatches substantially decreases the
T.sub.m of the LNA modified oligonucleotides for their DNA targets
(11-13, 18-20, and 26-28). A similar picture emerges with the
unmodified reference oligonucleotides (1-4 and 32-35)
[0440] The data with the low salt buffer shows that LNA modified
oligonucleotides exhibit a sensitivity to the ionic strength of the
hybridisation buffer similar to normal oligonucleotides. From the
T.sub.m data with the TMAC buffer we infer that TMAC exhibits a
T.sub.m equalising effect on LNA modified oligonucleotides similar
to the effect observed with normal DNA oligonucleotides. LNA
modified oligonucleotides retain their exquisite specificity in
both hybridisation buffers.
[0441] The fully modified LNA oligonucleotide containing all four
monomers (71 and 75), the almost fully modified LNA oligonucleotide
(except for a 3'-terminal DNA nucleoside) containing both Z.sup.G
and Z.sup.T (41 and 41A) and the partly modified oligonucleotide
containing a central block of Z.sup.T and Z.sup.G (40 and 40A) also
exhibit substantially increased affinity compared to the unmodified
control oligonucleotide (39 and 39A; 1 and 8). This shows that LNAs
of formula Z are very useful in the production of both fully and
partly modified oligomers. We note that the almost fully modified
oligomer (41 and 41A) exhibits an unprecedented high affinity for
both complementary RNA (>93.degree. C.) and DNA (83.degree. C.).
A similar extreme affinity (for both RNA and DNA) was observed with
the almost fully modified LNA oligomer containing exclusively
Z.sup.T (Table 1: 52 and 53) and the fully modified LNA oligomer
(71 and 75). The affinity of the partly modified poly-T
oligonucleotide depended on the positions and the number of Z.sup.T
monomers incorporated (44-51). Whereas the T.sub.m's with RNA
targets (45, 47, 49 and 51) in all cases were higher than the
corresponding unmodified oligonucleotides (43) one gave a lower
T.sub.m with the DNA target (46). Since mixed sequence
oligonucleotide containing 3 Z.sup.T residues exhibited a
substantially increased affinity for their DNA (10) and RNA target
(16) compared to the unmodified reference oligonucleotides (1 and
8) this suggests that other binding motifs than Watson-Crick (such
as for example the Hoogsteen binding motif) are open to poly-T
oligonucleotides and that these binding motifs are somewhat
sensitive to the precise architecture of the modified
oligonucleotide. In all cases introduction of single base
mismatches into the complex between the fully Z.sup.T modified
poly-T oligonucleotide and a DNA target (54-56) resulted in a
significant drop in T.sub.m .
[0442] Oligonucleotides containing either LNAs of structures V
(Table 2), X (Table 3) and Y (Table 4) were analysed in the context
of fully and partly modified poly-T sequences. The fully modified
oligonucleotides of structure V and Y exhibited an increase in
T.sub.m (albeit much lower than the Z.sup.T modified
oligonucleotides) with both RNA (Table 2, 14 and Table 4, 14) and
DNA targets (Table 2, 13, and Table 4, 13) compared to the
unmodifed oligonucleotides (Table 1, 42 and 43). The partly
modified oligonucleotides containing monomers of structure V and Y
behaved similarly to partly modified oligonucleotides containing
Z.sup.T and probably this is due to the homopolymer nature of the
sequence as outlined above. Oligonucleotides containing X.sup.T in
all cases exhibited a much reduced T.sub.m compared to the
reference DNA oligonucleotides.
EXAMPLE 130
[0443] A Fully Modified LNA Oligonucleotide Form Stable Hybrids
with Complementary DNA in Both the Anti-parallel and the Parallel
Orientation.
[0444] A full modified LNA oligonucleotide was hybridised to its
complementary DNA in both the anti-parallel and the parallel
orientation. Hybridisation solutions (1 mL) contained 10 mM
Na.sub.2HPO.sub.4 (pH 7), 100 mM NaCl and 0.1 mM EDTA and 1 .mu.M
of each of the two olignucleotides. As shown in Table 1 both the
anti-parallel (71) and the parallel binding orientation (77)
produces stable duplexes. The anti-parallel is clearly the most
stable of the two. However, even the parallel duplex is
significantly more stable than the corresponding anti-parallel
duplex of the unmodified DNA oligonucleotides (Table 1, 1).
EXAMPLE 131
[0445] LNA Monomers can Be Used To Increase the Affinity of RNA
Oligomers for their Complementary Nucleic Acids.
[0446] The thermostability of complexes between a 9-mer RNA
oligonucleotide containing 3 LNA-T monomers (Z.sup.T) and the
complementary DNA or RNA oligonucleotides were measured
spectirophotometrically. Hybridisation solutions (1 ml) containing
10 mM Na.sub.2HPO.sub.4, pH 7.0, 100 mM NaCl, 0.1 mM EDTA and
1.mu.M of each of the two oligonucleotides. Identical hybridisation
mixtures using the unmodified RNA oligonucleotides were measured as
references. As shown in Table 5 the LNA modified RNA
oligonucleotide hybridises to both its complementary DNA (1) and
RNA (3) oligonucleotide. As previously observed for LNA modified
DNA oligonucleotides, the binding affinity of the LNA modified RNA,
oligonucleotide is strongest to the RNA complement (3). In both
cases the affinity of the LNA modified RNA oligonucleotide is
substantially higher than that of the unmodified controls (2 and
4). Table 5 also shows that the specificity towards both DNA and
RINA targets are retained in LNA modified RNA oligonucleotides.
EXAMPLE 132
[0447] LNA-LNA Base Pairing.
[0448] RNA or DNA oligonucleotides containing three Z.sup.T LNA
monomers or an oligonucleotide composed entirely of LNA Z monomers
were hybridised to complementary unmodified DNA oligonucleotides or
DNA oligonucleotides containing three Z.sup.A LNA monomers and the
T.sub.m of the hybrids were measured spectrophotometrically.
Hybridisation solutions (1 ml) contained 10 mM Na.sub.2HPO.sub.4,
pH 7.0, 100 mM NaCl and 0.1 mM EDTA and 1 .mu.M of each of the two
oligonucleotides. As shown in Table 6 all the LNA modified
oligonucleotides hybridises to the complementary, unmodified DNA
oligonucleotides (2 and 3) as well as the complementary LNA
modified oligonucleotides (4, 5 and 6). As observed previously the
presence of LNA monomers in one strand of a hybrid (2 and 3)
increases the T.sub.M significantly compared to the unmodified
control hybrid (1). The presence of LNA-LNA base pairs in the
hybrid increases the T.sub.M even further (4 and 5) Moreover, a
highly stable hybrid can be formed between a fully modified LNA
oligonucleotide and a partly LNA-Z.sup.A modified DNA
oligonucleotide (6). This constitutes the first example of LNA-LNA
base pairs in a hybrid.
EXAMPLE 133
[0449] An LNA All-phosphoromonothioate Oligonucleotide Display
Relatively Less Decreased Thermostability Towards Complementary DNA
and RNA than the Corresponsing All-phosphorothioate DNA
Oligonucleotide.
[0450] The thermostability of an all-phosphoromonothioate DNA
oligonucleotide containing three Z.sup.T LNA monomers (LNA
oligonucleotide) and the corresponding all-phosphoromonothioate
reference DNA oligonucleotide towards complementary DNA and RNA was
evaluated under the same conditions as described in Example 132,
however without EDTA (Table 7). It was observed that the LNA
all-phosphoromonothioate oligonucleotide containing three LNA
Z.sup.T monomers displayed only weakly decreased thermostability
(Table 7, 3 and 4) when compared to the corresponding reference LNA
oligonucleotide (Table 1, 10 and 16). The corresponding
all-phosphoromonothioate DNA oligonucleotide (Table 7, 1 and 2)
displayed significantly decreased thermosetability when compared to
the corresponding reference DNA oligonucleotide (Table 1, 1 and 8).
This has important possible implications on the use of all--or
partially phosphoromonothioate LNA oligonucleotides in antisense
and other therapeutic applications. Thus, the compatibility of LNA
monomers and unmodified monomers in an phosphoromono-thioate
oligonucleotide has been demonstrated. It can be anticipated that
such constructs will display both Rnase H activity and nuclease
resistance in addition to the LNA enhanced hybridisation
characteristics
EXAMPLE 134
[0451] 2'-Thio-LNA Display Nucleic Acid Recognition Properties
Comparable with Those of LNA (Monomer Z).
[0452] The hybridisation conditions were as described in Example
132, however without EDTA. The results for the 2'-thio-LNAs (Table
8) clearly indicate a positive effect on the thermal stability of
duplexes towards both DNA and RNA by the introduction of
2'-thio-LNA monomer U.sup.S (The monomers correspond to formula Z
of FIG. 2 where the methyleneoxy bridge has been substituted with a
methylenethio bridge). This effect (.DELTA.T.sub.m.about.+5.degree.
C./modification towards DNA; .DELTA.T.sub.m.about.+8.degree.
C./modification towards RNA) is comparable with that observed for
parent LNA. The picture is complicated by the simultaneous
introduction of two modifications (the 2'-thio functionality and
uracil instead of thymine). However, as we have earlier observed
identical melting temperatures for the LNA thymine and uracil
monomers, and as the references containing 2'-deoxyuridine instead
of thymidine, if anything, would be expected to display lower
T.sub.m values, the comparison is relevant.
EXAMPLE 135
[0453] 2'-Amino-LNA (Monomer Z.sup.TNH) and 2'-Methylamino-LNA
(Monomer Z.sup.TNMe) Display Nucleic Acid Recognition Properties
Comparable with those of Parent LNA (Monomer Z).
[0454] The hybridisation conditions were as described in Example
132, however without EDTA. The melting results for the
2'-amino-LNAs (Table 9) clearly indicate a positive effect on the
thermal stability of duplexes towards DNA and RNA by introduction
of either 2'-amino-LNA monomers T.sup.NH or T.sup.NMe (The monomers
correspond to formula Z of FIG. 2 where the methyleneoxy bridge has
been substituted with a methyleneamino bridge or
methylene-(N-methyl)amino bridge, respectively). This effect
(.DELTA.T.sub.m.about.+3.degree. C./modification towards DNA and
.DELTA.T.sub.m.about.+6 to +8.degree. C./modification towards RNA)
is comparable to that of parent LNA. It is notheworthy, that the
increased thermal affinity is also observed with an oligo composed
of a mixture of 2'-alkylamino-LNA monomers and nonalkylated
2'-amino-LNA monomers.
[0455] LNA and LNA Modified Oligonucleotides as a Substrates for
Enzymes
EXAMPLE 136
[0456] 3'-Exonucleolytic Stability of Oligomers 5'-V.sup.T.sub.13T
and 5'-Z.sup.T.sub.13T.
[0457] A solution of oligonucleotides (0.2 OD) in 2 ml of the
following buffer (0.1 M Tris-HCl, pH 8.6, 0.1 M NaCl, 14 mM
MgCl.sub.2) was digested at 25.degree. C. with 1.2 U SVPDE (snake
venom phosphodiesterase). During digestion, the increase in
absorbance at 260 nm was followed. Whereas the unmodified control
T.sub.14 was fully degraded after 10 min of degradation,
5-Z.sup.T.sub.13T and 5'-V.sup.T.sub.13T remained intact for 60
min.
EXAMPLE 137
[0458] LNA Modified Oligos as Substrates for T4 Polynucleotide
Kinase.
[0459] 20 pmoles of each primer (FP2: 5'-GGTGGTTTGTTTG-3'; DNA
probe), (AL2: 5'-GGTGGTTTGTTTG-3', LNA nucleosides in bold) and
(AL3: 5'-GGTGGTTTGTTTG-3', LNA nucleosides in bold) was mixed with
T4 polynucleotide Kinase (5 Units; New England Biolabs) and 6 .mu.l
.gamma.-.sup.32PATP (3000 Ci/mmol, Amersham) in a buffer containing
70 mM Tris-HCl (pH 7.6), 10 mM MgCl.sub.2, 5 mM dithiotretiol
(final volume 20 .mu.l). The samples were incubated 40 min at
37.degree. C. and afterwards heated to 65.degree. C. for 5 min. To
each of the reactions were added 2 .mu.l of tRNA (1 .mu.g/.mu.l),
29 .mu.l of a 3M ammonium acetate and 100 .mu.l of ethanol. The
reactions were incubated at -20.degree. C. for 30 min. and the
labelled oligos were precipitated by centrifugation at 15000 g for
30 min. The pellet was resuspended in 20 .mu.l H.sub.2O. The
samples (1 .mu.l) were mixed with a loading buffer (formamide (pH
8.0), 0.1% xylene cyanol FF, 0.1% bromophenol blue and 10 mM EDTA)
and electrophoresed on a denaturing polyacrylamide gel (16%
acrylamide/-bisacrylamide solution, 7 M urea, 1.times.TBE and 0.1
mM EDTA) in a TBE running buffer (90 mM Tris-HCl (pH 8.3), 90 mM
boric acid and 2.5 mM disodium EDTA-2 H.sub.2O). The gel was dried
on a gel dryer (BioRad model 583) and autoradiographed to a X-ray
film (CL-XPosure film, Pierce 34075) for 20 min. The result is
shown in FIG. 6 (FP2: lane 1 and 2; AL2: lane 3 and 4; AL3: lane 5
and 6). Three conclusions can be drawn on the basis of this
experiment. Firstly, it can be concluded that partly and fully LNA
modified oligos are excellent mimics of natural nucleic acid in
their ability to act as substrate for a nucleic acid specific
enzyme like polynucleotide kinase. Secondly, it can be concluded
that LNA modified oligos can be efficiently precipitated by
procedures normally employed to precipitate standard nucleic acids.
