U.S. patent application number 10/423440 was filed with the patent office on 2004-05-20 for preparation of phosphorothioate oligomers.
This patent application is currently assigned to McGill University. Invention is credited to Jin, Yi, Just, George, Marsault, Eric, Xin, Zhili.
Application Number | 20040097722 10/423440 |
Document ID | / |
Family ID | 24179289 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040097722 |
Kind Code |
A9 |
Just, George ; et
al. |
May 20, 2004 |
Preparation of phosphorothioate oligomers
Abstract
Methods and intermediates for the preparation of oligomers
containing diastereomerically enriched phosphorothioate linkages
are disclosed.
Inventors: |
Just, George; (Ile Cadieux,
CA) ; Xin, Zhili; (Montreal, CA) ; Marsault,
Eric; (Montreal, CA) ; Jin, Yi; (Montreal,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE - 46TH FLOOR
PHILADELPHIA
PA
19103
US
|
Assignee: |
McGill University
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0229219 A1 |
December 11, 2003 |
|
|
Family ID: |
24179289 |
Appl. No.: |
10/423440 |
Filed: |
April 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10423440 |
Apr 25, 2003 |
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09452604 |
Dec 1, 1999 |
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6596857 |
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09452604 |
Dec 1, 1999 |
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08951132 |
Oct 15, 1997 |
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6031092 |
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08951132 |
Oct 15, 1997 |
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08546198 |
Oct 20, 1995 |
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5734041 |
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Current U.S.
Class: |
536/25.34 |
Current CPC
Class: |
C07F 9/65846 20130101;
C07H 9/06 20130101; C07H 19/10 20130101; C07H 9/04 20130101; C07H
21/00 20130101; C07F 9/1411 20130101 |
Class at
Publication: |
536/025.34 |
International
Class: |
C07H 021/04 |
Claims
What is claimed is:
1. A method for synthesizing an oligomer having a phosphorothioate
linkage comprising the steps of: reacting a first synthon of
Formula I: 96wherein: Q is independently O or S; R.sup.1 is a
hydroxyl protecting group; R.sup.2 is a chiral auxiliary of formula
--C(R.sup.8)R.sup.3--C(R.- sup.16)R.sup.5--CHR.sup.6--NHR.sup.7;
R.sup.3 is hydrogen, alkyl, cyanomethyl, monohalomethyl,
dihalomethyl, trigalomethyl, --CH.sub.2Si(R.sup.4).sub.3, or
--CH.sub.2--SO.sub.kR.sup.4 where k is 0, 1 or 2; R.sup.4 is
independently alkyl, aryl, aralkyl or alkaryl having up to 15
carbon atoms; R.sup.5 is H, --CN, --Si(R.sup.4).sub.3,
SO.sub.kR.sup.4 or halogen; or R.sup.8 and R.sup.16 are each H, and
R.sup.3 and R.sup.5, together, form one of the structures 97
wherein: R.sup.10 and R.sup.11 are H, alkyl having from 1 to about
10 carbons, --CH.sub.2C(.dbd.O)OR.sup.22, --CH.sub.2CN,
--CH.sub.2Si(CH.sub.3).sub.3, or o- or p-C.sub.6H.sub.4--R.sup.21;
R.sup.21 is hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having from 1
to about 10 carbons, --NO.sub.2, or --N(R.sup.22).sub.2; R.sup.22
is independently H or alkyl having from one to about 10 carbon
atoms; p is 1 or 2; Z.sup.1 and Z.sup.2 are independently halogen,
CN, --Si(CH.sub.3).sub.3, and --C(.dbd.O)OR.sup.22; R.sup.30 is
hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having from 1 to about 10
carbons, or --O--Si(R.sup.4).sub.3; R.sup.6 is H, alkyl or aralkyl
having up to 15 carbon atoms; or R.sup.5 and R.sup.6, together with
the atoms to which they are attached, form a 5 or 6 membered ring;
R.sup.7 is alkyl or aralkyl having up to 15 carbon atoms; or
R.sup.6 and R.sup.7, together, form one of the structures 98wherein
V, T, and Z are independently CH or N; R.sup.8 is H or methyl;
R.sup.16 is H, alkyl or aralkyl having up to 15 carbon atoms; B a
is a nucleobase; and n is an integer from 0 to 50; with a second
synthon of Formula II: 99 wherein: R.sup.9 is a hydroxyl protecting
group or a linker connected to a solid support; and m is an integer
from 0 to 50; for a time and under reaction conditions effective to
form a third synthon of Formula III: 100and contacting said third
synthon with a sulfurizing agent to form an oligomer of Formula IV:
101wherein D is said phosphorothioate linkage having the formula:
102
2. The method of claim 1 wherein said phosphorothioate linkage is
diastereomerically enriched.
3. The method of claim 1 wherein about 75% of said phosphorothioate
linkage is in a single stereoisomeric form.
4. The method of claim 1 wherein about 85% of said phosphorothioate
linkage is in a single stereoisomeric form.
5. The method of claim 1 wherein about 95% of said phosphorothioate
linkage is in a single stereoisomeric form.
6. The method of claim 1 wherein said phosphorothioate linkage is
in a single stereoisomeric form, substantially free of other
stereoisomeric forms.
7. The method of claim 1 wherein n is 0.
8. The method of claim 1 wherein said first synhon is in a single
stereoisomeric form, substantially free of other stereoisomeric
forms.
9. The method of claim 1 further comprising removing said R.sup.1
group from said phosphorothioate.
10. The method of claim 1 further comprising removing said chiral
auxiliaries.
11. The method of claim 1 wherein said compound of Formula IV
contains a plurality of said phosphorothioate linkages.
12. The method of claim 1 wherein said first and second synthons
are reacted at a temperature of from about -20.degree. C. to about
40.degree. C.
13. The method of claim 1 wherein said first and second synthons
are reacted at a temperature of from about -15.degree. C. to about
0.degree. C.
14. The method of claim 1 wherein said first synthon is formed by
reacting a compound of Formula V: 103with an azaphospholane of
Formula VIa: 104wherein R.sup.3-R.sup.8 are as defined above; and X
is halogen, dialkylamino, inidazols, triazole or substituted
phenoxy wherein said substituents are electron withdrawing.
15. The method of claim 14 wherein said electron withdrawing
substituents are halogen or nitro.
16. The method of claim 14 wherein said azaphospholane is produced
by reacting a reagent of formula
HO--C(R.sup.8)R.sup.3--C(R.sup.16)R.sup.5--- CHR.sup.6--NHR.sup.7
and a phosphorus trihalide, phosphorus tri(dialkylamide),
phosphorus triphenoxide, or phosphorus tri(imidazolides).
17. The method of claim 1 wherein said first synthon is formed by
reacting a compound of Formula VII: 105and a .gamma.-amino alcohol
of formula
HO--C(R.sup.8)R.sup.3--C(R.sup.16)R.sup.5--CHR.sup.6--NHR.sup.7;
wherein X and R.sup.1-R.sup.8 and R.sup.16 are as defined
above.
18. The method of claim 17 wherein X is chlorine.
19. The method of claim 17 wherein said reaction is
stereoselective.
20. The method of claim 18 wherein said first synthon is in a
single stereoisomeric form, substantially free of other
stereoisomeric forms.
21. The method of claim 1 wherein'said reaction is performed in the
presence of a catalyst.
22. The method of claim 21 wherein said catalyst has one of the
Formulas VIII or IX: 106wherein: R.sup.12 and R.sup.13 are
independently hydrogen, halogen, cyano, nitro, alkyl having from
one to 10 carbons, substituted alkyl having from one to 10 carbons,
and ester group, or R.sup.12 and R.sup.13 together with the carbon
atoms to which they are attached, form a substituted or
unsubstituted phenyl ring where said substituents are electron
withdrawing; and R.sup.14 is hydrogen, halogen, cyano, nitro, thio,
alkyl having from one to 10 carbons, substituted alkyl having from
one to 10 carbons, norbornyl, substituted norbornyl, aryl,
substituted aryl wherein said substituents are electron
withdrawing, or has the formula: 107wherein L is protecting
group.
23. The method of claim 21 wherein R.sup.14 is halogen or nitro,
and wherein R.sup.12 and R.sup.13 are each halogen or each
cyano.
24. The method of claim 22 wherein R.sup.14 is bromine, and
R.sup.12 and R.sup.13 are each cyano.
25. The method of claim 22 wherein R.sup.14, R.sup.12 and R.sup.13
are each bromo.
26. The method of claim 21 wherein R.sup.14 has one of the
formulas: 108wherein R.sup.15 is H, methyl, trialkylsilyl or
acetyl.
27. The method of claim 1 wherein R.sup.3 is cyanomethyl or
--CH.sub.2--SO.sub.kR.sup.4 where k is 0, 1 or 2.
28. The method of claim 27 wherein R.sup.7 is lower alkyl or
aralkyl.
29. The method of claim 1 wherein said first synthon has one of the
Formulas Xa, XIa, XIIa, XIIIa or XXa: 109wherein W has the formula:
110and R.sup.1-R.sup.16, V, T and Z are as defined above.
30. The method of claim 29 wherein said first synthon has the
Formula Xb or Xc: 111
31. The method of claim 29 where;n said first synthon has the
Formula XIVa: 112wherein p, Z.sup.1 and Z.sup.2 are as defined
above.
32. The method of claim 31 wherein said first synthon has the
Formula XVa or XVIa: 113wherein Y, R.sup.7, Z.sup.1, Z.sup.2, and p
are as defined above.
33. The method of claim 29 wherein said first synthon has the
Formula XVIIa or XVIIIa: 114wherein W, R.sup.7, R.sup.10 and
R.sup.11 are as defined above.
34. The method of claim 1 further comprising removing said R.sup.9
groups.
35. The method of claim 1 further comprising removing said R.sup.2
groups.
36. The oligomer produced by the method of claim 1.
37. An azaphospholane of Formula VIb: 115wherein: y is X or W
wherein X is halogen, dialkylamino, imidazole, triazole or
substituted phenoxy wherein said substituents are electron
withdrawing, and W has the formula: 116wherein: Q is independently
O or S; R.sup.1 is a hydroxyl Protecting group; R.sup.2 is a chiral
auxiliary of formula
--C(R.sup.8)R.sup.3--C(R.sup.16)R.sup.5--CHR.sup.6--NHR.sup.7;
R.sup.3 is hydrogen, alkyl, cyanomethyl, monohalomethyl,
dihalomethyl, trihalomethyl, --CH.sub.2Si(R.sup.4).sub.3, or
--CH.sub.2--SO.sub.kR.sup.- 4 where k is 0, 1 or 2; R.sup.4 is
independently alkyl, aryl, aralkyl or alkaryl having up to 15
carbon atoms; R.sup.5 is H, --CN, --Si(R.sup.4).sub.3,
SO.sub.kR.sup.4 or halogen; or R.sup.8 and R.sup.16 are each H, and
R.sup.3 and R.sup.5, together, form one of the structures 117
wherein: R.sup.10 and R.sup.11 are H, alkyl having from 1 to about
10 carbons, --CH.sub.2C(.dbd.O)OR.sup.22, --CH.sub.2CN,
--CH.sub.2Si(CH.sub.3).sub.3, or o- or p-C.sub.6H.sub.4--R.sup.21;
R.sup.21 is hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having from 1
to about 10 carbons, --NO.sub.2, or --N(R.sup.22).sub.2; R.sup.22
is independently H or alkyl having from one to about 10 carbon
atoms; p is 1 or 2; Z.sup.1 and Z.sup.2 are independently halogen,
CN, --Si(CH.sub.3).sub.3, and --C(.dbd.O)OR.sup.22; R.sup.30 is
hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having from 1 to about 10
carbons, or --O--Si(R.sub.4).sub.3; R.sup.6 is H, alkyl or aralkyl
having up to 15 carbon atoms; or R.sup.5 and R.sup.6, together with
the atoms to which they are attached, form a 5 or 6 membered ring;
R.sup.7 is alkyl or aralkyl having up to 15 carbon atoms; or
R.sup.6 and R.sup.7, together, form one of the structures
118wherein X, T, and Z are independently CH or NH; or Z and
R.sup.5, together with the atoms to which they are attached, form a
phenyl ring; R.sup.8 is H or methyl; R.sup.9 is a hydroxyl
protecting group or a solid support; R.sup.16 is H, alkyl or
aralkyl having up to 15 carbon atoms; B is a nucleobase; and n is
an integer from 0 to 50.
38. The azaphospholane of claim 37 wherein Y is W and n is 0.
39. The azaphospholane of claim 37 wherein Y is halogen.
40. The azaphospholane of claim 37 wherein Y is chlorine.
41. The azaphospholane of claim 37 wherein R.sup.7 is alkyl.
42. The azaphospholane of claim 37 wherein R.sup.7 R.sup.6 and the
atoms to which they are attached form a 5 or 6 membered ring.
43. The azaphospholane of claim 38 wherein R.sup.3 is cyanomethyl
or --CH.sub.2--SO.sub.kR.sup.6 where k is 0, 1 or 2.
44. The azaphospholane of claim 37 having one of the Formulas XVIc
or XVId: 119
45. The azaphospholane of claim 36 having one of the Formulas Xb,
XIb, XIIb, XIIIb or XXb: 120wherein R.sup.3-R.sup.16, Y, V, T, Z,
Z.sub.1, Z.sub.2 and p are as defined above.
46. The azaphosphalane of claim 45 having the Formula XIVb:
121wherein Y, R.sup.6, R.sup.7, R.sup.8, p, Z.sup.1 and Z.sup.2 are
as defined above.
47. The azaphosphalane of claim 44 having the Formula Xd or Xe:
122wherein Y, R.sup.3, R.sup.5, R.sup.8, and R.sup.16 are as
defined above.
48. The azaphospholane of claim 45 having the Formula XVIIb or
XVIIIb. 123wherein Y, R.sup.7, R.sup.10 and R.sup.11 are as defined
above.
49. The azaphospholane of claim 45 having the Formula XVb or XVIb:
124wherein Y, R.sup.7, Z.sup.1, Z.sup.2, and p are as defined
above.
50. The azaphospholane of claim 37 wherein said azaphospholane is
diastereomerically enriched.
51. The azaphospholane of claim 37 wherein 75% of said
azaphospholane is in a single stereoisomeric form.
52. The azaphospholane of claim 37 wherein 85% of said
azaphospholane is in a single stereoisomeric form.
53. The azaphospholane of claim 37 wherein 95% of said
azaphospholane is in a single stereoisomeric form.
54. The azaphospholane of claim 37 wherein said azaphospholane is
in a single stereoisomeric form, substantially free of other
stereoisomeric forms.
55. An oligomeric compound comprising a moiety having a phosphite
linkage of the Formula XXX: 125wherein: R.sup.3 is hydrogen, alkyl,
cyanomethyl, monohalomethyl, dihalomethyl, trihalomethyl,
--CH.sub.2Si(R.sup.4).sub.3, or --CH.sub.2--SO.sub.kR.sup.4 where k
is 0, 1 or 2; R.sup.4 is independently alkyl, aryl, aralkyl or
alkaryl having up to 15 carbon atoms; R.sup.5 is H, --CN,
--Si(R.sup.4).sub.3, SO.sub.kR.sup.4 or halogen; or R.sup.8 and
R.sup.16 are each H, and R.sup.3 and R.sup.3, together, form one of
the structures 126 wherein: R.sup.10 and R.sup.11 are H, alkyl
having from 1 to about 10 carbons, --CH.sub.2C(.dbd.O)OR.sup- .22,
--CH.sub.2CN, --CH.sub.2Si(CH.sub.3).sub.3, or o- or
p-C.sub.6H.sub.4--R.sup.21; R.sup.21 is hydrogen,
--O--C(.dbd.O)CH.sub.3, alkoxy having from 1 to about 10 carbons,
--NO.sub.2, or --N(R.sup.22).sub.2; R.sup.22 is independently H or
alkyl having from one to about 10 carbon atoms; p is 1 or 2;
Z.sup.1 and Z.sup.2 are independently halogen, CN,
--Si(CH.sub.3).sub.3, and --C(.dbd.O)OR.sup.22; R.sup.30 is
hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having from 1 to about 10
carbons, or --O--Si(R.sub.4).sub.3; R.sup.6 is H, alkyl or aralkyl
having up to 15 carbon atoms; or R.sup.5 and R.sup.6, together with
the atoms to which they are attached, form a 5 or 6 membered ring;
R' is alkyl or aralkyl having up to 15 carbon atoms; or R.sup.6 and
R.sup.7, together, form one of the structures 127wherein X, T, and
Z are independently CH or NH; or Z and R.sup.5, together with the
atoms to which they are attached, form a phenyl ring; R.sup.8 is H
or methyl; R.sup.9 is a hydroxyl protecting group or a solid
support; R.sup.16 is H, alkyl or aralkyl having up to 15 carbon
atoms; B is a nucleobase; and n is an integer from 0 to 50.
56. The oligomeric compound of claim 55 wherein 75% of said
phosphosphite linkage is in a single stereoisomeric form.
57. The oligomeric compound of claim 55 wherein 85% of said
phosphosphite linkage is in a single stereoisomeric form.
58. The oligomeric compound of claim 55 wherein 95% of said
phosphosphite linkage is in a single stereoisomeric form.
59. The oligomeric compound of claim 55 wherein said phosphosphite
linkage is in a single stereoisomeric form, substantially free of
other stereoisomeric forms.
Description
FIELD OF THE INVENTION
[0001] This invention relates to methods for the preparation of
diastereomerically enriched phosphorothioate linked
oligonucleotides, and to intermediates useful in their preparation.
This invention also relates to sequence-specific phosphorothioate
oligonucleotides having chiral phosphorus linkages and to a novel
chemical synthesis of these and other oligonucleotides.
BACKGROUND OF THE INVENTION
[0002] It is well known that most of the bodily states in
multicellular organisms, including most disease states, are
effected by proteins. Such proteins, either acting directly or
through their enzymatic or other functions, contribute in major
proportion to many diseases and regulatory functions in animals and
man. Classical therapeutics has generally focused upon interactions
with such proteins in efforts to moderate their disease-causing or
disease-potentiating functions. In newer therapeutic approaches,
modulation of the actual production of such proteins is desired. By
interfering with the production of proteins, the maximum
therapeutic effect might be obtained with minimal side effects. It
is the general object of such therapeutic approaches to interfere
with or otherwise modulate gene expression which would lead to
undesired protein formation.
[0003] One method for inhibiting specific gene expression is with
the use of oligonucleotides. Oligonucleotides complementary to a
specific target messenger RNA (mRNA) sequence are used. Several
oligonucleotides are currently undergoing clinical trials for such
use.
[0004] Transcription factors interact with double-stranded DNA
during regulation of transcription. Oligonucleotides can serve as
competitive inhibitors of transcription factors to modulate the
action of transcription factors. Several recent reports describe
such interactions (see, Bielinska, et. al., Science 1990, 250,
997-1000; and Wu, et al., Gene 1990, 89, 203-209.)
[0005] Oligonucleotides also have found use in diagnostic tests.
Such diagnostic tests can be performed using biological fluids,
tissues, intact cells or isolated cellular components. As with the
above gene expression inhibition, diagnostic use can take advantage
of an oligonucleotide's ability to hybridize with a complementary
strand of nucleic acid. Hybridization is the sequence specific
hydrogen bonding of oligonucleotides via Watson-Crick and/or
Hoogsteen base pairs to RNA or DNA. The bases of such base pairs
are said to be complementary to one anothar.
[0006] Oligonucleotides are also widely used as research reagents.
They are useful for understanding the function of many other
biological molecules as well as in the preparation of such other
biological molecules. One particular use, the use of
oligonucleotides as primers in the reactions associated with
polymerase chain reaction (PCR), has been the cornerstone for the
establishment of an ever expanding commercial business. The use of
such PCR reactions has seemingly "exploded" as more and more use of
this very important biological tool is made. The uses of PCR have
extended into many areas in addition to those contemplated by its
Nobel laureate inventor. Examples of such new areas include
forensics, paleontology, evolutionary studies and genetic
counseling to name just a few. Primers are needed for each of these
uses. Oligonucleotides, both natural and synthetic, serve as the
primers.
[0007] Oligonucleotides also are used in other laboratory
procedures. A number of these uses are described in common
laboratory manuals such as Molecular Cloning, A Laboratory Manual,
Second Ed., J. Sambrook, et al., Eds., Cold Spring Harbor
Laboratory Press, 1989; and Current Protocols In Molecular Biology,
F. M. Ausubel, et. al., Eds., Current Publications, 1993. Such uses
include Synthetic oligonucleotide Probes, Screening Expression
Libraries with Antibodies and Oligonucleotides, DNA Sequencing, In
Vitro Amplification of DNA by the Polymerase Chain Reaction and
Site-directed Mutagenesis of Cloned DNA from Book, 2 of Molecular
Cloning, A Laboratory Manual, ibid. and DNA-Protein Interactions
and The Polymerase Chain Reaction from Vol. 2 of Current Protocols
In Molecular Biology, ibid.
[0008] To supply the users of oligonucleotides, many scientific
journals now contain advertisements for either oligonucleotide
precursors or for custom-synthesized oligonucleotides. This has
become an important commercial use of oligonucleotides.
Oligonucleotides can be synthesized to have properties that are
tailored for the desired use. Thus, a number of chemical
modifications have been introduced into oligonucleotides to
increase their usefulness in diagnostics, as research reagents, and
as therapeutic entities. These modifications are designed, for
example, to increase binding to a target nucleic acid strand, to
assist in identification of the oligonucleotide or an
oligonucleotide-target complex, to increase cell penetration, to
provide stability against nucleases and other enzymes that degrade
or interfere with the structure or activity of the
oligonucleotides, to provide a mode of disruption (terminating
event) once sequence-specifically bound to a target, or to improve
the pharmacokinetic properties of the oligonucleotides.
[0009] Since they exist as diastereomers, phosphorothioate,
methylphosphqnate, phosphotriester, phosphoramidate and other
phosphorus oligonucleotides synthesized using known, automated
techniques result in mixtures of Rp and Sp diastereomers at the
individual phosphorothioate, methylphosphonate, phosphotriester,
phosphoramidate or other phosphorus linkages. Thus, a 15-mer
oligonucleotide containing 14 asymmetric linkages has 2.sup.14,
i.e. 16,384, possible stereoisomers. It is possible that oligomers
having diastereomerically enriched linkages could possess
advantages in hybridizing to a target mRNA or DNA. Accordingly,
there is a need for such oligomers.
[0010] Miller, P. S., McParland, K. B., Jayaraman, K., and Ts'o, P.