In fact, the relative signal intencities of the unmodified (lane 1,
2), partly (lane 3, 4) and fully modified oligos (lane 5, 6) in the
autoradiogram suggests that the more LNA nucleosides a standard DNA
oligo contains the more efficiently it can be precipitated by
salt/alcohol procedures. Thirdly, the similar positions of the
signal in the autoradiogram of the unmodified, partly and fully
modified oligos shows that incorporation of LNA nucleosides into a
DNA oligo does not alter its electrophoretic mobility in
polyacrylamide gels.
EXAMPLE 138
[0460] 3'-End Labelling of LNA-containing Oligonucleotides with
Terminal Deoxynucleotidyl Transferase.
[0461] Oligonucleotides containing LNA monomers were 3'end-labelled
using the enzyme terminal deoxynucleotidyl transferase. The
sequence and extent of LNA modification were as follows (where LNA
monomers are in bold):
1 Control 5' GGT GGT TTG TTT G 3' (1) 5' GGT GGT TTG TTT G 3' (2)
5' GGT GGT TTG TTT G 3' (3) 5' GGT GGT TTG TTT G 3'
[0462] Oligonucleotide (50 pmol) was incubated with 250 .mu.Ci
[.alpha.-.sup.32P]ddATP (3000 Ci/mmol) and 100 Units terminal
deoxynucleotidyl transferase in 250 .mu.l 100 mM cacodylate buffer
pH 7.2, 2 mM CoCl.sub.2 and 0.2 mM 2-mercaptoethanol at 37.degree.
C. for 2 hours. The reaction was then stopped by adding formamide
loading buffer and heating to 100.degree. C. for 5 min before
placing on ice. Samples (0.2 pmol) were run on a 19% acrylamide gel
containing 7M urea and the percentage incorporation of
radioactivity into the oligonucleotide bands was quantified by
means of a phosphorimager (Molecular Dynamics). The results show
incorporation of radioactivity in all cases, including the
oligonucleotide with a high LNA content: Control 94.9%, (1) 39.7%,
(2) 83.7%, (3) 31.7%. We conclude that LNA modified oligos are
substrates for the TdT enzyme.
EXAMPLE 139
[0463] The Ability of Terminal Deoxynucleotidyl Transferase (TdT)
to Tail LNA Modified Oligonucleotides Depends on the Design of the
Oligomer.
[0464] The following 15 mer primers and a mixture of 8 to 32 base
oligonucleotide markers were 5' end labelled with [.gamma..sup.33P]
ATP and T4 polynucleotide kinase (where LNA monomers are in
bold):
2 P1 5'-TGC ATG TGC TGG AGA-3' P2 5'-GC ATG TGC TGG AGA T-3' PZ1
5'-TGC ATG TGC TGG AGA-3' PZ2 5'-GC ATG TGC TGG AGA T-3'
[0465] Reactions were boiled for 5 min after labelling to remove
any PNK activity. Four picomoles of each labelled primer, 25 U
terminal deoxynucleotidyl transferase and 16 .mu.M dATP were
incubated in 25 .mu.l 100 mM cacodylate buffer pH 7.2, 2 mM
CoCl.sub.2 and 0.2 mM 2-mercaptoethanol for 90 min at 37.degree. C.
The reactions were stopped by the addition of formamide stop
solution and the reaction products run on a 19% polyacrylamide 7 M
urea gel with the labelled markers. Autoradiography using Biomax
film was carried out on the dry gel. As shown in FIG. 22, P1 (lane
2), P2 (lane 4) and PZ1 (lane 3) all gave a tail estimated at
greater than 70 bases long on the basis of the 8-32 base marker
(lanes 1 and 6). Primer PZ2 (lane 5) was not extended under these
reaction conditions. We conclude that the TdT enzyme will tolerate
LNA monomers within the oligonucleotide, but not at the extreme 3'
end.
EXAMPLE 140
[0466] LNA-thymidine-5'-triphosphate (LNA-TTP) as a Substrate for
Terminal Deoxynucleotidyl Transferase (TdT).
[0467] In order to test the ability of the triphosphate of LNA-TTP
(Example 123) to be accepted by terminal deoxynucleotidyl
transferase as a substrate, an oligonucleotide tailing reaction was
performed. A 15 mer primer (sequence: 5'-TGC ATG TGC TGG AGA-3')
and a mixture of 8 to 32 base oligonucleotide markers were 5' end
labelled with [.gamma..sup.33P] ATP and T4 polynucleotide kinase.
Reactions were boiled for 5 min after labelling to remove any PNK
activity. Four picomoles of the labelled primer, 25 U terminal
deoxynucleotidyl transferase and 32, 64 or 128 .mu.M dTTP or
LNA-TTP were incubated in 25 .mu.l 100 mM cacodylate buffer pH 7.2,
2 mM CoCl.sub.2 and 0.2 mM 2-mercaptoethanol for 90 min at
37.degree. C. The reactions were stopped by the addition of
formamide stop solution and the reaction products run on a 19%
polyacrylamide 7M urea gel with the labelled markers.
Autoradiography using Biomax film was carried out on the dry gel.
As shown in FIG. 10, reactions with either 32 .mu.M dTTP (lane B),
64 .mu.M dTTP (lane C) or 128 .mu.M dTTP (lane D) all produced
tailed oligonucleotides which on the basis on the 8-32
oligonucleotide marker (outermost left and rigth lanes) were
estimated at greater than 100 nucleotides. The LNA-TTP reactions
(32 .mu.M dTTP (lane E), 64 .mu.M dTTIP (lane F) or 128 .mu.M dTTP
(lane G)) all resulted in the primer being extended by one base and
.about.50% of this being extended by a further base. This result is
very similar to that obtained with ribonucleotides and TdT. We
conclude that LNA derived triphosphates can be recognised and
incorporated into a DNA oligonucleotide by the TdT enzyme. This
latter finding that LNA-TTP can bind to the polymerase underscores
the possibility of successfully using LNA-monomer derivatives as
nucleoside drugs.
EXAMPLE 141
[0468] Exonuclease Free Kienow Fragment DNA Polymerase I can
Incorporate LNA Adenosine, Cytosine, Guanosine and
Uridine-5'-triphosphates (LNA ATP, LNA CTP, LNA GTP, LNA UTP) into
a DNA Strand.
[0469] A primer extension assay was used to evaluate the LNA NTP's
(see Example 123), ribonucleotides, as substrates for exonuclease
free Klenow fragment DNA polymerase I (EFK). The assay used a
.sup.33P 5' end labelled 15 mer primer hybridised to one of four
different 24 mer templates. The sequences of the primer and
templates are (LNA monomer in bold):
3 Primer 5' TGCATGTGCTGGAGA 3' Template 1 3'
ACGTACACGACCTCTACCTTGCTA 5' Template 2 3' ACGTACACGACCTCTCTTGATCAG
5' Template 3 3' ACGTACACGACCTCTTGGCTAGTC 5' Template 4 3'
ACGTACACGACCTCTGAACTAGTC 5'
[0470] One picomole .sup.33P labelled primer was hybridised to 2
picomoles of template in .times.2 Klenow buffer. To this was added
either 4 .mu.M dNTP.alpha.S or 500 .mu.M LNA NTP or a mixture of 4
.mu.M dNTP.alpha.S and 500 .mu.M LNA NTP. Two units of EFK DNA
polymerase was added to each reaction. 2 mU inorganic
pyrophosphatase was added to each of the reactions. Primer plus
template plus enzyme controls were also carried out. All reactions
were carried out in a total volume of 20 .mu.l. The reactions were
incubated at 37.degree. C. for 3 min. Reactions were then stopped
by the addition of 10 .mu.l formamide EDTA stop solution. Reaction
products were separated on a 19% polyacrylamide 7M urea gel and the
product fragments sized by comparison with a .sup.33P labelled 8 to
32 base oligonucleotide ladder after exposure to Kodak Biomax
autoradiography film.
[0471] FIG. 20 shows the result with LNA-UTP using template 1. The
tracks (1-12) correspond to the following reactions: Incorporation
of LNA UTP by EFK. Lane 1--Primer, template and enzyme. Lane
2--plus dTTP.alpha.S. Lane 3--plus LNA UTP. Lane 4--plus
dTTP.alpha.S and dGTP.alpha.S. Lane 5--plus LNA UTP and
dGTP.alpha.S. Lane 6--plus dATP.alpha.S, dGTP.alpha.S and
dTTP.alpha.S. Lane 7--plus LNA UTP, dCTP.alpha.S, dGTP.alpha.S and
dTTP.alpha.S. Lane 8--plus dGTP.alpha.S. Lane 9--plus dCTP.alpha.S,
dGTP.alpha.S and dTTP.alpha.S. Lane 10--plus LNA UTP, dATP.alpha.S,
dCTP.alpha.S and dGTP.alpha.S. Lane 11--plus dATP.alpha.S,
dCTP.alpha.S and dGTP.alpha.S. Lane 12--all 4 dNTP.alpha.S. The
lanes either side show the 8-32 base oligonucleotide markers used
for sizing the products.
[0472] As is evident from FIG. 20, LNA UTP is specifically
incorporated as a "T". Further extension from an LNA UTP terminated
3' end with dNTP.alpha.S is very slow.
[0473] FIG. 21 shows the result with LNA-ATP, LNA CTP, and LNA GTP
using template 2-4. The tracks (1-21) correspond to the following
reactions: Lanes 1, 7, 13 and 17--primer, template and enzyme. Lane
2--plus dGTP.alpha.S. Lane 3--plus dATP.alpha.S and dGTP.alpha.S.
Lane 4--plus LNA GTP. Lane 5--plus dGTP.alpha.S and LNA ATP. Lane
6--plus LNA ATP and LNA GTP. Lane 8--plus dATP.alpha.S. Lane
9--plus dATP.alpha.S and dCTP.alpha.S. Lane 10--plus LNA ATP. Lane
11--plus dCTP.alpha.S and LNA ATP. Lane 12--plus dATP.alpha.S and
LNA CTP. Lane 14--plus dTTP.alpha.S. Lane 15--plus dGTP.alpha.S and
dTTP.alpha.S. Lane 16--plus dTTP.alpha.S and LNA GTP. Lane 18--plus
dCTP.alpha.S. Lane 19--plus dCTP.alpha.S and dTTP.alpha.S. Lane
20--plus LNA CTP. Lane 21--dTTP.alpha.S and LNA CTP. The lanes
either side show the 8-32 base oligonucleotide markers used for
sizing the products.
[0474] The experiments using template 2 (track 1-6), show that LNA
GTP is able to produce the +1 product with efficient extension of
the primer (track 4). The addition of dGTP.alpha.S and LNA ATP
results in mainly the +2 product (track 5). This is from the
incorporation of dGTP.alpha.S to give the +1 product followed by
extension with LNA ATP. There is evidence of a small amount of +3
product from the consecutive incorporation of LNA ATP. The
experiments using Template 3 (tracks 7-12) show that LNA ATP is
efficiently incorporated to give the +1 product (track 10).
Extension of this product with dCTP.alpha.S is slow (track 11). The
addition of dATP.alpha.S and LNA CTP results in the +2 and +3
products (track 12). The absence of any significant +1 product
shows that the addition of the first LNA CTP is efficient, but that
the addition of the second LNA CTP is slow. The results from
experiments on Templates 1 (tracks 13-16) and 4 (tracks 17-21) show
similar trends to those on the other templates. LNA CTP is
efficiently incorporated to give the +1 product on Template 4
(track 20). Extension of this product by dTTP.alpha.S is again slow
(track 21). The addition of LNA GTP and dTTP.alpha.S to reactions
on Template 1 results in the +2 product (track 16). Again this
shows that the addition of a single LNA triphosphate is quite
efficient, but that the addition of consecutive LNA triphosphates
is slow.
EXAMPLE 142
[0475] LNA Monomers can be Used to Enhance the Resistance of an
Oligonucleotide to Digestion by Exonuclease III.