O. P (1981), Biochemistry, 20:1874, found that small di-, tri- and
tetramethylphosphonate and phosphotriester oligonucleotides
hybridize to unmodified strands with greater affinity than natural
phosphodiester oligonucleotides. Similar increased hybridization
was noted for small phosphotriester and phosphoramidate
oligonucleotides; Koole, L. H., van Genderen, M. H. P., Reiners, R.
G., and Buck, H. M. (1987), Proc. K. Ned. Adad. Wet., 90:41;
Letsinger, R. L., Bach, S. A., and Eadie, J. S. (1986), Nucleic
Acids Res., 14:3487; and Jager, A., Levy, M. J., and Hecht, S. M.
(1988), Biochemistry, 27:7237. The effects of the diastereomers of
undefined stereochemistry on hybridization becomes even more
complex as chain length increases.
[0011] Bryant, F. R. and Benkovic, S. J. (1979), Biochemistry,
18:2825 studied the effects of diesterase on the diastereomers of
ATP. Published patent application PCT/US88/03634 discloses dimers
and trimers of 2',5'-linked diastereomeric adenosine units.
Niewiarowski, W., Lesnikowski, Z. J., Wilk, A., Guga, P., Okruszek,
A., Uznanski, B., and Stec, W. (1987), Acta Biochimica Polonia,
34:217, synthesized dimers of thymidine having high diastereomeric
excess, as did Fujii, M., Ozaki, K., Sekine, M., and Hata, T.
(1987), Tetrahedron, 43:3395.
[0012] Stec, W. J., Zon, G., and Uznanski, B. (1985), J.
Chromatography, 326:263, have reported the synthesis of certain
mixtures of phosphorothioates or methyphosphonate oligonucleotides
and have separated them by chromatography. However, they were only
able to separate the diastereomers of certain small oligomers
having a limited number of diastereomerically pure phosphorus
linkages.
[0013] In a preliminary report, J. W. Stec, Oligonucleotides as
antisense inhibitors of gene expression: Therapeutic implications,
meeting abstracts, Jun. 18-21, 1989, noted that a
non-sequence-specific thymidine homopolymer octamer--i.e. a
(dT).sub.8-mer, having "all-except-one" Rp configuration
methylphosphonate linkages--formed a thermodynamically more stable
hybrid with a 15-mer deoxyadenosine homopolymer--i.e. a
d(A).sub.15-mer--than did a similar thymidine homopolymer having
"all-except-one" Sp configuration methylphosphonate linkages. The
hybrid between the "all-except-one" Rp (dT).sub.8-mer and the
d(A).sub.15-mer had a Tm of 38.degree. C. while the Tm of the
"all-except-one" Sp (dT).sub.8-mer and the d(A).sub.15-mer was
<0.degree. C. The hybrid between a (dT).sub.8-mer having natural
phosphodiester linkages, i.e. octathymidylic acid, and the
d(A).sub.15-mer was reported to have a Tm of 14.degree. C. The
"all-except-one" thymidine homopolymer octamers were formed from
two thymidine methylphosphonate tetrameric units with high
diastereomeric excess linked by a natural phosphodiester
linkage.
[0014] Six or more nucleotides units are generally necessary for an
oligonucleotide to be of optimal use in applications involving
hybridization. It is often preferred to have even more nucleoside
units for best performance, often as many as 10 to 30. Because it
has not been possible to stereochemically resolve more than two or
three adjacent phosphorus linkages, the offsets of induced
chirality in the phosphnrus linkages of chemically synthesized
oligonucleotides has not been well assessed heretofore. This is
because with few limited exceptions, the sequence-specific
phosphorothioate, methylphosphonate, phosphotriester or
phosphoramidate oligonucleotides obtained utilizing known automated
synthetic techniques have been mixtures with no diastereomeric
excess.
[0015] Some aspects of the use of enzymatic methods to synthesize
oligonucleotides having chiral phosphorus linkages have been
investigated. Burgers, P. M. J. and Eckstein, F. (1979), J.
Biological Chemistry, 254:6889; and Gupta, A., DeBrosse, C., and
Benkovic, S. J. (1982) J. Bio. Chem., 256:7689 enzymatically
synthesized diastereomerically pure polydeoxy-adenylic acid having
phosphorothioate linkages. Brody, R. S. and Frey, P. S. (1981),
Biochemistry, 20:1245; Eckstein, F. and Jovin, T. M. (1983),
Biochemistry, 2:4546; Brody, R. S., Adler, S., Modrich, P., Stec,
W. J., Leznikowski, Z. J., and Frey, P. A. (1982) Biochemistry, 21:
2570-2572; and Romaniuk, P. J. and Eckstein, F. (1982) J. Biol.
Chem., 257:7684-7688 all enzymatically synthesized poly TpA and
poly ApT phosphorothioates while Burgers, P. M. J. and Eckstein, F.
(1978) Proc. Natl. Acad. Sci. USA, 75: 4798-4800 enzymatically
synthesized poly UpA phosphorothioates. Cruse, W. B. T., Salisbury,
T., Brown, T., Cosstick, R. Eckstein, F., and Kennard, O. (1986),
J. Mol. Biol., 192:891, linked three diastereomeric Rp GpC
phosphorothioate diners via natural phosphodiester bonds into a
hexamer. Most recently Ueda, T., Tohda, H., Chikazuni, N.,
Eckstein, R., and Watanabe, K. (1991) Nucleic Acids Research,
19:547, enzymatically synthesized RNA's having from several hundred
to ten thousand nucleotides incorporating Rp linkages of high
diastereomeric excess. Enzymatic synthesis, however, is
disadvantageous in that it depends on suitable polymerases that may
or may not be available, especially for modified nucleoside
precursors.
[0016] As reviewed by W. J. Stec and A. Wiek (1994), Angew. Chem.
Int. Ed. English 33:709, the oxathiaphospholane method has been
successful for the preparation of phosphorothioates with defined
stereochemistry. However, it suffers from disadvantages, such as
the non-trivial preparation of diastereomerically pure
oxathiaphospholane, and the difficulty in synthesizing and
isolating satisfactorily pure oligomers longer than 12-mers.
[0017] It would therefore be of great advantage to provide
oligonucleotides having phosphorus linkages with controlled
stereochemistry.
OBJECTS OF THE INVENTION
[0018] It is one object of this invention to provide
sequence-specific oligonucleotides having chirally pure
phosphorothioate linkages with high diastereomeric excess.
[0019] Another object is to provide phosphorus-linked
oligonucleotides having substantially all Rp or all Sp
linkages.
[0020] A further object is to provide research and diagnostic
materials for assaying bodily states in animals, especially
diseased states.
[0021] It is yet another object to provide new methods for
synthesizing sequence-specific oligonucleotides having chirally
pure phosphorothioate linkages, and useful intermediates
therefor.
SUMMARY OF THE INVENTION
[0022] The present invention provides stereoselective methods for
preparing sequence-specific oligonucleotides having chiral
phosphorus linkages. In certain preferred embodiments, these
methods comprise the steps of:
[0023] reacting a first synthon of Formula I: 1
[0024] wherein:
[0025] Q is independently O or S;
[0026] R.sup.1 is a hydroxyl protecting group;
[0027] R.sup.2 is a chiral auxiliary of formula --C
(R.sup.8)R.sup.3--C(R.sup.16)R.sup.5--CHR.sup.6--NHR.sup.7;
[0028] R.sup.3 is hydrogen, alkyl, cyanomethyl, monohalomethyl,
dihalomethyl, trihalomethyl, --CH.sub.2Si(R.sup.4).sub.3, or
--CH.sub.2--SO.sub.kR.sup.4 where k is 0, 1 or 2;
[0029] R.sup.4 is independently alkyl, aryl, aralkyl or alkaryl
having up to 15 carbon atoms;
[0030] R.sup.5 is H, --CN, --Si(R.sup.4).sub.3, SO.sub.kR.sup.4 or
halogen;
[0031] or R.sup.8 and R.sup.16 are each H, and R.sup.3 and R.sup.5,
together, form one of the structures: 2
[0032] wherein:
[0033] R.sup.10 and R.sup.11 are H, alkyl having from 1 to about 10
carbons, --CH.sub.2C(.dbd.O)OR.sup.22, --CH.sub.2CN,
--CH.sub.2Si(CH.sub.3).sub.3, or o- or
p-C.sub.6H.sub.4--R.sup.21;
[0034] R.sup.21 is hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having
from 1 to about 10 carbons, --NO.sub.2, or --N(R.sup.22).sub.Z;
[0035] R.sup.22 is independently H or alkyl having from one to
about 10 carbon atoms;
[0036] p is 1 or 2;
[0037] Z.sup.1 and Z.sup.2 are independently halogen, --CN,
--Si(CH.sub.3).sub.3, and --C(.dbd.O)OR.sup.22;
[0038] R.sup.30 is hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having
from 1 to about 10 carbons, or --O--Si(R.sub.4).sub.3;
[0039] R.sup.6 is H, alkyl or aralkyl having up to 15 carbon
atoms;
[0040] or R.sup.5 and R.sup.6, together with the atoms to which
they are attached, form a 5 or 6 membered ring;
[0041] R.sup.7 is alkyl or aralkyl having up to 15 carbon
atoms;
[0042] or R.sup.6 and R.sup.7, together, form one of the structures
3
[0043] wherein V, T, and Z are independently CH or N;
[0044] R.sup.8 is H or methyl;
[0045] R.sup.16 is H, alkyl or aralkyl having up to 15 carbon
atoms;
[0046] B is a nucleobase; and
[0047] n is an integer from 0 to 50;
[0048] with a second synthon of Formula II: 4
[0049] wherein:
[0050] R.sup.9
[0051] is a hydroxyl protecting group or a linker connected to a
solid support; and
[0052] m is an integer from 0 to 50;
[0053] for a time and under reaction conditions effective to form a
third synthon of Formula III: 5
[0054] and
[0055] contacting said third synthon with a sulfurizing agent to
form an oligomer of Formula IV: 6
[0056] wherein D is said phosphorothioate linkage having the
formula: 7
[0057] In preferred embodiments, said phosphorothioate linkage is
diastereomerically enriched. In other preferred embodiments about
75% of the phosphorothioate linkage is in a single stereoisomeric
form. In further preferred embodiments about 85% of the
phosphorothioate linkage is in a single stereoisomeric form. In
especially preferred embodiments about 95% of the phosphorothioate
linkage is in a single stereoisomeric form. Most preferably, the
phosphorothioate linkage is in a single stereoisomeric form,
substantially free of other stereoisomeric forms. Preferably, the
first synthon is in a single stereoisomeric form, substantially
free of other stereoisomeric forms.
[0058] In some preferred embodiments n is 0. In further preferred
embodiments, RL groups are subsequently removed to yield new second
synthons for iterative synthesis, and chiral auxiliaries are
removed after iterative synthesis is completed. In preferred
embodiments of the present methods the oligomer of Formula IV
contains a plurality of phosphorothioate linkages.
[0059] Preferably, first and second synthons are reacted at a
temperature of from about -20.degree. C. to about 40.degree. C.,
with from about -15.degree. C. to about 0.degree. C. being more
preferred.
[0060] In some preferred embodiments the first synthon is formed by
reacting a compound of Formula V: 8
[0061] with an azaphospholane of Formula VIa: 9
[0062] wherein R.sup.3-R.sup.8 are as defined above; and X is
halogen, dialkylamino, imidazole, triazole or substituted phenoxy
wherein said substituents are electron withdrawing, preferably
halogen or nitro.
[0063] In some embodiments the azaphospholane described above is
produced by reacting a reagent of formula
HO--C(R.sup.8)R.sup.3--C(R.sup.16)R.sup.- 5--CHR.sup.6--NHR.sup.7
and a phosphorus trihalide, phosphorus tri(dialkylamide),
phosphorus triphenoxide or phosphorus triimidazolide.
[0064] In more preferred embodiments the first synthon is formed by
reacting a compound of Formula VII: 10
[0065] and a .gamma.-amino alcohol of formula
HO--C(R.sup.8)R.sup.3--C(R.s- up.16)R.sup.5--CHR.sup.6--NHR.sup.7.
Preferably, X is chlorine, dialkylamino or diphenoxy, and said
reaction is stereoselective. It is especially preferred that the
first synthon is in a single stereoisomeric form, substantially
free of other stereoisomeric forms.
[0066] In some preferred embodiments the reaction of first and
second synthons is performed in the presence of a catalyst, said
catalyst preferably having one of the Formulas VIII or IX: 11
[0067] wherein:
[0068] R.sup.12 and R.sup.13 are independently hydrogen, halogen,
cyano, nitro, alkyl having from one to 10 carbons, substituted
alkyl having from one to 10 carbons, an ester group, or R.sup.12
and R.sup.13 together with the carbon atoms to which they are
attached form a substituted or unsubstituted phenyl ring where said
substituents are electron withdrawing; and
[0069] R.sup.14 is hydrogen, halogen, cyano, nitro, thio, alkyl
having from one to 10 carbons, substituted alkyl having from one to
10 carbons, norbornyl, substituted norbornyl, aryl, substituted
aryl wherein said substituents are electron withdrawing, or has the
formula: 12
[0070] wherein L is protecting group.
[0071] In some preferred embodiments R.sup.14 is halogen or nitro,
preferably bromine, and R.sup.12 and R.sup.13 are each halogen or
each cyano, with cyano being especially preferred.
[0072] Other preferred embodiments R.sup.14 has one of the
formulas: 13
[0073] wherein R.sup.15 is H, methyl, trialkylsilyl or acetyl.
[0074] In some preferred embodiments of the method R.sup.3 is
cyanomethyl or --CH.sub.2--SO.sub.kR.sup.4 where k is 0, 1 or 2,
and R.sup.7 is lower alkyl or aralkyl.
[0075] In further preferred embodiments said first synthon has one
of the Formulas Xa, XIa, XIIa, XIIIa or XXa: 14
[0076] wherein W has the formula: 15
[0077] and R.sup.1-R.sup.16, V, T and Z are as defined above.
[0078] Other preferred first synthons have the Formula Xb or Xc:
16
[0079] More preferred first synthons have the Formula XVIIa or
XVIIIa: 17
[0080] Particularly preferred first synthons have the Formula XIVa:
18
[0081] Especially preferred first synthons have the Formula XVa or
XVIa: 19
[0082] In preferred embodiments of the methods of the invention
R.sup.1 groups are removed from the oligomers, thus creating new
second synthons for further iterative synthesis.
[0083] Also provided according to the invention are
phosphorothioate oligomers produced by the method of claim 1, and
azaphospholanes having Formula VIb: 20
[0084] In preferred embodiments of the invention, 75% of said
azaphospholanes having Formula VIb are in a single stereoisomeric
form, with 85% being more preferred, and 95% being particualrly
preferred. In especially preferred embodiments, the azaphospholanes
having Formula VIb are in a single stereoisomeric form,
substantially free of other stereoisomeric forms.
[0085] In preferred embodiments the azaphospholane has one of the
Formulas Xb, XIb, XIIb, XIIIb, Xd, Xe or XXb: 21
[0086] In other preferred embodiments the azaphospholane has the
Formula XVIIb or XVIIIb: 22
[0087] In some particularly preferred embodiments the
azaphosphalane has the Formula XIVb: 23
[0088] Especially preferred embodiments the azaphospholane has the
Formula XVb or XVIb: 24
[0089] Also provided in accordance with the invention are
oligomeric compounds comprising a phosphite linkage having the
Formula XXX: 25
[0090] In preferred embodiments of the invention, 75% of said
phosphosphite linkage is in a single stereoisomeric form, with 85%
being more preferred, and 95% being particualrly preferred. In
especially preferred embodiments, the phosphosphite linkage is in a
single stereoisomeric form, substantially free of other
stereoisomeric forms.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0091] The present invention is directed to methods for the
synthesis of phosphorothioate compounds having diastereomerically
enriched phosphorothioate linkages, and to intermediates useful in
their preparation.
[0092] In one aspect, the invention provides methods for the
preparation of phosphorothioate linkages comprising the steps of
reacting a first synthon of Formula I: 26
[0093] wherein:
[0094] Q is independently O or S;
[0095] R.sup.1 is a hydroxyl protecting group;
[0096] R.sup.2 is a chiral auxiliary of formula
--C(R.sup.8)R.sup.3--C(R.s-
up.16)R.sup.5--CHR.sup.6--NHR.sup.7;
[0097] R.sup.3 is hydrogen, alkyl, cyanomethyl, monohalomethyl,
dihalomethyl, trihalomethyl, --CH.sub.2Si(R.sup.4).sub.3, or
--CH.sub.2--SO.sub.kR.sup.4 where k is 0, 1 or 2;
[0098] R.sup.4 is independently alkyl, aryl, aralkyl or alkaryl
having up to 15 carbon atoms;
[0099] R.sup.5 is H, --CN, --Si(R.sup.4).sub.3, SO.sub.kR.sup.4 or
halogen;
[0100] or R.sup.8 and R.sup.16 are each H, and R.sup.3 and R.sup.5,
together, form one of the structures 27
[0101] wherein:
[0102] R.sup.10 and R.sup.11 are H, alkyl having from 1 to about 10
carbons, --CH.sub.2C(.dbd.O)OR.sup.22, --CH.sub.2CN,
--CH.sub.2Si(CH.sub.3).sub.3, or o- or
p-C.sub.6H.sub.4--R.sup.21;
[0103] R.sup.21 is hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having
from 1 to about 10 carbons, --NO.sub.2, or --N(R.sup.22).sub.2;
[0104] R.sup.22 is independently H or alkyl having from one to
about 10 carbon atoms;
[0105] p is 1 or 2;
[0106] Z.sup.1 and Z.sup.2 are independently halogen, CN,
--Si(CH.sub.3).sub.3, and --C(.dbd.O)OR.sup.22;
[0107] R.sup.30 is hydrogen, --O--C(.dbd.O)CH.sub.3, alkoxy having
from 1 to about 10 carbons, or --O--Si(R.sup.4).sub.3;
[0108] R.sup.6 is H, alkyl or aralkyl having up to 15 carbon
atoms;
[0109] or R.sup.5 and R.sup.6, together with the atoms to which
they are attached, form a 5 or 6 membered ring;
[0110] R.sup.7 is alkyl or aralkyl having up to 15 carbon
atoms;
[0111] or R.sup.6 and R.sup.7, together, form one of the structures
28
[0112] wherein V, T, and Z are independently CH or N;
[0113] R.sup.8 is H or methyl;
[0114] R.sup.16 is H, alkyl or aralkyl having up to 15 carbon
atoms;
[0115] B is a nucleobase; and
[0116] n is an integer from 0 to 50;
[0117] with a second synthon of Formula II: 29
[0118] wherein:
[0119] R.sup.9 is a hydroxyl protecting group or a linker connected
to a solid support; and
[0120] m is an integer from 0 to 50;
[0121] for a time and under reaction conditions effective to form a
third synthon of Formula III: 30
[0122] and
[0123] contacting said third synthon with a sulfurizing agent to
form an oligomer of Formula IV: 31
[0124] wherein D is said phosphorothioate linkage having the
formula: 32
[0125] In accordance with the invention, first synthons are cyclic
phosphoramidites having the general Formula VIc: 33
[0126] in which W, R.sup.3, R.sup.5-R.sup.8 and R.sup.16 are as
defined above.
[0127] The reaction of first and second synthons is conducted in
the presence of a catalyst. The structures of the first synthon and
the catalyst are chosen such that the opening of the cyclic O--P--N
phosphoramidite (azaphospholane) ring proceeds by the
stereoselective breaking of the intracyclic P--N bond of the
azaphospholane, to yield a third synthon, which is
diastereomerically enriched at phosphorus. Accordingly, in
preferred embodiments of the methods of the invention, first
synthons are diastereomerically enriched, and more preferably in a
single stereochemical form, substantially free of other
stereochemical forms. It is also advantageous for the first synthon
and the catalyst to bear substituent groups which are of relatively
large size (i.e., bulky groups) to aid in the proper orientation of
reactants to achieve the desired stereoselectivity. As used herein,
the term stereoselective has its normal meaning as a process in
which one stereoisomer is produced or destroyed more rapidly than
another, resuting in a predominance of the favored
stereoisomer.
[0128] In preferred embodiments catalysts have one of the Formulas
VIII or IX: 34
[0129] wherein R.sup.12-R.sup.14 are as defined above.
[0130] It has been found in accordance with the present invention
that imidazole catalysts having electron-withdrawing substituents,
in addition to substituents of relatively large size, are
especially advantageous in production of stereochemically enriched
products. While not wishing to be bound by a particular theory, it
is believed that the catalyst first protonates the azaphospholane
nitrogen, creating a good leaving group, which is displaced by the
catalyst or its conjugate base. The imidazole or tetrazole attached
to the phosphorus is then displaced either by the 3'-hydroxyl of
the nucleosidic species, leading to a phosphite triester of high
stereochemical purity, or by the catalyst, leading to
epimerization.
[0131] It has been found in accordance with the present invention
that catalysts which have appreciable acidity (i.e., which have pKa
values of about 2 to 4) and which are relatively large can overcome
the tendency toward epimerization at phosphorus, and result in
stereoselective addition of the free 5'-hydroxyl of the nucleosidic
species to be added. Thus, preferred substituents for groups
R.sup.13, R.sup.14 and R.sup.15 are those which are electron
withdrawing, (and which therefore increase acidity), and of a size
sufficient to maintain stereoselectivity. It will be recognized,
however, that it is not necessary that all three groups R.sub.6,
R.sub.7 and R.sup.8 be of great bulk, so long as the overall size
of the catalyst is sufficient to afford the desired
stereoselectivity. Thus preferred R.sup.12 and R.sup.13 groups are
independently hydrogen, halogen, cyano, nitro, alkyl having from
one to 10 carbons, substituted alkyl having from one to 10 carbons,
an ester group, or R.sup.12 and R.sup.13, together with the carbon
atoms to which they are attached, form a substituted or
unsubstituted phenyl ring where said substituents are electron
withdrawing. Preferred R.sup.14 groups include hydrogen, halogen,
cyano, nitro, thio, alkyl having from one to 10 carbons,
substituted alkyl having from one to 10 carbons, norbornyl,
substituted norbornyl, aryl, substituted aryl wherein said
substituents are electron withdrawing, or has the formula: 35
[0132] wherein L is protecting group. In prefered embodiments of
the invention the catalyst is 2,4,5-tribromoimidazole,
dibromocyanoimidazole, or dicyanobromoimidazole. In particularly
preferred embodiments the catalyst is
4,5-dicyano-2-bromoimidazole.