[0476] In order to test the resistance of the LNA containing
oligonucleotides to Exonuclease III degradation the following
reaction was performed. The following 15 mer primers and 8 to 32
base oligonucleotide markers were 5' end labelled with
[.gamma..sup.33P] ATP and T4 polynucleotide kinase (LNA monomer in
bold):
4 P2 5'-GC ATG TGC TGG AGA T-3' PZ2 5'-GC ATG TGC TGG AGA T-3'
[0477] Reactions were boiled for 5 min after labelling to remove
any PNK activity. 8 picomoles of each primer was hybridised to 25
pmoles Template (sequence: 3'-ACG TAC ACG ACC TCT ACC TTG CTA-5')
in .times.2 Klenow buffer. 10 Units of Exonuclease III was added to
each of the reactions. Controls were also set up which had 1 .mu.l
water added in place of the enzyme. The reactions were incubated at
37.degree. C. for 5 min. The reactions were stopped by the addition
of 10 .mu.l formamide/EDTA stop solution. The reactions were heated
at 95.degree. C. for 3 min before loading onto a 19% polyacrylamide
7M urea gel. The gel was fixed in 10% acetic acid/10% methanol
before transferring to 3 MM paper and drying. The dried gel was
exposed to a phosphor screen for 3 hours. The phosphor screen was
analysed on the Molecular Dynamics Storm 860 instrument using
ImageQuant software. The, phosphor screen analysis showed that in
the absence of the enzyme the P2 full length band was 99% of the
signal and PZ2 full length band was 96% of the signal. In the
presence of the enzyme only 20% of the P2 full length product was
left after the 5 minute incubation. However, 62% of the full length
PZ2 product remained after the same treatment. This shows that a
single LNA monomer at the 3' end of an oligonucleotide can enhance
the resistance to degradation by exonuclease III.
[0478] PCR Applications
EXAMPLE 143
[0479] LNA monomers can be used to significantly increase the
performance of biotinylated-DNA oligos in the sequence specific
capture of PCR ampflicons in a MTP format. Two DIG labelled
amplicons from pUC19 were generated by PCR amplification as
follows:
[0480] PCR Reaction Mixture for Amplicon 1
[0481] 1 .mu.l pUC19 (1 ng/.mu.l),
[0482] 1 .mu.l reverse primer (5'-AACAGCTATGACCATG-3') (20
.mu.M),
[0483] 1 .mu.l forward primer (5'-GTAAAACGACGGCCAGT-3') (20
.mu.M),
[0484] 10 .mu.l dUTP-mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 6 mM
dUTP),
[0485] 1.5 .mu.l DIG-11-dUTP (1 mM)
[0486] 10 .mu.l 10.times. Taq buffer (Boehringer Mannheim incl
MgCl.sub.2)
[0487] 1 .mu.l Taq polymerase (Boehringer Mannheim) 5 U/.mu.l
[0488] H.sub.2O ad 100 .mu.l
[0489] PCR Reaction Mixture for Amplicon 2
[0490] 1 .mu.l pUC19 (1 ng/.mu.l),
[0491] 0.4 .mu.l primer 3 (5'-GATAGGTGCCTCACTGAT-3') (50
.mu.M),
[0492] 0.4 .mu.l primer 4 (5 '-GTCGTTCGCTCAAGCTG-3') (50
.mu.M),
[0493] 10 .mu.l dUTP-mix (2 mM dATP, 2 mM dCTP, 2 mM dGTP and 6 mM
dUTP),
[0494] 1.5 .mu.l DIG-11-dUTP (1 mM)
[0495] 10 .mu.l 10.times. Taq buffer (Boehringer Mannheim incl
MgCl.sub.2)
[0496] 1 .mu.l Taq polymerase (Boehringer Mannheim) 5 U/.mu.l
[0497] H.sub.2O ad 100 .mu.l
[0498] PCR reaction: (Cycler: Perkin Elmer 9600) 94.degree. C. 5
min; add polymerase; 94.degree. C. 1 min, 45.degree. C. 1 min,
70.degree. C. 2 min (29 cycles) 72.degree. C. 10 min.
[0499] 10 .mu.l from each PCR reaction was analysed on a standard
agarose gel and the expected fragments of approximately 100 bp and
500 bp were observed. 10 .mu.l of DIG-labelled amplicon 1 or
amplicon 2 was mixed with 5 pmol of 5' biotinylated capture probe
in 1.times.SSC (0.15 M NaCl, 15 mM citrate, pH 7.0) in a total
volume of 450 .mu.l. The following capture probes were used: B-DNA1
(biotin-ATGCCTGCAGGTCGAC-3'; DNA probe specific for amplicon 1),
B-DNA2 (biotin-GGTGGTTTGTTTG-3'; DNA probe specific for amplicon 2)
and B-LNA2 (biotin-GGTGGTTTGTTTG-3', LNA nucleosides in bold; LNA
probe specific for amplicon 2). Reactions were heated to 95.degree.
C. for 5 min in order to denature amplicons and allowed to cool at
25.degree. C. for 15 min to facilitate hybridisation between the
probe and the target amplicon strand. After hybridisation 190 .mu.l
of each reaction were transferred to a streptavidin coated micro
plate (Pierce, cat. no. 15124) and incubated for one hour at
37.degree. C. After washing the plate with phosphate buffered
saline (PBST, 0.15 M Na.sup.+, pH 7.2, 0.05% Tween 20, 3.times.300
.mu.l), 200 .mu.l of peroxidase labelled anti-DIG antibodies were
added (Boehringer Mannheim, diluted 1:1000 in PBST). Plates were
incubated for 30 min at 37.degree. C. and washed (PBST, 3.times.300
.mu.l). Wells were assayed for peroxidase activity by adding 100
.mu.l of substrate solution (0.1 M citrate-phosphate buffer pH 5.0,
0.66 mg/ml ortho-pheylenediamine dihydrochloride, 0.012%
H.sub.2O.sub.2). The reaction was stopped after 8 min by adding 100
.mu.l H.sub.2SO.sub.4 (0.5 M) and the absorbance at 492 nm was read
in a micro plate reader. As shown in FIG. 3, the unmodified
bio-DNAs capture probes (B-DNA1 and B-DNA2) both behave as
expected, i.e. they each capture only their target PCR amplicon.
Cornpared to the B-DNA1 probe the B-DNA2 probe is rather
inefficient in capturing its cognate amplicon. The capture
efficiency of the B-DNA2 probe, however, can be dramatically
improved by substituting 12 of its 13 DNA nucleosides by the
corresponding LNA nucleosides. As shown in FIG. 3 the use of the
B-LNA2 probe in place of the B-DNA2 probe leads to a more that 10
fold increase in the sensitivity of the assay. At the same time the
B-LNA2 retains the ability of the un-modified B-DNA2 to efficiently
discriminate between the related and non-related amplicon,
underscoring the excellent specificity of LNA-oligos. We conclude
that 1) biotin covalently attached to an LNA modified oligo retains
its ability to bind to streptavidin, 2) that LNA modified oligos
works efficiently in a MTP based amplicon capture assay and that 3)
LNA offers a means to dramatically improve the performance of
standard DNA oligos in the affinity capture of PCR amplicons.
EXAMPLE 144
[0500] An LNA Substituted Oligo is Able to Capture its Cognate PCR
Amplicon by Strand Invasion.
[0501] Two identical sets of 10 .mu.l reactions of amplicon or 2
(prepared as in Example 143) were mixed with either 1, 5 or 25 pmol
of the B-LNA2 capture probe (biotin-GGTGGTTTGTTTG-3', LNA
nucleosides in bold; probe specific for amplicon 2) in 1.times.SSC
(0.15 M NaCl, 15 mM citrate, pH 7.0) in a total volume of 450
.mu.l. One set of reactions were heated to 95.degree. C. for 5 min
in order to denature amplicons and allowed to cool to 25.degree. C.
to facilitate hybridisation between the probe and the target
amplicon strand. The other set of reactions were left without
denaturation. From each of the reactions 190 .mu.l were transferred
to a streptavidin coated micro plate (Pierce, cat. no.15124) and
incubated for one hour at 37.degree. C. After washing the plate
with phosphate buffered saline (PBST, 0.15 M Na.sup.+, pH 7.2,
0.05% Tween 20, 3.times.300 .mu.l ), 200 .mu.l of peroxidase
labelled anti-DIG antibodies were added (Boehringer Manheim,
diluted 1:1000 in PBST). Plates were incubated for 30 min at
37.degree. C. and washed (PBST, 3.times.300 .mu.l). Wells were
assayed for peroxidase activity by adding 100 .mu.l of substrate
solution (0.1 M citrate-phosphate buffer pH 5.0, 0.66 mg/ml
ortho-pheylenediamine dihydrochloride, 0.012% H.sub.2O.sub.2). The
reaction was stopped after 10 min by adding 100 .mu.l
H.sub.2SO.sub.4 (0.5 M) and the absorbance at 492 nm was read in a
micro plate reader. When amplicons are denaturated prior to
hybridisation with the capture probe (FIG. 4A) we observe an
efficient and sequence specific amplicon capture similar to that
shown in Example 143. Increasing the concentration of the B-LNA2
from 1 to 5 pmol leads to an increase in capture efficiency. A
further increase to 25 pmol of probe results in a decreased signal.
This observation is consistent with saturation of the available
biotin binding sites on the streptavidin MTP. When amplicons are
not denaturated prior to hybridisation with the capture probe (FIG.
4B) we also observe an efficient and sequence specific amplicon
capture. In fact, the data shows that amplicon capture without
denaturation are as effective and specific as amplicon capture with
denaturation. This strongly indicates that the Bio-LNA2 probe is
capable of binding to its target sequence by strand invasion. To
our knowledge, this constitutes the first example ever of sequence
specific targeting of dsDNA under physiological salt conditions by
a mixed purine/pyrimidine probe. Aside from its potential to
significantly simplify a range of basic research and DNA diagnostic
procedures this unexpected property of LNA modified oligos can be
foreseen to be of major importance in the development of efficient
new drugs by the antisense, and in particular anti-gene
approach.
EXAMPLE 145
[0502] An LNA Substituted Oligo, Immobilised on a Solid Surface
Function Efficiently in the Sequence Specific Capture of a PCR
Amplicon.
[0503] Wells of a streptavidin coated micro-titer plate (Boehringer
Mannheim) were incubated for 1 hour with either 5 pmol of the
B-DNA2 probe (biotin-GGTGGTTTGTTTG-3'; DNA probe specific for
amplicon 2) or the B-LNA2 probe (biotin-GGTGGTTTGTTTG-3', LNA
nucleosides in bold; LNA probe specific for amplicon 2) in a total
volume of 100 .mu.l.times.SSC (0.15 M NaCl, 15 mM citrate, pH 7.0).
In total, four wells were incubated with the B-DNA2 probe, four
wells with the B-LNA2 probe and four wells were incubated with
buffer alone. After incubation the wells were washed three times
with 1.times.SSC. DIG-labelled amplicon1 (60 .mu.l) or amplicon2
(60 .mu.l) (prepared as in Example 143) were mixed with 540 .mu.l
of 1.times.SSC, heat denaturated at 95.degree. C. for 5 min., and
transferred (100 .mu.l) to the micro plate wells. Two of the wells
containing either B-DNA2, B-LNA2 or no capture probe received
amplicon and two of the wells containing B-DNA2, B-LNA2 or no
capture probe received amplicon2. After 1 hour at 37.degree. C. the
plate was washed 3 times with phosphate buffered saline (PBST, 0.15
M Na.sup.+, pH 7.2, 0.05% Tween 20, 3.times.300 .mu.l) and 200
.mu.l of peroxidase labelled anti-DIG antibodies were added
(Boehringer Mannheim, diluted 1:1000 in PBST). Plates were
incubated for 30 min at 37.degree. C. and washed 3 times with 300
.mu.l PBST. Wells were assayed for peroxidase activity by adding
100 .mu.l of substrate solution (0.1 M citrate-phosphate buffer pH
5.0, 0.66 mg/ml ortho-pheylenediamine dihydrochloride, 0.012%
H.sub.2O.sub.2). The reaction was stopped after 6 min by adding 100
.mu.l H.sub.2SO.sub.4 (0.5 M) and the absorbance at 492 nm was read
in a micro plate reader. As shown in FIG. 5, the LNA modified
capture probe (B-LNA2) captures its specific amplicon (amplicon2)
very efficiently and significantly better (approx. five fold
increase in sensitivity) than the corresponding unmodified DNA
capture probe (B-DNA2). No signal is obtained when the B-LNA2 probe
is incubated with the unrelated amplicon (amplicon1) underscoring
the exquisite specificity of the B-LNA2 probe. We conclude that LNA
modified oligos function efficiently in the sequence specific
capture of PCR amplicons when immobilised on a solid surface. We
further conclude that the use of LNA modified oligos in place of
standard DNA oligos provide for a better signal to noise ratio.
Thus, LNA offers a means to significantly improve the performance
of current DNA based assays that utilises immobilised capture
probes, like for instance the array format wherein multiple
immobilised probes are used to simultaneously detect the occurrence
of several different target sequences in a sample.
EXAMPLE 146
[0504] Fully Mixed LNA Monomers can be Used to Significantly
Increase the Performance of Immobilised Biotinylated-DNA Oligos in
the Sequence Specific Capture of PCR Amplicons in a MTP Format.
[0505] Three DIG labelled amplicons from Nras sequence (ref.:
Nucleic Acid Research, 1985, Vol. 13, No. 14, p 52-55) were
generated by PCR amplification as follows:
5 PCR primers: Forward primer: 5'-GCAGCTCTCAGTAGTTTAGTACA- -3'
bases 701-723 according to the NAR reference. 910 bp reverse
primer: 5'-GTAGAGCTTTCTGGTATGACACA-3' bases 1612-1590 (reverse
sequence according to NAR ref.). 600 bp reverse primer:
5'-TAAGTGACAGACGTATCTCAGAC-3' bases 1331-1303 (reverse sequence
according to NAR ref.). 200 bp reverse primer:
5'-CTCTGTTTCAGACATGAACTGCT-3' bases 909-886 (reverse sequence
according to NAR ref.).