[0133] It has been found in accordance with the present invetion
that the dicyanoimidazole, bromoimidazole, and tribromoimidazole
catalysts described in accordance with the present invention are
useful as substitutes for tetrazole catalysts in standard solid
phase oligonucleotide synthetic regimes. Such synthetic procedures
are well known in the art, and are extensively described in the
literature. See for example, Caruthers U.S. Pat. Nos. 4,415,732;
4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and
Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069, and
Oligonucleotides and Analogues, A Practical Approach, Eckstein, F.,
IRL Press, New York (1991). The use of the catalysts of the
invention in these synthetic methologies provides significant
advantages over tetrazole catalysts, including, for example,
significantly lower cost.
[0134] In other preferred embodiments R.sup.14 has one of the
formulas: 36
[0135] wherein R.sup.15 is H, methyl, trimethylsilyl or acetyl.
[0136] In some preferred embodiments R.sup.6 and R.sup.7, together
with the atoms to which they are attached, form an heterocyclic
(i.e., imidazole, triazole or tetrazole) ring, which performs the
function of the catalyst. Preferred first synthons which
incorporate the catalyst therein have the general Formula Xa or
XIIIa: 37
[0137] wherein V, T and Z are each independently N or CH. In
especially preferred embodiments the first synthons incorporate
imidazole rings, and have the Formula Xb or Xc: 38
[0138] In further preferred embodiments of the invention the
imidazole portions of the first synthons are further substituted,
for example, by having a phenyl ring fused thereto. Thus in another
preferred embodiment first synthons have the Formula XIa: 39
[0139] In further preferred embodiments, first synthons incorporate
other relatively large substituent groups which facilitate the
stereoselective opening of the azaphospholane ring. In particularly
preferred embodiments first synthons have the Formula XIIa, and
particularly Formula XVIIa or XVIIIa: 40
[0140] in which R.sup.10 and R.sup.11 are as defined above.
[0141] In especially preferred embodiments, first synthons have the
Formula XVb or XVIb: 41
[0142] In some preferred embodiments, the first synthon is obtained
by reaction of a compound of Formula V: 42
[0143] with an azaphospholane of Formula VIa: 43
[0144] wherein R.sup.3-R.sup.8 are as defined above; and X is
halogen, preferably chlorine, dialkylamino, imidazole or
substituted phenoxy wherein said substituents are electron
withdrawing, and preferably are halogen or nitro.
[0145] In more preferred embodiments the first synthon is obtained
by reaction of a compound of Formula VII: 44
[0146] and a .gamma.-amino alcohol of formula
HO--C(R.sup.8)R.sup.3--C(R.s- up.16)R.sup.5--CHR.sup.6--NHR.sup.7;
wherein X and R.sup.1-R.sup.16 are as defined above.
[0147] R.sub.2 is a chiral auxiliary, which has the formula
--C(R.sup.8)R.sup.3--C(R.sup.16)R.sup.5--CHR.sup.6--NHR.sup.7, and
which is formed as a consequence of the opening of the cyclic
phosphite ring. The chiral auxiliary functions as a protecting
group for the phosphorus linkage during the course of the synthesis
of oligomeric phosphorothioates. Accordingly, chiral auxiliaries
are allowed to remain on the growing chain, and are removed at the
end of the iterative synthetic regime. Removal of chiral
auxiliaries can be conveniently accomplished in a single treatment
after the completion of the iterative synthesis by treatment with
either acidic reagents or by base catalyzed .beta.-elimination.
Suitable reagants include, for example, 70% trifluoroacetic acid,
ammonia, and fluoride ion. Removal of chiral auxiliaries via
.beta.-elimination should be particularly advantageous where first
synthons have the Formula XXa.
[0148] After reacting first and second synthons to form a third
synthon, the third synthon is sulfurized to form a phosphorothioate
linkage having the formula: 45
[0149] Sulfurization may be accomplished by any of the several
sulfurizing agents known in the art to be suitable for conversion
of phosphites into phosphorothioates. Useful sulfurizing agents
include Beaucage reagent described in e.g., Iyer, R. P.; Egan, W.;
Regan, J. B.; Beaucage, S. L., 3H-1,2-Benzodithole-3-one
1,1-Dioxide as an Improved Sulfurizing Reagent in the Solid-Phase
Synthesis of Oligodeoxyribonucleoside Phosphorothioates, Journal of
American Chemical Society, 1990, 112, 1253-1254 and Iyer, R. P.;
Phillips, L. R.; Egan, W.; Regan J. B.; Beaucage, S. L., The
Automated Synthesis of Sulfur-Containing Oligodeoxyribonucleotides
Using 3H-,12-Benzodithiol-3-one 1,1-Dioxide as a Sulfur-Transfer
Reagent, Journal of Organic Chemistry, 1990, 55, 4693-4699.
Tetraethyl-thiuram disulfide can also be used as described in Vu,
H.; Hirschbein, B. L., Internucleotide Phosphite Sulfurization With
Tetraethylthiuram Disulfide, Phosphorothioate Oligonucleotide
Synthesis Via Phosohoramidite Chemistry, Tetrahedron Letters, 1991,
32, 3005-3007. Further useful reagents for this step are dibenzoyl
Tetrasulfide, Rao, M. V.; Reese, C. B.; Zhengyun, Z., Dibenzoyl
Tetrasulphide--A Rapid Sulphur Transfer Agent in the Synthesis of
Phosphorthioate Analogues of Oligonucleotides, Tetrahedron Letters,
1992, 33, 4839-4842; di(phenylacetyl)disulfide, Kamer, R. C. R.;
Roelen, H. C. P. F.; van den Eist, H.; van der Marel, G. A.; van
Boom, J. H., An Efficient Approach Toward the Synthesis of
Phosphorothioate Diesters Va the Schonberg Reaction. Tetrahedron
Letters, 1989. 30, 6757-6760; sulfur; and sulfur in combination
with ligands like triaryl, trialkyl or triaralkyl or trialkaryl
phosphines.
[0150] The methods of the present invention can also be used to
produce analogs of phosphorothioates, including phosphoroselenoates
and phosphoroboronates. For example, phosphoroselenoates can be
prepared by the methods of the invention by utilizing potassium
selenocyanate in place of the sulfurizing agents described above.
Phosphoroboronates can be prepared by similar adaptation of
oxidizing agents known known in the art. See, for example,
Antisense Research and Applications, Crooke, S. T., and Lebleu, B.,
Eds. CRC Press, Boca Raton, Fla. (1993).
[0151] R.sub.9 and R.sub.1 can each be a hydroxyl protecting group.
Protecting groups are known per se as chemical functional groups
that can be selectively appended to and removed from
functionalities, such as hydroxyl groups and carboxyl groups. These
groups are present in a chemical compound to render such
functionality inert to chemical reaction conditions to which the
compound is exposed. The tert-butyldimethylsilyl (TBDMS) group is
representative of protecting groups useful for protecting the
hydroxyl functionality. A preferred protecting group for R.sup.1 is
the dimethoxytrityl group. Other representative groups may be found
in Greene, T. W. and Wuts, P. G. M., "Protective Groups in Organic
Synthesis" 2d. Ed., Wiley & Sons, 1991. Typically, protecting
groups are removed at the end of the iterative synthesis.
[0152] R.sub.9 may alternatively be a linker connected to a solid
support. Solid supports are substrates which are capable of serving
as the solid phase in solid phase synthetic methodologies, such as
those described in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066;
4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S.
Pat. Nos. 4,725,677 and Re. 34,069. Linkers are known in the art as
short molecules which serve to connect a solid support to
functional groups (e.g., hydroxyl groups) of initial synthon
molecules in solid phase synthetic techniques. Suitable linkers are
disclosed in Oligonucleotides And Analogues A Pratical Approach,
Ekstein, F. Ed., IRL Press, New York, 1991.
[0153] Alkyl groups according to the invention include straight
chain, branched, and cyclic carbon and hydrogen containing groups
such as methyl, isopropyl, and cyclohexyl groups. Preferred alkyl
groups have 1 to about 6 carbon atoms.
[0154] Aralkyl groups according to the invention include both alkyl
and aryl portions, although the point of attachment of such groups
is through an alkyl portion thereof. Benzyl groups provide one
example of an aralkyl group. Alkaryl groups include both alkyl and
aryl portions, and are attached through their aryl portions. The
term aryl is intended to denote monocyclic and polycyclic aromatic
groups including, for example, phenyl, naphthyl, xylyl, pyrrole,
and furyl groups. Although aryl groups (e.g., imidazo groups) can
include as few as 3 carbon atoms, preferred aryl groups have 6 to
about 14 carbon atoms, more preferably 6 to about 10 carbon atoms.
The alkyl, alkaryl, and aryl groups may be substituted (e.g., i.e,
bear halogens and hydroxy groups) or unsubstituted moieties.
[0155] Certain substituent groups of compounds of the invention
bear electron withdrawing groups. As used herein, the term
"electron wihdrawing" has its normal meaning as a chemical
functionality which electronically or inductively causes the
withdrawal of electron density form the moiety to which the
electron withdrawing groups is attached. Representative electron
withdrawing groups include nitro groups and halogens. Other
electron withdrawing groups will be apparent to those of skill in
the art, once armed with the present disclosure.
[0156] Substituent B is a nucleobase. The term nucleobase as used
herein is intended to include naturally occurring nucleobases
(i.e., heterocyclic bases found in naturally occurring nucleic
acids) and their non-naturally occurring analogs. Thus, nucleobases
according to the invention include naturally occurring bases
adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U),
both in their unprotected state and bearing protecting or masking
groups. Examples of nucleobase analogs include
N.sup.4,N.sup.4-ethanocytosine, 7-deazaxanthosine,
7-deazaguanosine, 8-oxo-N.sup.6-methyladenine, 4-acetylcytosine,
5-(carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl
uracil, inosine, N.sup.6-isopentyladenine, 1-methyladenine,
2-methylguanine, 5-methylcytosine, N.sup.6-methyladenine,
7-methylguanine, 5-methylaminomethyl uracil,
5-methoxyaminomethyl-2-thiou- racil, 5-methoxyuracil, pseudouracil,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-thiouracil,
5-(1-propynyl)-2-thiocytosine, 2-thiocytosine, and
2,6-diaminopurine. Other suitable base analogs, for example the
pyrimidine analogs 6-azacytosine, 6-azathymidine and
5-trifluoromethyluracil, may be found in Cook, D. P., et al.,
International Publication No. 92/02258, which is herein
incorporated by reference.
[0157] The compounds of the invention are preferably up to 50
nucleobases in length, with 10 to 30 nucleobases being more
preferred, and 15 to 25 nucleobases being especially preferred.
[0158] In preferred embodiments the phosphorothioate linkage
produced by the method of the invention is diastereomerically
enriched. The term "diastereomerically enriched" denotes the
predominance of one stereochemical form over the other. In
preferred embodiments the phosphorothioate linkage is 75% in a
single stereochemical form. In further preferred embodiments the
phosphorothioate linkage is 85% in a single stereochemical form,
with 90% being further preferred and 95% being especially
preferred. In further preferred embodiments the phosphorothioate
linkage is in a single stereochemical form, substantially free of
other stereochemical forms.
[0159] Preferably, following sulfurization, the phosphorothioate is
next converted to a new first synthon. This is first accomplished
by the removal of the 5'-hydroxyl protecting group R.sub.1, under
conditions which will necessarily depend upon the chemical identity
of the specific R.sub.1 group. After removal of the protecting
group, the unprotected 5'-alcohol may be employed as a new second
synthon in the iternative method. Libraries of dimeric and higer
synthons may be prepared and stored to facilitate the iterative
synthesis of desired nucleobase sequences.
[0160] Also provided according to the invention are azaphospholanes
of Formula VIb: 46
[0161] wherein Y is X or W, wherein X is halogen, dialkylamino,
imidazole, or substituted phenoxy wherein said substituents are
electron withdrawing, and W has the formula: 47
[0162] wherein constituent members are as defined above.
Preferably, the azaphospholanes of Formula VIb are
diastereomerically enriched. In particular, it is advantageous to
have defined stereochemistry around phosphorus atom, to afford
diastereomerically enriched products upon stereoselective opening
of the azaphospholane ring.
[0163] In preferred embodiments, compounds of the invention have
one of the Formulas Xb, XIb, XIIb, XIIIb or XXb: 48
[0164] wherein R.sup.3-R.sup.16, Y, V, T, Z, Z.sub.1, Z.sub.2 and p
are as defined above.
[0165] Paticularly preferred embodiments of the compounds of the
invention have the Formula XIVb, Xd, Xe, XVIIb, XVIIIb, XVb or
XVIb: 4950
[0166] As used herein, the term "contacting" means directly or
indirectly causing placement together of moieties to be contacted,
such that the moieties come into physical contact with each other.
Contacting thus includes physical acts such as placing the moieties
together in a container. The term "reacting" as used herein means
directly or indirectly causing placement together of moieties to be
reacted, such that the moieties chemically combine or
transform.
[0167] The method of the invention is performed in the presence of
a solvent, for example chloroform or acetonitrile. Other solvents
suitable for use in the present method will be readily apparent to
those skilled in the art, once having been made aware of the
present disclosure.
[0168] In general, it is preferred that the molar ratio of the
catalyst to the first synthon starting material be from about 1 to
about 50; preferably from about 2.5 to about 10.
[0169] The method of the present invention can be carried out in
any suitable vessel which provides efficient contacting between the
first and second synthons, and the catalyst. The reaction vessel
used should be resistant to the components of the reaction mixture.
Glass-lined vessels would be suitable for this purpose. Additional
vessel materials will be apparent to those skilled in the art.
[0170] The reagents of the present method may be added in any
order. The method is preferably carried out under an inert
atmosphere, any should be carried out in a dry atmosphere. Any
suitable inert gas may be employed, such as nitrogen, helium and
argon.
[0171] Preferably, the method is carried out at temperatures
ranging between about -20.degree. C. and about 40.degree. C., with
temperatures ranging from about -15.degree. C. to about 0.degree.
C. being more preferred.
[0172] Reaction time is generally from about one minute to about
two hours, with reaction times of from about one minute to about 10
minutes being preferred.
[0173] Product can be recovered by any of several methods known to
those of skill in the art. Preferably, products are recovered by
chromatography. Additional separation of isomers can be
accomplished by techniques known in the art including high
performance liquid chromatography.
[0174] When R.sup.9 is a solid support, purification is carried out
after removal of the oligonucleotide from the solid support using
methods known in the art.
[0175] The invention is further illustrated by way of the following
examples. These examples are illustrative only and are not intended
to limit the scope of the appended claims.
EXAMPLES
General Methods
[0176] Melting points (m.p.) were determined using an
Electrothermal MP apparatus and are uncorrected. Optical rotation
measurements were carried out in the indicated solvents employing a
Jasco DIP-140 digital polarimeter. Mass spectra (CI or EI) were
obtained on an HP 5980A quadrupole mass spectrometer in the
direct-inlet mode.
[0177] NMR spectra were recorded on Varian XL200, XL300, or Unity
500 spectrometers. Chemical shifts are given in the .delta. scale
in parts per million. The assignments of proton spectra are based
on COSY experiments. The residual proton signals of
deuteriochloroform (.delta. 7.24 ppm), methanol (.delta. 3.30 ppm)
and acetonitrile (.delta. 1.93 ppm) were used as reference in these
solvents. The multiplicities are recorded using the following
abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet. .sup.31P NMR spectra were obtained on either a Varian
XL200, XL300 or Unity 500 instrument, and chemical shifts are given
with respect of aqueous phosphoric acid. Peak assignments of
.sup.13C-NMR spectra were, in some cases, made with the aid of APT,
HMQC or HETCOR experiments.
[0178] Tetrahydrofuran (THF) was distilled from sodium benzophenone
ketyl. Dichloromethane was distilled from P.sub.2O.sub.5.
Triethylamine and acetonitrile were distilled from CaH.sub.2.
N,N-Dimethyl formamide was dried by shaking with KOH, followed by
distillation. Thin-layer chromatography (TLC) was performed using
Kieselgel 60 F.sub.254 aluminium-backed plates (0.2 mm thickness)
and visualized by UV and/or dipping in a solution of ammonium
molybdate (2.5 g) and ceric sulfate (1 g) in 10% v/v aqueous
sulphuric acid (100 ml), followed by heating. Kieselgel 60 (Merck
230-400 mesh) silica gel was employed for column
chromatography.
Example 1
3R-Hydroxy-N-iso-propylbutanoamide (10)
[0179] 51
[0180] A 2 M solution of trimethylaluminium in hexane (50 ml, 100
mmol) was slowly added to a solution of 8.5 ml (100 mmol)
isopropylamine in 100 ml dichloromethane under nitrogen at room
temperature. The mixture was stirred for 30 min and then cooled to
0.degree. C. before 6.6 g (50 mmol) of ethyl-3R-hydroxybutanoate
was added. The reaction mixture was stirred at room temperature for
2 hours for completion, carefully quenched with dilute HCl and
extracted with chloroform. The organic extract was dried over
MgsO.sub.4 and concentrated to affored 7 g N-isopropyl 3R-hydroxy
butanoamide. After recrystallization, 5.5 g pure amide 10 was
obtained (yield 76%).: m.p. 62.degree. C.; .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 6.41 (m, 1H, NH), 4.35 (b, 1H, OH), 3.78-4.15
(m, 2H, NeCH, Me.sub.2CH), 2.08-2.32 (m, 2H, CH.sub.2), 1.12 (d,
J=6.3 Hz, 3H, Me), 1.07 (d, J=6.6 Hz, 6H, NCHMe.sub.2); .sup.13C
NMR (50 MHz, CDCl.sub.3) .delta. 171.5 (C.dbd.O), 64.7 (CHOH), 43.8
(NHCH), 41.0 (CH.sub.2), 22.7 (Me), 22.4 (NCHMe.sub.2); MS(EI) m/e
145 ([M.sup.+], 25%), 130 (27), 112 (4), 101 (6), 86 (34), 69 (8),
58 (22), 44 (100); HRMS (EI) m/e calc'd for C.sub.7H.sub.15O.sub.2N
[M.sup.+]: 145.1103, found 145.1109.
Example
2R-Hydroxy-4-(N-isopropyl)aninobutane (11)
[0181] 52
[0182] To a solution of 32 ml 1 M borane (32 mmol) in THF was added
2.32 g (16 mmol) 3R-hydroxy-N-iso-propyl-butyl butanoamide 10 in 20
ml THF at 0.degree. C. under nitrogen. The solution was then
brought to roflux and maintaned there for one hour. The reaction
mixture was cooled to room temperature and 1 N HCl was added slowly
to quench the reaction. THF was removed in vacuo, and true aqueous
solution was saturated with solid NaOH and then was extracted three
times with a total 300 ml chloroform. The combined chloroform phase
was dried, filtered and distilled to afford 1.4 g
3-(N-isopropylamino)butan-2-ol 11 as a clear, colorless liquid
(yield 67%).: .sup.1H NMR (200 MHz, CDCl.sub.3) .delta. 3.80-3.96
(m, 1H, CHOH), 2.86-2.98 (m, 1H, MeCH), 2.56-2.75 (m, 2H,
NCH.sub.2), 1.24-1.60 (m, 2H, CH.sub.2), 1.08 (d, J=6.1 Hz, 3H,
Me), 0.98 (d, J=6.2 Hz, 6H, Me.sub.2); .sup.13C NMR (200 MHz,
CDCl.sub.3) .delta. 69.5 (OCH), 48.6 (NHCH.sub.2), 46.0 (NHCH),
37.2 (CH.sub.2), 23.5, 22.9, 22.5; MS (EI) m/e 131 ([M.sup.+],
10%), 116 (81), 98 (35), 72 (100), 58 (30), 56 (45), 44 (37); HRMS
(EI) m/e calc'd for C.sub.7H.sub.17ON [.sup.+]: 131.1310, found
131.1311; [.alpha.].sub.D.sup.20=32.5.degree. (c=0.21,
chloroform).
Example 3
2R-Hydroxy-4-(N-tert-butyl)aminobutane (31)
[0183] 53
[0184] To a solution of 26.4 ml 1 M (26.4 mmol) borane in THF was
added 2.1 g (13.2 mmol) 3R-hydroxy-N-tert-butylbutanoamide in 20 ml
THF at 0.degree. C. under nitrogen. The solution was then brought
to reflux and maintained there for one hour. The reaction mixture
was cooled down to room temperature and 1 N HCl was added slowly to
quench the reaction. THF was removed in vacuo, and the aqueous
solution was saturated with solid NaOH and then was extracted three
times with a total 250 ml diethyl ether. The combined organic phase
was dried, filtered and distilled to afford 398 mg
2R-hydroxy-4-(N-tert-butyl)aminobutane 31 as a clear, colorless
liquid (yield 20.1%): .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.
3.80-3.92 (m, 1H, CHOH), 3.40-3.80 (b, 2H, OH, NH), 2.50-2.90 (m,
2H, NCH.sub.2), 1.28-1.61 (m, 2H, CH.sub.2), 1.07 (d, 3H, Me), 1.03
(5, 9H, Me.sub.3)
Example 4
5'-O-(tert-Butyldimethylsilyl)thymidine (1)
[0185] 54
[0186] To a solution of 2.42 g (10 mmol) thymidine in 15 ml DMF was
added 1.7 g (25 mmol) imidazole and 1.6 g (10.6 mmol)
tert-butyldimethyl silyl chloride. The solution was stirred at room
temperature for 3 hours. DMF was then removed in vacuo and the
residue was dissolved in 150 ml of ethyl acetate. The solution was
washed with water and the organic layer was dried over MgSO.sub.4.
After removing the solvent, the solid was recrystallized with ethyl
acetate/pentane to obtain 2.5 g pure
5'-O-(tert-butyldimethylsilyl)thymidine 1 (70% yield).: m.p.
193-194.degree. C.; .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 9.0
(s, 1H, NH), 7.50 (s, 1H, H-6), 6.36 (dd, J=5.8, 8.1 Hz, 1H, H-1'),
4.44 (m, 1H, H-3'), 4.03 (m, 1H, H-4'), 3.85 (m, 2H, H-5'), 2.66
(d, J=3.8 Hz, 1H, OH), 2.35 (m, 1H, H-2'), 2.07 (m, 1H, H-2'), 1.89
(s, 3H, C.dbd.CMe), 0.89 (s, 9H, CMe.sub.3), 0.09 (s, 6H,
SiMe.sub.2); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 163.8
(C-4), 150.4 (C-2), 135.4 (C-6), 110.9 (C-5), 87.2 (C-4'), 85.0
(C-1'), 72.6 (C-3'), 63.6 (C-5'), 41.1 (C-2'), 25.9 (SiCMe.sub.3),
18.3 (SiCMe.sub.3), 12.5 (C.dbd.CMe), -5.4 (SiMe.sub.2), -5.5
(SiMe.sub.2).