[0506] PCR reaction mixture for Nras amplicons: 2.3 .mu.l human
placental genomic DNA (440 nl/.mu.l), 50 .mu.l 10.times.PCR buffer
(without MgCl.sub.2 Perkin Elmer), 30 .mu.l 25 mM MgCl.sub.2, 50
.mu.l dNTP-mix (2 mM dATP, dCTP, dGTP and 1.8 mM dTTP), 10 .mu.l 1
mM Dig-11-dUTP, 10 .mu.l 25 .mu.M forward primer, 10 .mu.l 25 .mu.M
reverse primer, 5 .mu.l 5 U/.mu.l AmpliTaq Gold (Perkin Elmer) and
water ad 500 .mu.l. PCR reaction: The above mixture was made for
all the Nras PCR products. The only difference being reverse primer
910 bp, 600 bp or 200 bp added once at a time. The PCR mixtures
were aliquoted into ten PCR tubes each and cycled in a Perkin Elmer
9600 at the following conditions: 95.degree. C. 3 min; 55.degree.
C. 2 min, 72.degree. C. 3 min, 95.degree. C. 1 min (30 cycles);
55.degree. C. 2 min, 72.degree. C. 10 min and 4.degree. C. soak. 10
.mu.l from each PCR reaction was analysed on a standard agarose gel
and the expected fragments of approximately 910 bp, 600 bp and 200
bp were observed. Assay conditions: Wells of a streptavidin coated
micro-titer plate (Boehringer Mannheim; binding capacity of 20 pmol
biotin per well) were incubated for 1 hour in 5.times.SSCT (0.75 M
NaCl, 75 mM citrate, pH 7.0, 0.1% Tween 20) at 37.degree. C. with
either 1 pmol of DNA Nras Cap A (biotin-5'-TTCCACAGCACAA-3'),
LNA/DNA Nras Cap A (biotin-5'-TTCCACAGCACAA-3'), LNA Nras Cap A
(biotin-5'-TTCCACAGCACAA-3')- , DNA Nras Cap B
(biotin-5'-AGAGCCGATAACA-3'), LNA/DNA Nras Cap B
(biotin-5'-AGAGCCGATAACA-3') or LNA Nras Cap B
(biotin-5'-AGAGCCGATAACA-3- '); LNA nucleosides in bold. The Nras
Cap A capture probes capture amplicons Nras 910, Nras 600 and Nras
200. Nras Cap B capture probes capture specific amplicons Nras 910
and Nras 600. After incubation with the different capture probes,
the wells were washed in 5.times.SSCT and 5 .mu.l native or
denatured (95.degree. C. 5 min and 10 min on ice) DIG-labelled
amplicons (Nras 910, Nras 600 or Nras 200) in 95 .mu.l 1.times.SSCT
(0.15 M NaCl, 15 mM citrate, pH 7.0, 0.1% Tween 20) were added per
well and incubated for 1 hour at 37.degree. C. The wells were
washed three times in phosphate buffered saline (1.times.PBST, 0.15
M Na.sup.+, pH 7.2, 0.05% Tween 20) and incubated 30 min at
37.degree. C. with 200 .mu.l peroxidase labelled anti-DIG
antibodies (Boehringer Mannheim, diluted 1:1000 in 1.times.PBST).
Finally the wells were washed three times in 1.times.PBST and
assayed for peroxidase activity by adding 100 .mu.l of substrate
solution (0.1 M citrate-phosphate buffer pH 5.0, 0.66 mg/ml
ortho-pheylenediamine dihydrochloride, 0.012% H.sub.2O.sub.2) the
reaction was stopped after 9 min by adding 100 .mu.l 0.5 M
H.sub.2SO.sub.4 and diluted 4 times in H.sub.2SO.sub.4 before the
absorbance at 492 nm was read in a micro-titer plate reader. As
shown in FIG. 23A, capture probes spiked with 12 LNA nucleosides
(LNA Nras Cap A and LNA Cap B) capture very efficiently the
specific amplicons without prior denaturation (native amplicons).
Capture probes spiked with 4 LNA nucleosides (LNA/DNA Nras Cap A
and LNA/DNA Nras Cap B) capture the same amplicons with a lower
efficiency and the DNA capture probes (DNA Nras Cap A and DNA Nras
Cap B) do not capture the specific amplicons at all. The control
amplicon, Nras 200, are not captured by the LNA Cap B or the
LNA/DNA Nras Cap B probes demonstrating the exquisite specificity
of the LNA spiked capture probes. FIG. 23B shows the same
experiment performed with denatured amplicons. Essentially the same
picture emerges with the essential difference that capture
efficiencies are generally increased. We conclude that LNA modified
oligos containing mixed LNA nucleosides (A, T, G or C LNA
nucleosides) function efficiently in sequence specific capture of
PCR amplicons when immobilised on a solid surface. We further
conclude that LNA offers a means to construct capture probes that
will function efficiently in amplicon capture without prior
denaturation i.e. capture by strand displacement. This ability
facilitates a significant simplification of current amplicon
detection formats based on DNA.
EXAMPLE 147
[0507] LNA Modified Oligos Function as Primers for Nucleic Acid
Polymerases.
[0508] The ability of an LNA modified oligo (5'-GGTGGTTTGTTTG-3',
LNA nucleosides in bold) to serve as primer in template dependent,
enzymatic elongation were investigated with 3 different classes of
polymerases. A reverse transcriptase M-MuLV (Boehringer Mannheim)
which can use both RNA and DNA as template, the Klenow polymerase
which is representative of standard DNA polymerases and a
thermostable polymerase, BM-TAQ (Boehringer Mannheim). As control
the extension reactions were conducted using the identical
unmodified DNA primer (5'-GGTGGTTTGTTTG-3'). The LNA and DNA
primers were labelled with .sup.32P-.gamma.-ATP as previously
described in Example 137. A 50 mer DNA oligo
(5'-AAAAATCGACGCTCAAGTCAGAAA- AGCATCTCACAAACAAACAAACCACC-3') was
used as template. The reaction with M-MuLV (Boehringer Mannheim,)
contained 2 .mu.l of either labelled LNA-primer or DNA primer (10
.mu.M), 2 .mu.l of DNA template (10 .mu.M), 2 .mu.l of 2 mM dNTP, 2
.mu.l of 10.times.buffer (500 mM Tris-HCl, 300 mM KCl, 60 mM
MgCl.sub.2, 100 mM DTT, pH 8.3 (37.degree. C.)), 1 .mu.l of enzyme
(20 U/.mu.l) and water to 20 .mu.l. The reactions were incubated at
37.degree. C. for 60 min. The reaction with Klenow polymerase (USB)
contained 2 .mu.l of either labelled LNA or DNA primer (10 .mu.M),
2 .mu.l of DNA template (10 .mu.M), 2 .mu.l of 2 mM dNTP, 2 .mu.l
of 10.times.buffer (100 mM Tris-HCl, 50 mM MgCl.sub.2, 75 mM DTT,
pH 7.5), 1 .mu.l of enzyme (10 U/.mu.l) and water to 20 .mu.l. The
reactions were incubated at 37.degree. C. for 60 min. The reaction
with BM-Taq (Boehringer Mannheim) contained 2 .mu.l of either
labelled LNA or DNA-primer (10 .mu.M), 2 .mu.l of DNA template (10
.mu.M), 2 .mu.l of 2 mM dNTP, 2 .mu.l of 10.times.buffer (100 mM
Tris-HCl, 15 mM MgCl.sub.2, 50 mM KCL, pH 8.3), 1 .mu.l of enzyme
(5 U/.mu.l) and water to 20 .mu.l. The reactions were incubated at
a starting temperature of 37.degree. C. and ramped at 1.degree.
C./min to 60.degree. C. where they were maintained for 30 min. At
the end of the incubation period the reactions were stopped by the
addition of 10 .mu.l of loading buffer (0.25% (w/v) bromophenol
blue, 0.25% (w/v) xylene cyanol, 80% (v/v) formamid). The samples
were heated to 95.degree. C. for 1 min., placed on ice and 2 .mu.l
was loaded onto a 8% sequencing polyacrylamide gel and
electroplhoresed on a Life Technologies Inc. BRL model 52. After
electrophoresis the gel was dried on the glass plate and subjected
to autoradiography (X-ray film: Kodak X-Omat AR). As shown in FIG.
7, clear and similar extension products are observed with both the
LNA and DNA primer when either the Klenow polymerase (lanes 3) or
the BM-Taq polymerase (lanes 5) is used. When M-MuLV reverse
transcriptase is used (lanes 2) an extension product can be
detected only in the case of the LNA-primer. The labelled LNA and
DNA primer that have not been subjected to enzymatic elongation are
present in lanes 1, 4 and 6. We conclude that the incorporation of
LNA nucleosides into standard DNA oligos does not prevent
recognition of the oligo/template duplex by nucleic acid
polymerases. We further conclude that LNA modified oligos act as
efficiently as primers as unmodified DNA oligos.
EXAMPLE 148
[0509] LNA Modified Oligo Functions as Primers in Target
Amplification Processes.
[0510] The ability of LNA modified oligos to act as primers in PCR
amplification was analysed with three oligos differing only in the
number of LNA nucleosides they contained: 4 LNA nucleosides (AL2
primer: 5'-GGTGGTTTGTTTG-3', LNA nucleosides in bold), 1 LNA
nucleoside (AL10 primer: 5'-GGTGGTTTGTTTG-3', LNA nucleoside in
bold) and no LNA nucleoside (FP2 primer: 5'-GGTGGTTTGTTTG-3'). The
PCR reactions (100 .mu.l) contained either no template (control),
0.01 ng, 0.1 ng or 1 ng of template (pUC19 plasmid), 0.2 .mu.M
reverse primer (5'-GTGGTTCGCTCCAAGCTG-3'), 0.2 .mu.M of either the
AL2, AL10 or FP2 forward primer, 200 .mu.M of dATP, dGTP, dCTP and
dTTP, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl.sub.2, 50 mM KCl and 2.5 U
of the BM-Taq polymerase. A total of 50 cycles each consisting of
94.degree. C. 1 min. -45.degree. C. 1 min. -72.degree. C. 1.5 min.
were conducted (with an additional 2.5 U of Taq polymerase added
after the first 30 cycles) on a Techne Genius thermocycler. After
the final cycle the reactions were incubated at 72.degree. C. 3
min. and then at 4.degree. C. overnight. To 30 .mu.l of each
reaction was added 6 .mu.l of loading buffer (0.25% (w/v)
bromophenol blue and 40% (v/v) glycerol) and the samples (together
with a Amplisize.TM. size marker) were loaded onto a 2% agarose gel
and electrophoresed for 45 min. at 150V. Finally, the gel was
stained with ethidiumbromid and photographed. As shown in FIG. 8
the PCR reactions using the unmodified forward primer FP2 and
unmodified reverse primer generates detectable amplicons of the
correct sizes with all amounts of template used (lane 9: 0.01 ng
template, lane 10: 0.1 ng and lane 11: 1 ng). No signal is obtained
in the control reaction without template (lane 12). When the FP2
forward primer is replaced by the primer containing 1 central LNA
nucleoside (AL10) amplicons are also detected with all amounts of
template used (lane 5: 0.01 ng, lane 6: 0. 1 ng and lane 7: 1 ng).
This clearly indicates that the AL10 primer sustains an exponential
amplification. ie. the AL10 primer can be both extended and used as
template in its entirety. Again, the control reaction without
template (lane 8) does not produce an amplicon. When the FP2
forward primer is replaced by the primer containing 4 central LNA
nucleosides (AL2), amplicons of the correct size cannot be detected
in any of the reactions. (lane 1: 0.01 ng template, lane 2: 0.1 ng,
lane 3: 1 ng and lane 4: no template). With the highest
concentration of template (1 ng), however, a high molecular weight
band appears in the gel (lane 3). This, however, is an artefact of
the RP1 primer as indicated by the control reaction wherein each of
the primers AL2 (lane A), AL10 (lane B), FP2 (lane C) and RP1 (lane
D) were tested for their ability to produce an amplicon with the
highest amount of template (1 ng). Since AL2 was shown to act as a
primer in Example 147, the absence of detectable amplicons strongly
indicates that it lacks the ability to act as a template, i.e. the
block of 4 consecutive LNA nucleosides blocks the advance of the
polymerase thereby turning the reaction into a linear amplification
(the product of which would not be detectable by the experimental
set-up used). We conclude that LNA modified oligos can be used as
primers in PCR amplification. We further conclude that the degree
of amplification (graded from fully exponential to linear
amplification) can be controlled by the design of the LNA modified
oligo. We note that the possibility to block the advance of the
polymerase by incorporating LNA nucleosides into the primer
facilitates the generation of amplicons carrying single stranded
ends. Such ends are readily accessible to hybridisation without
denaturation of the amplicon and this feature could be useful in
many applications.
EXAMPLE 149
[0511] An LNA Modified Oligomer Carrying a 5' Anthraquinone can be
Covalently Immobilised on a Solid Support by Irradiation and the
Immobilised Oligomer is Efficient in the Capture of a Complementary
DNA Oligo.