Example 5
5'-O-(4,4'-Dimethoxytrityl)thymidine (16)
[0187] 55
[0188] Triethylamine (10 ml) in 200 ml THF was injected into a
solid mixture of 6.8 g (28.0 mmol) thymidine and 10.2 g (28.6 mmol)
4,4'-dimethoxytrityl chloride under nitrogen with stirring. The
solution was stirred at room temperature for 2 hours. After
completion of the reaction, 10 ml methanol was added to consume the
excess DMTrCl. The mixture was stirred for 5 minutes and the
solvent removed by rotary evaporation. The residue was dissolved in
250 ml of ethyl acetate and the solution was washed with saturated
NaHCO.sub.3 and dried over MgSO.sub.4. The solid was recrystallized
from ethyl acetate/hexane to obtain 13.0 g 5'-protected thymidine
16 (85.6%).: m.p. 124-126.degree. C.; .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 8.97 (s, 1H, NH), 7.60 (m, 1H, H-6), 6.72-7.42
(m, 13H, Ph), 6.42 (m, 1H, H-1'), 4.56 (m, 1H, H-3'), 4.05 (m, 1H,
H-4'), 3.78 (s, 6H, OMe.sub.2), 3.41 (m, 2H, H-5'), 2.62 (m, 1H,
OH), 2.46 (m, 2H, H-2'), 1.46 (s, 3H, Me); .sup.13C NMR (50 MHz,
CDCl.sub.3) .delta. 163.0 (C-4), 157.6, 149.8 (C-2), 143.5, 135.0
(C-6), 134.7, 134.6, 129.4, 127.4, 127.3, 126.5, 112.8, 110.9
(C-5), 86.7 (C-4'), 86.2, 84.7 (C-1'), 72.5 (C-3'), 63.8 (C-5'),
55.5 (OCH.sub.3), 41.3 (C-2'), 12.5 (CH.sub.3).
Example 6
5'-O-(4,4'-Dimethoxytrityl)-3'-O-(tert-Butyldimethlsilyl)thymidine
(17)
[0189] 56
[0190] To a solution of 13.0 g (23.9 mmol)
5'-O-(4,4'-dimethoxytrityl)thym- idine 16 in 50 ml DMF was added
3.0 g (44 mmol) imidazole and 3.6 g (23.9 mmol)
tert-butyl-dimethylsilyl chloride. The solution was stirred at room
temperature for 3 hours. DMF was then removed in vacuo and the
residue was dissolved in 300 ml of ethyl acetate. The solution was
washed with water and the organic layer was dried over MgSO.sub.4.
After concentration of the solution and recrystallization from
ethyl acetate/hexane, the solid product
5'-O-(4,4'-dimethoxy)-3'-(tert-butyldim- ethylsilyl)thymidine 17
was used directely for the next reaction.
Example 7
3'-O-(tert-Butyldimethylsilyl)thymidine (18)
[0191] 57
[0192] A solution of 5 g (7.6 mmol)
5'-O--(4,4'-dimethoxytrityl)-3'-O-(ter-
t-butyldimethylsilyl)thymidine 17 in 100 ml 80% aq. acetic acid was
stirred until the removal of dimethoxytrityl group was completed.
Saturated Na.sub.2CO.sub.3 was then added to adjust the pH of the
solution to 6-7. The solution was then extracted with ethyl
acetate. The extract was dried and the mixture was chromatographed
on a silica gel column (CH.sub.2Cl.sub.2:MeOH=20:1) to give 2.5 g
of 3'-O-(tert-butyldimethylsilyl)thymidine 18 (92.6%).: m.p.
93-95.degree. C. (lit. 83-84.degree. C.); H NMR (200 MHz,
CDCl.sub.3) .delta. 9.18 (s, 1H, NH), 7.36 (b, 1H, H-6), 6.12 (t,
J=6.8 Hz, 1H, H-1'), 4.44-4.48 (m, 1H, H-3'), 3.69-3.91 (m, 3H,
H-4', H-5'), 2.87 (m, 1H, OH), 2.15-2.35 (m, 2H, H-2'), 1.87 (s,
3H, C.dbd.CMe), 0.86 (s, 9H, CMe.sub.3), 0.05 (s, 6H, SiMe.sub.2);
.sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 163.9 (C-1), 150.4
(C-2), 137.1 (C-6), 110.9 (C-5), 87.6 (C-4'), 86.8 (C-1'), 71.5
(C-3'), 61.9 (C-5'), 40.4 (C-2'), 25.7 (SiCMe.sub.3), 17.9 (SiCMe),
12.5 (C.dbd.CMe), -4.7 (SiMe.sub.2), -4.9 (SiMe.sub.2).
Example 8
2-Chloro-3-iso-propyl-6R-methyl-1-oxa-3-aza-2-phosphacyclohexane
(12)
[0193] 58
[0194] To a solution of 2.2 ml (3.45 g, 25 mmol) phosphorus
trichloride in 30 ml dichloromethane was added a solution of 2.89 g
(22 mmol) 2R-hydroxy-4-(N-iso-propyl)aminobutane 11 and 5.0 g (6.9
ml, 50 mmol) triethylamine in 20 ml dichloromethane with vigorous
stirring under nitrogen at 0.degree. C. Stirring was continued at
room temperature for 0.5 hour. The solvent was removed by
evaporation under reduced pressure and the residue was extracted
with diethyl ether (3.times.50 ml). Distillation gave 2.9 g of
product 12 (74.4% yield).: .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 4.48-4.68 (m, 1H, OCH), 3.40-3.61 (m, 1H, NCH), 3.15-3.38
(m, 1H, NCH.sub.2), 2.80-2.96 (m, 1H, NCH.sub.2), 1.71-1.85 (m, 2H,
CH.sub.2), 1.25 (d, J=6.4 Hz, 3H, Me), 1.13 (dd, 6H, Me.sub.2);
.sup.13C NMR (50 MHz, CDCl.sub.3) .delta. 69.5 (d, J=4.3 Hz, OCMe),
49.6 (d, J=34 Hz, NCMe.sub.2), 37.4 (d, J=5.5 Hz, NCH.sub.2), 33.4
(CH.sub.2), 22.2 (d, J=4.3 Hz, Me), 21.1 (d, J=13.3 Hz, Me.sub.2),
19.2 (d, J=5.0 Hz, Me.sub.2); .sup.31P NMR (81 MHz, CDCl.sub.3)
.delta. 160.6.
Example 9
Phosphoramidite (13a)
[0195] 59
[0196] To a solution of 215 mg (1.1 mmol) of
2-chloro-3-isopropyl-6R-methy- l-1-oxa-3-aza-2-phosphacyclohexane
12 in 40 ml CH.sub.2Cl.sub.2 was added a solution of 356 mg (1.0
mmol) of 5-O-(tert-butyldimethylsilyl)thymidine and 0.154 ml
triethylamine (111 mg, 1.1 mmol) in 20 ml CH.sub.2Cl.sub.2 with
vigorous stirring under nitrogen at 0.degree. C. The mixture was
stirred at room temperature until TLC indicated that the reaction
had gone to completion. The reaction mixture was transferred to 150
ml ethyl acetate, which was previously washed with a saturated
NaHCO.sub.3 solution. Saturated NaHCO.sub.3 was then added to wash
the solution. The separated organic phase was dried over
MgSO.sub.4. A white foam (quantitative yield) was formed after the
solvent was removed by evaporation under reduced pressure. Two
components were found from .sup.31P NMR spectra in a ratio of 1:3
(135.0 ppm:133.6 ppm). The reaction mixture in CDCl.sub.3 was then
refluxed for about 4 hours to get a ratio of up to 1:12 (135.0
ppm:133.6 ppm). After chromatography on a silica gel column
(hexane:ethyl acetate:triethylamine=5:3:2), the fast eluting
component (133.6 ppm) was separated as a pure diastereomer.:
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.50 (m, 1H, H-6), 6.36
(dd, J=8.5, 5.8 Hz, .sup.1H, H-1'), 4.56 (m, 1H, H-3'), 4.30 (m,
1H, HCMe), 4.04 (m, 1H, H-4'), 3.75-3.90 (m, 2H, H-5'), 3.39 (hept,
J=6.3 Hz, 1H, NCH), 3.24 (m, 1H, NCH.sub.2), 2.72 (m, 1H,
NCH.sub.2), 2.35 (m, 1H, H-2'), 2.06 (m, 1H, H-2'), 1.89 (d, J=0.9
Hz, 3H, CH.sub.3), 1.64 (m, 2H, CH.sub.2), 1.16 (d, J=6.4 Hz, 3H,
OCHMe), 1.08 (dd, J=2.0 Hz, 6.3Hz, 6H, NCMe.sub.2), 0.89 (s, 9H,
SiCMe.sub.3), 0.09 (d, J=1.5 Hz, 6H, SiMe.sub.2); .sup.13C NMR (125
MHz, CDCl.sub.3) .delta. 163.9 (C-4), 150.5 (C-2), 135.3 (C-6),
110.9 (C-5), 86.5 (d, J=2.8 Hz, C-4'), 84.8 (C-1'), 73.3 (d, J=19.2
Hz, C-3'), 66.0 (d, J=2.7 Hz, OCH), 63.2 (C-5'), 49.2 (d, J=34.8
Hz, NCH), 40.4 (d, J=4.6 Hz, C-2'), 36.6 (d, J=5.5 Hz, NCH.sub.2),
34.6 (CH.sub.2), 25.9 (SiCMe.sub.3), 22.9 (d, J=4.6 Hz, OCMe), 21.8
(d, J=10 Hz, NCMe.sub.2), 21.0 (d, J=4.6 Hz, NCMe.sub.2), 18.3
(SiCMe.sub.3), 12.5 (C.dbd.CMe), -5.5 (SiMe.sub.2), -5.4
(SiMe.sub.2); .sup.31P NMR (81 MHz, CDCl.sub.3) .delta. 133.6; MS
(CI, NH.sub.3)) m/e 516 ([M+H.sup.+], 37%), 390 (21), 339 (92), 178
(100), 160 (55); HRMS (CI, NH.sub.3) m/e calc'd for
C.sub.23H.sub.43N.sup.3O.sub.6PS- i [M+H.sup.+]: 516.2659, found
516.2664
Example 10
Phosphoramidite (13b)
[0197] 60
[0198] To a solution of 215 mg (1.1 mmol)
2-chloro-3-isopropyl-6R-methyl-1- -oxa-3-aza-2-phosph ocyclohexane
12 in 40 ml CH.sub.2Cl.sub.2 was slowly added a solution of 356 mg
(1.0 mmol) 5'-O-(tert-butyldimethylsilyl)thymi- dine 1 and 0.154 ml
triethylamine (111 mg, 1.1 mmol) in 20 ml CH.sub.2Cl.sub.2 with
vigorous stirring under nitrogen at -78.degree. C. The mixture was
stirred at -78.degree. C. until TLC indicated that the reaction had
gone to completion. The reaction mixture was quickly transferred to
150 ml ethyl acetate, which was previously washed with a cold
saturated NaHCO.sub.3 solution. Cold saturated NaHCO.sub.3 was then
added to wash the solution. The separated organic phase was dried
over MgSO.sub.4. A white foam (quantitative yield) was formed after
the solvent was removed by evaporation under reduced pressure at
0.degree. C. Two components were found from .sup.31P NMR spectra in
a ratio of 5:1 (135.1 ppm:133.6 ppm). The reaction mixture in
CDCl.sub.3 was immediately chromatographed on a silica gel column
(hexane:ethylacetate:triethylamine- =5:3:2) and the slow eluting
component (135.1 ppm) was separated as an enriched diastereomer
(92% pure).: .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.48 (m, 1H,
H-6), 6.32-6.35 (dd, J=5.4, 7.8 Hz, 1H, H-1'), 4.55-4.60 (m, 1H,
H-3'), 4.02-4.10 (m, 1H, OCH), 4.00 (m, 1H, H-4'), 3.74-3.89 (m,
2H, H-5'), 3.36-3.45 (m, 1H, NCH), 3.09-3.14 (m, 1H, NCH.sub.2),
2.85-2.93 (m, 1H, NCH.sub.2), 2.40-2.44 (m, 1H, H-2'), 2.12-2.21
(m, 1H, CH.sub.2), 2.00-2.06 (m, 1H, H-2'), 1.88 (s, 3H,
C=CCH.sub.3), 1.81-1.86 (m, 1H, CH.sub.2), 1.28 (d, J=6.3 Hz, 3H,
OCMe), 1.11 (dd, J=6.3, 11.7 Hz, 6H, NCMe.sub.2), 0.89 (s, 9H,
Me.sub.3), 0.08 (d, J=2.0 Hz, 6H, SiMe.sub.2); .sup.13C NMR (125
MHz, CDCl.sub.3) .delta. 163.7 (C-4), 150.2 (C-2), 135.5 (C-6),
110.8 (C-5), 87.0 (d, J=6.4 Hz, C-4'), 84.8 (d, J=5.5 Hz, C-1'),
72.8 (d, J=19.2 Hz, OCHMe), 70.3 (d, 7.3, C-3'), 63.1 (C-5'), 49.5
(d, J=39.4 Hz, NCHMe.sub.2), 40.1 (d, J=1.8 Hz, C-2'), 36.9 (d,
J=5.5 Hz, NCH.sub.2), 31.5 (d, J=7.3 Hz, CH.sub.2), 25.9
(SiCMe.sub.3), 23.3 (OCMe), 21.7 (d, J=11.0 Hz, NCHMe.sub.2), 21.1
(d, J=5.5 Hz, NCHMe.sub.2), 18.4 (SiCMe.sub.3), 12.5 (C.dbd.CMe),
-5.4 (SiMe.sub.2), -5.5 (SiMe.sub.2); .sup.31P NMR (81 MHz,
CDCl.sub.3) .delta. 135.1
Example 11
Phosphoramidite (14)
[0199] 61
[0200] To a solution of 0.2 ml (315 mg, 2.3 mmol) phosphorus
trichloride in 10 ml dichloromethane was added a solution of 320 mg
(2.2 mmol) butanol 31 and 0.7 ml triethylamine in 5 ml
dichloromethane with vigorous stirring under nitrogen at 0.degree.