[0512] Either 25 pmol/.mu.l or 12.5 pmol/.mu.l of an anthraquinone
DNA oligo (5'-AQ-CAG CAG TCG ACA GAG-3') or an anthraquinone LNA
modified DNA oligo (5'-AQ-CAG CAG TCG ACA GAG-3'; LNA monomer is
underlined) was spotted (1 .mu.l/spot) in 0.2 M LiCl on a
polycarbonate slide (Nunc). The oligos were irradiated for 15 min
with soft UV light. After irradiation the slide was washed three
times in Milli-Q water and air-dried. 25 ml of 0.5 pmol/.mu.l of
complimentary biotinylated oligomer (5'-biotin-CTC TGT CGA CTG
CTG-3') was hybridised to the immobilised oligomers in 5.times.SSCT
(75 mM Citrate, 0.75 M NaCl, pH 7.0, 0.1% Tween 20) at 50.degree.
C. for 2 hours. After washing four times with 1.times.SSCT and one
time phosphate buffered saline (PBST, 0.15 M Na.sup.+, pH 7.2,
0.05% Tween 20), 25 ml PBST containing 0.06 .mu.l/ml streptavidin
conjugated horse radish peroxidase and 1 .mu.g/ml streptavidin were
added to the slide. The slide was incubated for 30 min and washed 4
times with 25 ml PBST. The slide was visualised by using
chemo-luminescent substrate (SuperSignal; Pierce) as described by
the manufacturer and X-ray film (CL-XPosure film, Pierce 34075). As
shown in FIG. 9 both the AQ-DNA oligo and the AQ-LNA modified DNA
oligo yields a clearly detectable signal. We conclude that
anthraquinone linked LNA modified DNA oligos can be efficiently
attached to a solid surface by irradiation and that oligos attached
in this ways are able to hybridise to their complementary target
DNA oligos.
EXAMPLE 150
[0513] Hybridisation and Detection on an Array with Different LNA
Modified Cy3-labelled 8 Mers.
[0514] Slide preparation: Glass slides were aminosilanised using a
10% solution of amino propyl triethoxy silane in acetone followed
by washing in acetone. The following oligonucleotides were spotted
out onto the slides:
6 Pens Sequence Oligo used Oligo sequence 1 + 2 + 3 cf. probes Seq.
3 5'-GTA TGG AG-3' 1 pmol/.mu.l 1 internal mismatch Seq. 6 5'-GTA
TGA AG-3' 1 pmol/.mu.l match
[0515] Ten repeat spots, approximately 1 nl each spot, were
performed for each oligonucleotide from each pen on each of 12
slides.
7 Probes (LNA monomers in bold): a) 5'-Cy3-CTT CAT AC-3' Seq.
No.aZ1 b) 5'-Cy3-CTT CAT AC-3' Seq. No.aZ2 c) 5'-Cy3-CTT CAT AC-3'
Seq. No.aZ3 d) 5'-Cy3-CTT CAT AC-3' Seq. No.16
[0516] Slides and Conditions for Hybridisation:
[0517] Slides 1, 2 and 3 hybridised with aZ1 probe @ 300
fmol/.mu.l, 30 fmol/.mu.l, 3 fmol/.mu.l
[0518] Slides 4, 5 and 6 hybridised with aZ2 probe @ 300
fmol/.mu.l, 30 fmol/.mu.l, 3 fmol/.mu.l
[0519] Slides 7, 8 and 9 hybridised with aZ3 probe @ 300
fmol/.mu.l, 30 fmol/.mu.l, 3 fmol/.mu.l
[0520] Slides 10, 11 and 12 hybridised with seq. 16 probe @ 300
fmol/.mu.l, 30 fmol/.mu.l, 3 fmol/.mu.l
[0521] A probe diluted in 30 .mu.l hybridisation buffer
(5.times.SSC, 7% sodium lauryl sarcosine) was pipetted along the
length of each slide, covered with a coverslip, and placed into a
plastic box on top of a plastic insert, which was lying on a paper
towel wetted with water. Boxes were covered with aluminium foil to
keep light out, and incubated at +4.degree. C. overnight.
[0522] Slide Washes: Coverslips were removed and the slides
inserted in racks (6 slides per rack) which were placed in glass
slide dishes, wrapped in foil:
8 Slide Number Wash buffer (4.degree. C.) Wash time Probe sequence
1, 2, 3 5 .times. SSC, 0.1% Tween-20 2 .times. 5 min Seq. No. aZ1
4, 5, 6 5 .times. SSC, 0.1% Tween-20 2 .times. 5 min Seq. No. aZ2
7, 8, 9 5 .times. SSC, 0.1% Tween-20 2 .times. 5 min Seq. No. aZ3
10, 11, 12 5 .times. SSC, 0.1% Tween-20 2 .times. 5 min Seq. No.
16
[0523] After washing, slides were blow-dried and scanned. The
fluorescence was imaged on a slide scanner and the data analysed
from ImageQuant software (Molecular Dynamics). As shown in FIG. 11,
no binding of the Cy3 labelled probes is observed to the mismatched
oligo 3 with either the unmodified probe (slide 10-12), single LNA
modified probe aZ1 (slide 1-3) single LNA modified probe aZ2 (slide
4-6) or triple LNA modified probe aZ3 (slide 7-9) (i.e. the
obtained signal with the mismatched oligo 3 is comparable to the
background signal). With complementary oligo 6, specific signals
are observed in all cases. The intensity of these signals clearly
correlates with the number of LNAs present in the probes and with
the concentration of the probes. Each LNA T residue approximately
increased the signal strength by about a factor of 2 over that of
the normal DNA oligo probe, i.e. aZ1 and aZ2=2.times. signal of
sequence 16, and aZ3=8.times. signal of sequence 16. The
match/mismatch discrimination is good with the LNA T base
replacements, and with the increased signal strength, the mismatch
discriminations appear to be easier.
EXAMPLE 151
[0524] Hybridisation and Detection of End Mismatches on an Array
with LNA Modified Cy3-labelled 8 Mers.
[0525] Slide preparation: Glass slides were aminosilanised using a
10% solution of amino propyl triethoxy silane in acetone followed
by washing in acetone. The following oligonucleotides were spotted
out at 1 pmol/.mu.l onto the slides:
9 5'-GTGTGGAG-3' Seq No.9 5'-GTGTGGAA-3' Seq No.15 5'-GTGTGGAT-3'
Seq No.131 5'-GTGTGGAC-3' Seq No.132 5'-ATGTGGAA-3' Seq No.133
5'-CTGTGGAA-3' Seq No.134 5'-TTGTGGAA-3' Seq No.135
[0526] Ten repeat spots, approximately 1 nl each spot, were
performed for each oligonucleotide from each of 6 pens on each of
12 slides.
[0527] Probes (LNA Monomers in Bold):
10 DNA Probe No.1: 5'-Cy3-TTCCACAG-3' Probe No.2:
5'-Cy3-GTCCACAC-3' Probe No.3: 5'-Cy3-ATCCACAC-3' Probe No.4:
5'-Cy3-CTCCACAC-3' Probe No.5: 5'-Cy3-TTCCACAT-3' Probe No.6:
5'-Cy3-TTCCACAG-3' LNA Probe No.35Z-1: 5'-Cy3-TTCCACAC-3' Probe
No.35Z-2: 5'-Cy3-GTCCACAC-3' Probe No.35Z-3: 5'-Cy3-ATCCACAC-3'
Probe No.35Z-4: 5'-Cy3-CTCCACAC-3' Probe No.35Z-5:
5'-Cy3-TTCCACAT-3' Probe No.35Z-6: 5'-Cy3-TTCCACAG-3'
[0528] Probes with LNA monomers are prefixed with 35Z--as part of
the sequence number. Specific LNA monomers are indicated in
itailcs/bold and are situated at the 3' and 5' ends of the LNA
oligos.
[0529] Slides and conditions for hybridisation: Each probe sequence
was hybridised on a separate slide, and all probe concentrations
were 1 fmol/.mu.l. Each probe was diluted in hybridisation buffer
(5.times.SSC, 7% sodium lauryl sarcosine), of which 30 .mu.l was
pipetted along the length of each slide, covered with a coverslip,
and placed into a plastic box on top of a plastic insert, which was
lying on a paper towel wetted with water. Boxes were covered with
aluminium foil to keep light out, and incubated at +4.degree. C.
overnight.
[0530] Slide Washes: Coverslips were removed and the slides
inserted in racks (8 slides per rack) which were placed in glass
slide dishes, wrapped in foil. All slides were washed in
5.times.SSC for 2.times.5 min at +4.degree. C. After washing,
slides were blow-dried and scanned. The fluorescence was imaged on
a slide scanner and the data analyzed from ImageQuant software
(Molecular Dynamics).
[0531] Conclusions: As shown in FIGS. 12 and 13, probes containing
LNA nucleosides at their 5' and 3' ends are in the majority of
cases significantly better in discriminating between matched and
mismatched target sequences than their corresponding unmodified
oligonucleotides.
[0532] For DNA oligos, C=T mismatches were the most difficult to
distinguish, for example, where probe sequence 1 hybridised to
target sequence 132 and where probe sequence 5 hybridised to target
sequence 134. Other mismatches were visible such as T=T and G=T
mismatches, but these spots were less intense, for example where
probe sequences 5 and 6 respectively hybridised to target sequence
135. The LNA oligos, significantly reduced these C=T and T=T
mismatch spot intensites, to comparable levels to other mismatches.
The relative spot intensities of probe sequences 1, 2 and 3 were
similar for the DNA and LNA oligos. However, with probe sequences
4, 5 and 6, the LNA oligos gave a significantly increased spot
intensity when hybridised to their match target sequences 9, 133
and 134 respectively.
EXAMPLE 152
[0533] Hybridization and Detection of End Mismatches on an Array
with AT and All LNA Modified Cy3-labelled 8 Mers.
[0534] Slide preparation: Glass slides were aminosilanized using a
10% solution of amino propyl triethoxy silane in acetone followed
by washing in acetone. The following oligonucleotides were spotted
out at 1 pmol/.mu.l onto the slides:
11 5'-GTGTGGAG-3' Seq No.9 5'-GTGTGGAA-3' Seq No.15 5'-GTGTGGAT-3'
Seq No.131 5'-GTGTGGAC-3' Seq No.132 5'-ATGTGGAA-3' Seq No.133
5'-CTGTGGAA-3' Seq No.134 5'-TTGTGGAA-3' Seq No.135
[0535] Ten repeat spots, approximately 1 nl each spot, were
performed for each oligonucleotide from each of 6 pens on each of
36 slides.
12 Probes: (LNA monomers in bold): DNA: Probe No.1:
5'-Cy3-TTCCACAC-3' Probe No.2: 5'-Cy3-GTCCACAC-3' Probe No.3:
5'-Cy3-ATCCACAC-3' Probe No.4: 5'-Cy3-CTCCACAC-3' Probe No.5:
5'-Cy3-TTCCACAT-3' Probe No.6: 5'-Cy3-TTCCACAG-3' AT LNA: Probe
No.ATZ-1: 5'-Cy3-TTCCACAC-3' Probe No.ATZ-2: 5'-Cy3-GTCCACAC-3'
Probe No.ATZ-3: 5'-Cy3-ATCCACAC-3' Probe No.ATZ-4:
5'-Cy3-CTCCACAC-3' Probe No.ATZ-5: 5'-Cy3-TTCCACAT-3' Probe
No.ATZ-6: 5'-Cy3-TTCCACAG-3' All LNA: Probe No.AIIZ-1:
5'-Cy3-TTCCACAC-3' Probe No.AIIZ-2: 5'-Cy3-GTCCACAC-3' Probe
No.AIIZ-3: 5'-Cy3-ATCCACAC-3' Probe No.AIIZ-4: 5'-Cy3-CTCCACAC-3'
Probe No.AIIZ-5: 5'-Cy3-TTCCACAT-3' Probe No.AIIZ-6:
5'-Cy3-TTCCACAG-3'
[0536] Probes with LNA monomers are prefixed with ATZ- or AIIZ- as
part of the sequence number. Specific LNA monomers are indicated in
italics for the LNA oligos.
[0537] Slides and conditions for hybridization: Each probe sequence
was hybridized on a separate slide, and all probe concentrations
were 1 fmol/.mu.l. Each probe was diluted in hybridization buffer
(5.times.SSC, 7% sodium lauryl sarcosine), of which 30 .mu.l was
pipetted along the length of each slide, covered with a coverslip,
and placed into a plastic box on top of a plastic insert, which was
lying on a paper towel wetted with water. Boxes were covered with
aluminium foil to keep light out, and incubated at room temperature
overnight.
[0538] Slide Washes: Coverslips were removed and the slides
inserted in racks (9 slides per rack) which were placed in glass
slide dishes, wrapped in foil. All slides were washed in
5.times.SSC for 2.times.5 minutes at RT. After washing, slides were
blow-dried and scanned. The fluorescence was imaged on a slide
scanner and the data analyzed from ImageGuant software (Molecular
Dynamics).
[0539] Conclusion: As shown in FIGS. 15A, 15B and 15C, The average
intensity of DNA hybridization at room temperature was about 10% of
the intensity achieved with the AT or all LNA modified oligos. No
spots were seen on slides hybridized with DNA probes 5 and 6. These
conditions were therefore not optimal for the DNA probes. However,
the match/mismatch discrimination is very good with the LNA
nucleoside replacements at the A and T bases. The stringency for
the all LNA oligos may not be great enough as the match/mismatch
discrimination was not as good as for the AT LNA oligos.
[0540] The oligos with LNA modifications worked very well, and the
mismatches that were the most difficult to discriminate were;
[0541] Probe 1 to target 135=CT mismatch
[0542] Probe 2 to target 131=GT mismatch
[0543] Probe 3 to target 15=AA mismatch
[0544] Probe 4 to target 131=CT mismatch
[0545] Probe 5 to target 135=TT mismatch
[0546] Probe 6 to target 135=GT mismatch
[0547] Probe 6 to target 133=GA mismatch
[0548] The AT LNA oligos gave good discrimination where these
mismatch spot intensities were typically at the most 50% of the
intensity of the match spots. For these mismatches, the all LNA
oligos gave mismatch spot intensities about 50 to 70% of the match
spot intensities. Overall, LNA modifications allows the use of
higher temperatures for hybridizations and washes, and end
mismatches can be discriminated. These results are at least as good
as those from DNA probes hybridised at 4.degree. C. (see example
151).
EXAMPLE 153
[0549] Use of [.alpha..sup.33P] ddNTP's and ThermoSequenase.TM. DNA
Polymerase to Sequence DNA Templates Containing LNA T Monomers.
[0550] Radiolabelled terminator sequencing reactions were set up in
order to test the ability of the LNA T monomer to be accepted as a
template for DNA polymerases. The 15 mer primer (sequence: 5'-TGC
ATG TGC TGG AGA -3') was used to prime the following short
oligonucleotide sequences (LNA monomer in bold):
13 Template 1 3'-ACG TAC ACG ACC TCT ACC TTG CTA -5' Template TZ1
3'-ACG TAC ACG ACC TCT ACC TTG CTA -5'
[0551] The following reaction mixes were made:
[0552] Template 1 Mix:
[0553] 2 .mu.l.times.16 ThermoSequenase Buffer
[0554] 6 .mu.l Primer 2 pmole/.mu.l
[0555] 6 .mu.l Template 1 1 pmole/.mu.l
[0556] 4 .mu.l Water
[0557] 2 .mu.l ThermoSequenase DNA Polymerase (4 U/.mu.l)
[0558] 20 .mu.l Total volume
[0559] Template TZ1 Mix
[0560] 2 .mu.l.times.16 ThermoSequenase Buffer
[0561] 6 .mu.l Primer 2 pmole/.mu.l
[0562] 6 .mu.l Template TZ1 1 pmole/.mu.l
[0563] 4 .mu.l Water
[0564] 2 .mu.l ThermoSequenase DNA Polymerase (4 U/.mu.l)
[0565] 20 .mu.l Total volume
[0566] 2.mu.l Nucleotide mix (7.5 .mu.M each dNTP) was added to
each of 8 Eppendorf tubes. 0.5 .mu.l [.alpha..sup.33P] ddATP was
added to tubes 1 and 5. 0.5 .mu.l [.alpha..sup.33P] ddCTP was added
to tubes 2 and 6. 0.5 .mu.l [.alpha..sup.33P] ddGTP was added to
Tubes 3 and 7. 0.5 .mu.l [.alpha..sup.33 P] ddTTP was added to
tubes 4 and 8. 4.5 .mu.l of Template 1 mix was added to each of
tubes 1-4. 4.5 .mu.l of Template TZ1 mix was added to each of tubes
5-8. All the reactions were incubated at 60.degree. C. for 3 min.
The reactions were stopped by the addition of 4 .mu.l
formamide/EDTA stop solution. Reactions were heated at 95.degree.
C. for 3 min before loading onto a 19% polyacrylamide 7M urea gel.
The gel was fixed in 10% acetic acid 10% methanol before
transferring to 3 MM paper and drying. The dried gel was exposed to
Kodak Biomax autoradiography film.
[0567] The results are depicted in FIG. 18 (track 1-4) and FIG. 19
(5-8). The tracks correspond to the following reactions: (FIG. 18):
Lane 1--ddATP track. Lane 2--ddCTP track. Lane 3--ddGTP track, Lane
4--ddTTP track. Lane 5--8-32 base oligo markers; FIG. 19: Lane
A--8-32 base oligo markers. Lane 5--ddATP track. Lane 6--ddCTP
track. Lane 7--ddGTP track. Lane 8--ddTTP track.
[0568] As is evident from FIGS. 18 and 19, the full sequence of
both templates can easily be read from the autorad. The sequence is
5'-TGG AAC GTA-3' which corresponds to the template sequence 3'-ACC
TTG CTA-5'. This shows that a single LNA T monomer can act as a
template for DNA polymerases. The LNA T monomer is specifically
copied as "T" with ddATP being incorporated.
[0569] Therapeutic Applications
EXAMPLE 154
[0570] LNA Modified Oligos can be Transferred into Cells.
Experiment with Radiolabelled LNA Oligos.
[0571] 10 pmol of a oligodeoxynucleotide (ODN) (ODN#10: 5'-TTA ACG
TAG GTG CTG GAC TTG TCG CTG TTG TAC TT-3', a 35-mer complementary
to human Cathepsin D) and 10 pmoles of two LNA oligos: AL16
(5'-d(TGT GTG AAA TTG TTA T)-3', LNA nucleosides in bold) and AL17
(5'-d(ATA AAG TGT AAA G)-3', LNA nucleosides in bold) were mixed
with T4 polynucleotide Kinase (10 units, BRL cat. no. 510-8004SA),
5 .mu.l gamma-.sup.32P-ATP 5000 Ci/mmol, 10 uCi/.mu.l (Amersham) in
kinase buffer (50 mM Tris/HCl pH 7,6, 10 mM MgCl.sub.2, 5 mM DTT,
0.1 mM EDTA). The samples were incubated for 45 min at 37.degree.
C. and afterwards heated to 68.degree. C. for 10 min, and then
moved to +0.degree. C. Unincorporated nucleotides were removed by
passage over Chroma Spin TE-10 columns (Clontech cat. no. K1320-1).
The yields were 5.times.10.sup.5 cpm/.mu.l, 2.times.10.sup.5
cpm/.mu.l and 0.8.times.10.sup.5 cpm/.mu.l for ODN#10, AL16 and
AL17, respectively. MCF-7 human breast cancer cells originally
obtained from the Human Cell Culture Bank (Mason Research
Institute, Rockville) were cultured in DME/F12 culture medium (1:1)
supplemented with 1% heat inactivated fetal calf serum (Gibco BRL),
6 ng/ml bovine insulin (Novo) and 2.5 mM glutamax (Life
Technologies) in 25 cm.sup.2 cell culture flasks (Nunclon, NUNC)
and incubated in a humified incubator at 37.degree. C., 5%CO.sub.2,
20%O.sub.2, 75%N.sub.2. The MCF-7 cells were approximately 40%
confluent at the time of the experiment. A small amount (less than
0.1 pmol) of the kinased oligos were mixed with 1.5 .mu.g pEGFP-NI
plasmid (Clontech cat. no. 60851) and mixed with 100 .mu.l diluted
FuGENE6 transfection agent (Boehringer Mannheim cat no. 1 814 443),
dilution: 5 .mu.l FuGENE6 in 95 .mu.l DME/F12 culture medium
without serum. The FuGENE6/DNA/oligo-mixture were added directly to
the culture medium (5 ml) of adherent growing MCF-7 cells and
incubated with the cells for 18 hours, closely following the
manufacturers directions. Three types of experiments were set up.
1) ODN#10+pEGFP-NI; 2) AL16+PEGFP-NI; 3) AL17+pEGFP-NI. Cellular
uptake of DNA/LNA material were studied by removing
FuGENE6/DNA/oligo-mixture containing medium (an aliquot was
transferred to a scintillator vial). Cells were rinced once with
phosphate buffered saline (PBS), fresh culture medium was added and
cells inspected by fluorescence microscopy. Approximately 30% of
the transfected cells contained green fluorescent material,
indicating that approximately 30% of the cells have taken up the
pEGFP-NI plasmid and expressed the green fluorescent protein coded
by this plasmid. Following fluorescence microscopy the adherent
MCF-7 cells were removed from the culture flasks. Briefly, the
culture medium was removed, then cells were rinsed with a solution
of 0.25% trypsin (Gibco BRL) 1 mM EDTA in PBS (without Mg.sup.2+
and Ca.sup.2+), 1 ml trypsin/EDTA was added and cells were
incubated 10 min at 37.degree. C. During the incubation the cells
loosened and were easily resuspended and transferred to
scintillator vials. The cells were then completely dissolved by
addition of 10 ml Optifluor scintillation coctail (Packard cat. no.
6013199), and the vials were counted in a Wallac 1409 scintillation
counter. The results were as follows: 1) ODN#10+pEGFP-NI:
approximately 1.4% of the added radioactivity were associated with
cellular material; 2) AL16+pEGFP-NI: approximately 0.8% of the
added radioactivity were associated with cellular material; and 3)
AL17+pEGFP-NI: approximately 0.4% of the added radioactivity were
associated with cellular material. We conclude that 0.4-0.8% of the
added LNA oligos were taken up by the cells.
EXAMPLE 155
[0572] LNA is Efficiently Delivered to Living Human MCF-7 Breast
Cancer Cells.
[0573] To increase the efficiency of LNA-uptake by human MCF-7
cells different transfection agents were tested with various
concentrations of 5'FITC-labelled LNAs and DNA. The
oligonucleotides described in the table below were tested.
14TABLE: Oligonucleotides tested Sequence (LNA monomers Character-
Name in bold) istics AL16 5'-TGT GTG AAA TTG TTA T-3' LNA, enzym.
FITC-labeled AL17 5'-ATA AAG TGT AAA G-3' LNA, enzym. FITC-labeled
EQ3009-01 5'-TGC CTG CAG GTC GAC T-3' LNA-FITC-labeled EQ3008-01
5'-TGC CTG CAG GTC GAC T-3' DNA-FITC-labeled
[0574] AL16 and AL17 were enzymatically labelled with FITC as
described in Example 128. EQ3009-01 and EQ3008-01 were labelled
with FITC by standard solid phase chemistry. Three transfection
agents were tested: FuGENE-6 (Boehringer Mannheim cat. no. 1 814
443), SuperFect (Quiagen cat. no. 301305) and Lipofectin (GibcoBRL
cat. no. 18292-011). Human MCF-7 breast cancer cells were cultured
as described previously (Example 154). Three days before the
experiments the cells were seeded at a cell density of approx.
0.8.times.10.sup.4 cells per cm.sup.2. Depending on the type of
experiment the MCF-7 cells were seeded in standard T25 flasks
(Nunc, LifeTechnologies cat. no. 163371A), 24 wells multidish
(Nunc, LifeTechnologies cat. no. 143982A) or slide flasks (Nunc,
LifeTechnologies cat. no. 170920A). The experiments were performed
when cells were 30-40% confluent. Cellular uptake of LNA and DNA
was studied at serum-free conditions, i.e. the normal serum
containing DME/F12 medium was removed and replaced with DME/F12
without serum before the transfection-mixture was added to the
cells. Under these conditions SuperFect proved to be toxic to the
MCF-7 cells. Transfection mixtures consisting of SuperFect and
either plasmid DNA (pEGFP-N1, Clontech cat. no. 6085-1), oligo DNA
or oligo LNA was equally toxic to MCF-7 cells. In contrast to
SuperFect, FuGene6 and Lipofectin worked well with plasmid DNA
(pEGFP-N1). However, only lipofectin was capable of efficient
delivery of oligonucleotides to living MCF-7. Briefly, efficient
delivery of FITC-labelled LNA and DNA to MCF-7 cells was obtained
by culturing the cells in DME/F12 with 1% FCS to approx. 40%
confluence. The Lipofectin reagent was then diluted 40.times. in
DME/F12 medium without serum and combined with the oligo to a
concentration of 750 nM oligo. The oligo-Lipofectin complex was
allowed to form for 15 min at r.t., and further diluted with
serum-free medium to at final concentration of 250 nM oligo, 0.8
ug/ml Lipofectin. Then, the medium was removed from the cells and
replaced with the medium containing oligo-Lipofectin complex. The
cells were incubated at 37.degree. C. for 6 hours, rinsed once with
DME/F12 medium without serum and incubated for a further 18 hours
in DME/F12 with 1% FCS at 37.degree. C. The result of the
experiment was evaluated either directly on living cells in culture
flasks or in 24 wells multidishes or on cells cultured in slide
flasks and fixed in 4%, ice-cold PFA. In all cases a Leica DMRB
fluorescence microscope equipped with a high resolution CCD camera
was used. The result with living cells is shown in FIG. 16 and the
result with fixed cells cultured in slide flask is shown in FIG.
17. Both the cells in FIGS. 16 and 17 was transfected with the
FITC-labelled AL16 LNA molecule. By counting total number of cells
and green fluorescent cells in several fields we observe that
FITC-labelled AL16 LNA was transfected into approximately 35% of
the MCF-7 cells. Importantly, we saw that the LNA predominantly was
localised in the nuclei of the cells (FIG. 17). This is noteworthy,
since nuclear uptake of fluorescent oligos correlates with their
antisense activity (Stein C. A. et al. (1997) Making sense of
antisense: A debate. In HMS Beagle: A BioMedNet Publication
(http://hmsbeagle.com/06/cutedge/overwiev- .htm)). Increasing the
amount of oligo and lipofectin up to a final concentration of 1250
nM oligo and 4 ug/ml lipofectin only increased the percentage of
green fluorescent cells marginally. Increasing the concentration
even further was toxic for the cells. Similar results were obtained
with the other LNAs and the FITC-labelled oligo DNA (see the table
above). We conclude that: 1) LNA can be efficiently delivered to
living MCF-7 breast cancer cells by Lipofectin-mediated
iransfection. 2) A consistent high fraction, 30% or more of cells,
is transfected using ai final concentration of 250 nM LNA, 0,8 ug
Lipofectin pr. ml growth medium without serum. Increasing the
concentrations of LNA and Lipofectin up to 5 times only increased
the transfection yield marginally. 3) The procedure transfected the
LNA into the nuclei of the cells, which according to literature is
a good indication that such transfected LNAs may exhibit antisense
effects on cells.
EXAMPLE 156
[0575] LNA Modified Oligos can be Transferred into Cells.
Experiment with Fluorescein Labelled LNA Oligos.
[0576] Two LNA oligos: AL16 (5'-TGT GTG AAA TTG TTA T-3', LNA
nucleosides in bold) and AL17 (5'-ATA AAG TGT AAA G-3', LNA
nucleosides in bold) were labeled with fluorescein as described in
Example 128. MCF-7 human breast cancer cells were cultured as
described in Example 154. Three types of experiments were set up.
1) approximately 1.5 .mu.g FITC-labelled AL16; 2) approximately 1.5
.mu.g FITC-labelled AL17; and 3) approximately 0.75 .mu.g
FITC-labelled AL16 and 0.75 .mu.g pRSV.beta.gal plasmid (a plasmid
expressing the bacterial lac Z gene coded enzyme
.beta.-galactosidase, Tulchinsky et. al. (1992) PNAS, 89, 9146-50).
The two LNA oligos and the LNA-plasmid mix were mixed with FuGENE6
and added to MCF-7 cells as described in Example 154. After
incubation for 18 hours cellular uptake of the LNA oligos were
assessed by fluorescence microscopy of the cell cultures. A part of
the treated cells contained green fluorescent material (see FIG.
16), indicating that cells take up the fluorescein labelled LNA.
The fluorescein labelled AL16 appeared superior to fluorescein
labelled AL17 in this respect. After fluorescence microscopy the
culture medium were removed from the cells treated both with
fluorescein labelled AL16 and pRSVpgal. The cells were washed once
with PBS, fixed in 2% (v/v) formaldehyde, 0.2% (v/v) glutaraldehyde
at 4.degree. C. for 5 min and .beta.-galactosidase containing cells
were stained blue with X-gal (5-bromo-4-chloro-3-indoyl
.beta.-D-galactopyranosid) which turns from colorless to blue in
the presence of .beta.-galactosidase activity. The X-gal staining
showed that the pRSV.beta.gal effectively had been transferred into
cells. We conclude that the fluorescein LNA oligos where taken up
by the cells.
EXAMPLE 157
[0577] LNA Modified Oligos are Relatively Stable Under Cell Culture
Conditions.
[0578] Following fluorescence microscopy as described in Example
156 cells treated only with the fluorescein labelled AL16 LNA were
allowed to incubate for an additional 3 days. During this period of
time the number of green fluorescent cells appeared unaltered. We
conclude that fluorescein labelled LNA oligos has a good stability
under the conditions prevailing in cell culture.
EXAMPLE 158
[0579] Blockade by Antisense Locked Nucleic Acids (LNA) of
[D-Ala2]Deltorphin-Induced Antinociception in the Warm Water Tail
Flick Test in Conscious Rats.
[0580] Male Sprague-Dawley rats (300 g) were implanted with an
intrathecal (i.th). polyethylene catheter and allowed to recover
for at least 5 days before start of injections (including
controls). The antisense LNA compounds (12.5 and 2.5 .mu.g per
injection) were administered in a 5 .mu.l volume twice-daily (08.00
and 17.00 h) for 3 days. No signs of non-specific effects or
toxicity could be detected, as shown by observations of locomotor
behavior and measurements of body weight. The day after the last
injection the rats were injected with [D-Ala2]deltorphin (60 .mu.g,
i.th) and tested in the warm water (52.degree. C.) tail flick test
for .delta. opioid receptor-mediated antinociception. Data are
presented in FIG. 14 as medians based on 6-8 animals per group
(data converted to percent maximum possible response, % MFPE).
Statistical analyses were performed by means of Kruskal-Wallis
1-way ANOVA by ranks, followed by comparisons of treatments versus
control. As shown in FIG. 14, deltorphin produced a robust
antinociceptive effect in saline-treated controls. This response
was statistically significantly suppressed in both antisense LNA
groups (12.5 and 2.5 .mu.g) as compared with saline-treated
controls.
[0581] LNA Solid Supports
EXAMPLE 159
[0582] General Method for DMT-LNA Nucleoside Succinates.
[0583] Base protected DMT-LNA nucleoside and succinic anhydride
(1.5 equivalents) were taken in anhydrous ethylene dichloride
(.about.10 ml/g of nucleoside). To the mixture, triethylamine (2
equivalents) was added and the mixture was stirred at room
temperature. Reaction was followed by HPLC (conditions same as for
tritylation). After complete reaction (>95%), reaction mixture
was concentrated, coevaporated with ethylene dichloride and
acetonitrile, and dried in vacuo to remove triethylamine. Residue
was dissolved in ethylene dichloride or ethyl acetate (.about.100
ml/g of starting nucleoside), washed with cold 10% citric acid
(3.times.80 ml/g) and cold water (3.times.80 ml/g). Organic layer
was dried over anhydrous sodium sulfate, filtered and concentrated
with or without addition of 1-2 equivalents of triethylamine.
Residual solid was coevaporated with anhydrous acetonitrile
(2-3.times.) and dried in vacuo to give pure product as white
solid. General method for LNA nucleoside supports. Base protected
DMT-LNA-nucleoside succinate (free acid or triethylammonium salt,
65 micromol/g of support), amino derivatised support (Primer
Support.TM. 30HL, 160 micromol amino groups/g of support), DMAP (3
mg/g of support) and 1-(3-[dimethylamino]propyl)-3-ethy-
lcarbodimide hydrochloride (80 mg/g of support) were taken in
anhydrous pyridine (6 ml/g of support). To this mixture,
triethylamine (16 microliter/g of support) was added and the
mixture was kept on a shaker at 150 rpm overnight. Support was
filtered, washed with methanol (3.times.10 ml/g of support) and
dichloromethane (3.times.10 ml/g of support). After air drying,
support was dried in vacuo for 0.5 h. To this 6% DMAP in anhydrous
acetonitrile (Cap A, .about.3 ml/g of support) and a mixture of 20%
acetic anhydride/30% 2,4,6-collidine/50% acetonitrile (Cap B,
.about.3 ml/g of support) were added. The micture was kept on
shaker for 5 h. Support was filtered, washed with anhydrous
dichloromethane (2.times.10 ml/g of support) and dried as above. It
was resuspended in a mixture of Cap A and Cap B (total vol. 6 ml/g
of support) and kept on shaker overnight. Support was filtered,
washed with methanol (6.times.10 ml/g of support), dichloromethane
(3.times.10 ml/g of support) and dried in air. It was further dried
in vacuo for 5-6 h. Loading was determined by dimethoxytrityl assay
and was found to be approx. 40 .mu.mol/g.
EXAMPLE 160
[0584] First Strand cDNA Synthesis Using Poly dT Primers Containing
LNA T Monomers.
[0585] Reactions were set up in order to test the ability of poly
dT primers containing LNA T residues to prime 1.sup.st strand cDNA
synthesis. The following poly dT primers were tested (LNA monomers
are in bold):
15 RTZ1 5'-TTT TTT TTT TTT TT-3' RTZ2 5'-TTT TTT TTT TTT TT-3' RTZ3
5'-TTT TTT TTT TTT TT-3' RTZ4 5'-TTT TTT TTT TTT TT-3' RTZ5 5'-TTT
TTT TTT T-3'
[0586] Anchored poly dT primer from RPK0140 kit Cy Dye cDNA
labelling kit (Amersham Pharmacia Biotech) was as a control.
[0587] Reactions were set up as follows for each of the primers
above:
16 1 .mu.l Arabidopsis mRNA 0.5 .mu.g/.mu.l 2 .mu.l poly dT primer
8 pmoles/.mu.l 4 .mu.l x5 AMV Reverse Transcriptase buffer 1 .mu.l
Water 8 .mu.l Total volume
[0588] This mix was then heated to 75.degree. C. for 3 min and then
allowed to cool at room temperature for at least 10 min.
[0589] The following was then added to each of the reactions:
17 1 .mu.l 80 mM Sodium Pyrophosphate 1 .mu.l Human Placental
Ribonuclease Inhibitor 20 U/.mu.l 7 .mu.l 0.5 mM dNTP solution 2
.mu.l [.alpha..sup.33P] dATP 10 mCi/ml 3000 Ci/mmole 1 .mu.l AMV
Reverse Transcriptase 20 U/.mu.l 20 .mu.l Total volume
[0590] The reactions were incubated at 42.degree. C. for 2 hours.
The reactions were then heated at 95.degree. C. for 3 min before
loading onto a 6% polyacrylamide 7M urea gel. The gel was fixed in
10% acetic acid/10% methanol before transferring to 3 MM paper and
drying. The dried gel was exposed to Kodak Biomax autoradiography
film overnight.
[0591] The autoradiograph clearly showed that the LNA containing
oligonucleotide primers RTZ1-4 were able to efficiently prime cDNA
synthesis. The amount and intensity of the cDNA products produced
in these reactions was equal to that produced with the anchored
poly dT control oligonucleotide. RTZ 5 did produce some cDNA, but
the yield was significantly lower than that produced with the
control oligo primer.
EXAMPLE 161
[0592] LNA-modified Oligonuclotides Covalently Attached to Separose
Beads Function Efficiently in the Sequence Specific Capture of RNA
Molecules.
[0593] Three oligos were synthesised by chemistry (Amy Mueller) for
evaluation in poly (rA) binding.
18 NH.sub.2(T8)-T Control NH.sub.2(T15)-T Control
NH.sub.2(LNA-T8)-T Test
[0594] 200 nmol of each oligo were coupled to 50 mg of prepared
CNBr-activated Separose 4B (Pharmacia) per booklet instructions.
Unreacted binding sites on the resin were blocked in 100 nM Tris pH
8.0.
19 Table of Oligo Binding Data T9 oligo T16 oligos LNA T9 oligo No
oligo Steps A.sub.260 units A.sub.260 units A.sub.260 units Control
Oligo reacted 14.7 26.0 14.7 0 (200 nM) (200 nM) (200 nM) Unbound
oligo 5.50 10.43 4.20 -- .thrfore. Bound oligo 9.20 15.57 10.50 --
% Bound 62.6% 59.9% 71.4% --
[0595] Oligo bound resins were divided into two portions (.about.25
mg resin each) for poly (rA) binding analysis in duplicate. Poly
(rA) Pharmacia #27-4110-01 (dissolved at 28.2 A.sub.260 units/ml in
binding buffer) was used for binding. Five (5) A.sub.260 units were
bound to duplicate 25 mg portions of each oligo bound resin per SOP
QC 5543. Unbound "breakthrough" poly (rA) was quantitated by
A.sub.260 absorbance and used to calculate bound. The fate of the
bound poly (rA) was tracked through Low salt buffer wash and
several elutions. As shown in Table 10 both the LNA and DNA coated
beads function efficiently in the capture of poly (rA) target
molecules. The LNA coated beads, however, bind the poly (rA) target
much more tightly than the DNA coated beads as evidenced by the
poly (rA) elution profiles of the different beads. We conclude that
1) an LNA T9 oligo is efficient in the capture of RNA molecules
containing a strech of A residues and that 2) the captured RNA
molecules are bound much more tightly to the LNA T9 oligo beads
than to the control DNA T9 and DNA T16 oligo.
20TABLE 1 MONOMER Z T.sub.m T.sub.m (.degree. C.) T.sub.m (.degree.
C.) T.sub.m (.degree. C.) Oligo Target No. Na.sub.2HPO.sub.4/EDTA
Na.sub.2HPO.sub.4/NaCl/EDTA Na.sub.2HPO.sub.4/TMAC
5'-d(GTGTTATGC)-3' 5'-d(GCATATCAC)-3' 1 28 42 5'-d(GCATTTCAC)-3' 2
12 31 5'-d(GCATGTCAC)-3' 3 19 23 5'-d(GCATCTCAC)-3' 4 11 30
5'-d(GCATAACAC)-3' 5 12 5'-d(GCATAGCAC)-3' 6 <10
5'-d(GCATACCAC)-3' 7 <10 5'-(GCAUAUCAC)-3' 8 28
5'-(GCAUCUCAC)-3' 9 10 5'-d(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 10 44
56 5'-d(GCATTTCAC)-3' 11 27 43 5'-d(GCATGTCAC)-3' 12 30 43
5'-d(GCATCTCAC)-3' 13 23 38 5'-d(GCATAACAC)-3' 14 28
5'-d(GCATAGCAC)-3' 15 28 5'-d(GCATACCAC)-3' 15A 29
5'-(GCAUAUCAC)-3' 16 50 5'-(GCAUCUCAC)-3' 17 33 5'-d(GTGAGATGC)-3'
5'-d(GCATATCAC)-3' 18 26 39 5'-d(GCATTTCAC)-3' 19 33 44
5'-d(GCATGTCAC)-3' 20 28 38 5'-d(GCATCTCAC)-3' 21 49 57
5'-d(GCATAACAC)-3' 22 <15 5'-d(GCATAGCAC)-3' 23 <15
5'-d(GCATACCAC)-3' 24 <15 5'-(GCAUAUCAC)-3' 24A 34
5'-(GCAUCUCAC)-3' 24B 59 5'-d(GTGAUATGC)-3' 5'-d(GCATATCAC)-3' 25
44 56 5'-d(GCATTTCAC)-3' 26 25 44 5'-d(GCATGTCAC)-3' 27 32 43
5'-d(GCATCTCAC)-3' 28 24 37 5'-d(GCATAACAC)-3' 29 27
5'-d(GCATAGCAC)-3' 30 28 5'-d(GCATACCAC)-3' 31 20
5'-d(GTGAGATGC)-3' 5'-d(GCATATCAC)-3' 32 17 34 5'-d(GCATTTCAC)-3'
33 16 30 5'-d(GCATGTCAC)-3' 34 15 28 5'-d(GCATCTCAC)-3' 35 33 44
5'-d(GCATAACAC)-3' 36 9.0 5'-d(GCATAGCAC)-3' 37 <5
5'-d(GCATACCAC)-3' 38 <5 5'-(GCAUCUCAC)-3' 38A 33
5'-d(GGTGGTTTGTTTG)-3' 5'-d(CAAACAAACCACA)-3' 39 31 47 55
5'-(CAAACAAACCACA)-3' 39A 32 52 5'-d(GGTGGTTTGTTTG)-3'
5'-d(CAAACAAACCACA)-3' 40 40 57 67 5'-(CAAACAAACCACA)-3' 40A 50 70
d(GGTGGTTTGTTTG-3' 5'-d(CAAACAAACCACA)-3' 41 67 83 >90
5'-(CAAACAAACCACA)-3' 41A 85 >93 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 42 36 5'-(AAAAAAAAAAAAAA)-3' 43 32
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 44 36
5'-(AAAAAAAAAAAAAA)-3' 45 32 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 46 34 5'-(AAAAAAAAAAAAAA)-3' 47 40
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 48 42
5'-(AAAAAAAAAAAAAA)-3' 49 52 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 50 47 5'-(AAAAAAAAAAAAAA)-3' 51 53
5'-d(TTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 52 80
5'-(AAAAAAAAAAAAAA)-3' 53 70 5'-d(AAAACAAAA)-3' 54 63
5'-d(AAAAGAAAA)-3' 55 55 5'-d(AAAATAAAA)-3' 56 65
5'-d(GTGAAATGC)-3' 5'-d(GCATATCAC)-3' 57 26 5'-d(GCATTTCAC)-3' 58
45 5'-d(GCATGTCAC)-3' 59 23 5'-d(GCATCTCAC)-3' 60 25
5'-d(GTGA.sup.McCATGC)-3' 5'-d(GCATATCAC)3' 61 <15
5'-d(GTGA.sup.McCATGC)-3' 5'-d(GCATATCAC)-3' 63 32
5'-d(GCATTTCAC)-3' 64 27 5'-d(GCATGTCAC)-3' 65 53
5'-d(GCATCTCAC)-3' 66 32 5'-d(GTGACATGC)-3' 5'-d(GCATATCAC)-3' 67
32 5'-d(GCATGTCAC)-3' 69 52 5'-d(GTGATATG.sup.MeC)-3'
5'-d(GCATATCAC)-3' 71 64 5'-d(GCATGTCAC)-3' 73 52 5'-(GCAUAUCAC)-3'
75 74 5'-(GCAUCUCAC)-3' 76 60 5'-d(CACTATACG)-3' 77 40
5'-d(GTGTTTTGC)-3' 5'-d(GCAAAACAC)-3' 78 52
[0596]
21TABLE 2 Monomer V T.sub.m T.sub.m (.degree. C.) T.sub.m (.degree.
C.) T.sub.m (.degree. C.) Oligo Target No. Na.sub.2HPO.sub.4/EDTA
Na.sub.2HPO.sub.4/NaCl/EDTA Na.sub.2HPO.sub.4/TMAC
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 32
5'-(AAAAAAAAAAAAAA)-3' 27 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 31 5'-(AAAAAAAAAAAAAA)-3' 28
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 30
5'-(AAAAAAAAAAAAAA)-3' 23 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 23 5'-(AAAAAAAAAAAAAA)-3' 31
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 23
5'-(AAAAAAAAAAAAAA)-3' 16 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' <10 5'-(AAAAAAAAAAAAAA)-3' 42
5'-(AAAAAAGAAAAAAA)-3' 37 5'-d(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 26
5'-(GCAUAUCAC)-3' 27
[0597]
22TABLE 3 Monomer X T.sub.m T.sub.m (.degree. C.) T.sub.m (.degree.
C.) T.sub.m (.degree. C.) Oligo Target No. Na.sub.2HPO.sub.4/EDTA
Na.sub.2HPO.sub.4/NaCl/EDTA Na.sub.2HPO.sub.4/TMAC
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 23
5'-(AAAAAAAAAAAAAA)-3' 23 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 19 5'-(AAAAAAAAAAAAAA)-3' 23
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 9
5'-(AAAAAAAAAAAAAA)-3' 15 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 5 5'-(AAAAAAAAAAAAAA)-3' 14
[0598]
23TABLE 4 Monomer Y T.sub.m T.sub.m (.degree. C.) T.sub.m (.degree.
C.) T.sub.m (.degree. C.) Oligo Target No. Na.sub.2HPO.sub.4/EDTA
Na.sub.2HPO.sub.4/NaCl/EDTA Na.sub.2HPO.sub.4/TMAC
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 36
5'-(AAAAAAAAAAAAAA)-3' 37 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 35 5'-(AAAAAAAAAAAAAA)-3' 37
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA)-3' 35
5'-(AAAAAAAAAAAAAA)-3' 36 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 32 5'-(AAAAAAAAAAAAAA)-3' 33
5'-d(TTTTTTTTTTTTTT)-3' 5'-d(AAAAAAAAAAAAAA) -3' 36
5'-(AAAAAAAAAAAAAA)-3' 36 5'-d(TTTTTTTTTTTTTT)-3'
5'-d(AAAAAAAAAAAAAA)-3' 58 5'-(AAAAAAAAAAAAAA)-3' 58
5'-d(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 35 5'-(GCAUAUCAC)-3' 35
[0599]
24TABLE 5 Monomer Z Melting temperature (T.sub.m/.degree. C.) Oligo
Target No. Y.dbd.A Y.dbd.C Y.dbd.T Y.dbd.G 5'-r(GTGATATGC)-3'
5'-d(GCATYTCAC)-3' 1 55 34 38 37 5'-r(GUGAUAUGC)-3'
5'-d(GCATYTCAC)-3' 2 27 <10 <10 <10 5'-r(GTGATATGC)-3'
5'-r(GCAUYUCAC)-3' 3 63 45 -- -- 5'-r(GUGAUAUGC)-3'
5'-r(GCAUYUCAC)-3' 4 38 22 -- --
[0600]
25TABLE 6 Monomer Z T.sub.m Oligo Target No. Melting temperature
(T.sub.m/.degree. C.) 5'-d(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 1 28
5'-d(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 2 44 5'-d(GTGATATGC)-3'
5'-d(GCATATCAC)-3' 3 40 5'-d(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 4 63
5'-r(GTGATATGC)-3' 5'-d(GCATATCAC)-3' 5 74 5'-(GTGATATG.sup.MeC)-3'
5'-d(GCATATCAC)-3' 6 85
[0601]
26TABLE 7 Monomer Z (all-phosphoromonothioate oligonucleotides) Tm
Oligo Target No. Melting temperature (T.sub.m/.degree. C.)
5'-d(G.sup.ST.sup.SG.sup-
.SA.sup.ST.sup.SA.sup.ST.sup.SG.sup.SC)-3' 5'-d(GCATATCAC)-3' 1 21
5'-d(G.sup.ST.sup.SG.sup.SA.sup.ST.sup.SA.sup.ST.sup.SG.sup.SC)-3-
' 5'-r(GCAUAUCAC)-3' 2 17 5'-d(G.sup.ST.sup.SG.sup.SA.sup.-
ST.sup.SA.sup.ST.sup.SG.sup.SC)-3' 5'-r(GCATATCAC)-3' 3 41
5'-d(G.sup.ST.sup.SG.sup.SA.sup.ST.sup.SA.sup.ST.sup.SG.sup.SC)-3'
5'-r(GCAUAUCAC)-3' 4 47
[0602]
27TABLE 8 Monomer thio-Z (U.sup.S) Tm Oligo Target No. Melting
temperature (T.sub.m/.degree. C.) 5'-d(GTGAU.sup.SATGC)-3'
5'-d(GCATATCAC)-3' 1 34 5'-d(GTGAU.sup.SATGC)-3' 5'-(GCAUAUCAC)-3'
2 36 5'-d(GU.sup.SGAU.sup.SAU.sup.SGC)-3' 5'-d(GCATATCAC)-3' 3 42
5'-d(GU.sup.SGAU.sup.SAU.sup.SGC)-3' 5'-(GCAUAUCAC)-3' 4 52
5'-d(GTGTTTTGC)-3' 5'-(GCAAAACAC)-3' 5 27
5'-d(GU.sup.SGU.sup.SU.sup.SU.sup.SU.sup.SGC)-3' 5'-d(GCAAAACAC)-3'
6 51
[0603]
28TABLE 9 Monomers amino-Z (T.sup.NH) and methylamino-Z (T.sup.NMe)
Tm Oligo Target No. Melting temperature (T.sub.m/.degree. C.)
5'-d(GTGAT.sup.NHATGC)-3- ' 5'-d(GCATATCAC)-3' 1 33
5'-d(GTGAT.sup.NHATCG)-3+ 5'-(GCAUAUCAC)-3' 2 34
5'-d(GT.sup.NHGAT.sup.NHAT.sup.NHG- C)-3' 5'-d(GCATATCAC)-3' 3 39
5'-d(GT.sup.NHGAT.sup.NHAT.s- up.NHCG)-3' 5'-(GCAUAUCAC)-3' 4 47
5'-d(GTGAT.sup.NMeATGC)- -3' 5'-d(GCATATCAC)-3' 5 33
5'-d(GTGAT.sup.NMeATGC)-3' 5'-(GCAUAUCAC)-3' 6 36
5'-d(GT.sup.NMeGAT.sup.NMeAT.sup.N- MeGC)-3' 5'-d(GCATATCAC)-3' 7
39 5'-d(GT.sup.NMeGAT.sup.NM- eAT.sup.NMeGC)-3' 5'-(GCAUAUCAC)-3' 8
49 5'-d(GT.sup.NMeGT.sup.NHT.sup.NMeT.sup.NHT.sup.NMeGC)-3'
5'-d(GCAAAACAC)-3' 9 47 5'-d(GT.sup.NMeGT.sup.NHT.sup.NMe-
T.sup.NHT.sup.NMeGC)-3' 5'-(GCAAAACAC)-3' 10 63
[0604]
29TABLE 10 LNA T9 oligo T16 oligo T9 oligo No oligo Steps A.sub.260
units A.sub.260 units A.sub.260 units Control poly (rA) added
5.0/5.0 5.0/5.0 5.0/5.0 5.0/5.0 poly (rA) breakthrough 1.75/1.61
1.84/1.78 1.83/1.82 5.09/5.14 .thrfore. poly (rA) bound 3.25/3.39
3.16/3.22 3.17/3.18 0.0/0.0 % poly (rA) bound 65.0%/ 63.2%/ 63.4%/
0.0%/ 67.8% 64.4% 63.6% 0.0% Low Salt Wash/Elute 0.24/0.24
0.11/0.12 .053/.055 0.14/0.13 TE Elute 15 min RT 2.37/2.72
0.83/0.93 0.02/0.04 0.01/0.02 TE Elute O.N. RT 0.38/0.37 1.76/1.69
0.11/0.07 .003/.004 TE Elute 30 min 65.degree. C. .047/.040
0.38/0.46 1.62/1.70 .005/.004 10 mM Tris pH 10 Elute .002/.002
0.03/0.03 0.10/0.10 0.01/0.01 1 mM HCl pH 4.0 Elute 0.07/0.06
0.06/0.04 0.26/0.23 0.01/0.01 Ave. A.sub.260 Recovered 3.20 3.14
2.18 -- Ave. % A.sub.260 Recovered 96.4% 98.4% 68.7% --
* * * * *
References