C. and then at room temperature for 0.5 hour. The solvent was
removed by evaporation under reduced pressure and the residue was
extracted with diethyl ether (2.times.40 ml). After removing the
ether, 512 mg residual oil was obtained. Then 209 mg (1.0 mmol) of
this residual oil was dissolved in 10 ml dichloromethane, a
solution of 200 mg (0.56 mmol)
5'-O-(tert-butyldimethylsilyl)thymidine 1 and 0.16 ml triethylamine
(1.1 mmol) in 10 ml CH.sub.2Cl.sub.2 was added with vigorous
stirring under nitrogen at room temperature. The mixture was
stirred at room temperature until TLC indicated that the reaction
had gone to completion. The reaction mixture was transferred to 100
ml ethyl acetate, which was previously washed with saturated
NaHCO.sub.3 solution. Saturated NaHCO.sub.3 and then water was
added to wash the solution. The separated organic phase was dried
over MgSO.sub.4. A white foam (quantitative yield) was formed after
the solvent was removed by evaporation under reduced pressure. Two
components were found from .sup.31P NMR spectra in a ratio of 1:5
(132.4 ppm:130.8 ppm). The reaction mixture in C.sub.6D.sub.6 was
then refluxed for about 4 hours to get a ratio of up to 1:9 (d
137.8 ppm:136.1 ppm in benzene).
Example 12
Phosphoramidite (15)
[0201] 62
[0202] To a solution of 6.30 g (4.0 ml, 46 mmol) phosphorus
trichloride in 100 ml dichloromethane was added a solution of 6.61
g (40 mmol) (1R,2S) ephedrine and 10.1 g (14 ml, 100 mmol)
triethylamine in 50 ml dichloromethane with vigorous stirring under
nitrogen at 0.degree. C. and then at room temperature for 0.5 hour.
The solvent was removed by evaporation under reduced pressure and
the residue was extracted with diethyl ether (3.times.100 ml).
After removing the ether, 9.3 g residual oil was obtained. Then 345
mg (1.5 mmol) of this residual oil was dissolved in 40 ml
dichloromethane, a solution of 356 mg (1.0 mmol)
5'-O-(tert-butyldimethylsilyl)thymidine 1 and 0.154 ml
triethylamine (111 mg, 1.1 mmol) in 20 ml CH.sub.2Cl.sub.2was added
with vigorous stirring under nitrogen at -78.degree. C. The mixture
was stirrred at -78.degree. C. until TLC indicated that the
reaction had gone to completion. The reaction mixture was quickly
transferred to 150 ml ethyl acetate, which was previously washed
with cold saturated NaHCO.sub.3 solution. Cold saturated
NaHCO.sub.3 was added to wash the solution. The separated organic
phase was dried over MgSO.sub.4. A white foam (quantitative yield)
was formed after the solvent was removed by evaporation tinder
reduced pressure at 0.degree. C. Two components were found from the
.sup.31P NMR spectrum in a ratio of 1:2 (147.3 ppm:141.6 ppm). The
reaction mixture in CDCl.sub.3 was then refluxed for about 4 hours
to get a ratio of up to 1:5 (151.5 ppm:143.5 ppm). After
chromatography on silica gel column (hexane:ethyl
acetate:triethylamine=5:3:2), the slow eluting component (143.5
ppm) was separated as a pure diastereomer.: .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 7.23-7.50 (m, 5H, Ph), 6.35 (m, 1H, H-1'), 5.60
(d, J=6.8 Hz, 1H, PhCH), 4.71-4.75 (m, 1H, H-5'), 4.01-4.02 (m, 1H,
H-4'), 3.78-3.87 (m, 2H, H-5'), 3.52-3.58 (m, 1H, MeCH), 2.64 (d,
J=12.2 Hz, 3H, NMe), 2.31-2.36 (m, 1H, H-2'), 2.05-2.11 (m, 1H,
H-2'), 1.90 (s, 3H, C.dbd.CMe), 0.90 (s, 9H, CMe.sub.3), 0.60 (d,
J=6.4 Hz, 3H, CHMe), 0.09 (s, 6H, SiMe.sub.2); .sup.13C NMR (125
MHz, CDCl.sub.3) .delta. 163.9 (C-4), 150.5 (C-2), 137.9 (Ph),
135.3 (C-6), 128.1 (Ph), 127.7 (Ph), 126.7 (Ph), 111.0 (C-5), 86.7
(d, J=1.8 Hz, C-4'), 84.7 (OCPh), 84.6 (d, J=9.2 Hz, C-1'), 73.1
(d, J=18.3 Hz, C-3'), 63.0 (C-5'), 57.5 (d, J=5.6 Hz, NCMe), 40.6
(d, J=5.5 Hz, C-2'), 28.8 (d, J=17.4, NCH.sub.3), 25.9
(SiCMe.sub.3), 18.3 (SiCMe.sub.3), 14.6 (d, J=3.7 Hz, NCHKe), 12.5
(C.dbd.CMe), -5.4 (SiMe.sub.2), -5.5 (SiMe.sub.2); .sup.31P NMR (81
MHz, CD.sub.3CN) .delta. 143.5
Example 13
Phopshite Triester (20a) From Phosphoramidite 13a With MeOH
[0203] 63
[0204] Diastereomeric pure phosphoramidite 13a (10 mg, fast eluting
component) in 0.5 ml CDCl.sub.3 was put in 5 mm NMR tube, and 50
.mu.l of dry MeOH (a large excesses) was then added by syringe,
followed by 1 mg of dicyanoimidazole. The reaction was monitored by
.sup.31P NMR until the reaction went to completion. The phosphite
triesters formed in a ratio of 10:1 (139.4 ppm:138.8 ppm) and were
purified on a silica gel column.: .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 7.48 (s, 1H, H-6), 6.35 (dd, J=5.4, 8.8 Hz, 1H, H-1'),
4.78-4.82 (m, 1H, H-3'), 4.24-4.33 (m, 1H, OCH), 4.10 (d, J=2.4 Hz,
1H, H-4'), 3.76-3.89 (m, 2H, H-5'), 3.50 (d, J=10.7 Hz, 3H,
CH.sub.3), 2.78 (m, 1H, NCH), 2.65 (m, 2H, NCH.sub.2), 2.40 (m, 1H,
H-2'), 2.04 (m, 1H, H-2'), 1.89 (d, J=1.0 Hz, 3H,
C.dbd.C--CH.sub.3), 1.64-1.78 (m, 2H, OCHCH.sub.2), 1.25 (d, J=6.4
Hz, 3H, OCHCH.sub.3), 1.03 (d, J=6.3 Hz, 6H, NCHe.sub.2), 0.91 (s,
9H, SiCMe.sub.3), 0.10 (d, J=2.0 Hz, 6H, SiMe.sub.2); .sup.13C NMR
(125 MHz, CDCl.sub.3) .delta. 163.5 (C-4), 150.1 (C-2), 135.3
(C-6), 110.9 (C-5), 86.5 (d, J=2.7 Hz, C-4'), 84.8 (C-1'), 72.1 (d,
J=8.2 Hz, C-3'), 69.5 (OCH), 63.0 (C-5'), 49.2 (d, J=9.2 Hz,
OCH.sub.3), 48.8 (Me.sub.2CNH), 40.3 (d, J=3.7 Hz, C-2'), 38.7 (d,
J=4.6 Hz, CH.sub.2), 25.9 (SiCMe.sub.2), 22.9 (d, J=3.7 Hz,
NCHMe.sub.2), 18.3 (SiCMe.sub.2), 12.5 (C.dbd.CMe), -5.4
(SiMe.sub.2); .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 139.4
Example 14
Phophite Triester (20b) From Phosphoramidite 13b With Methanol
[0205] 64
[0206] Diastereomeric enriched (75%) phosphoramidite 13b (10 mg,
slow eluting component) in 0.5 ml CDCl.sub.3 was put in 5 mm NMR
tube, and 50 .mu.l dry MeOH (a large excess) was then added by
syringe, followed by 1 mg of dichloroimidazole. The reaction was
monitored by .sup.31P NMR until the reaction went to completion.
The phosphite triesters formed in a ratio of 1:2 (139.4 ppm:138.8
ppm) were purified on a silica gel column. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 7.48 (s, 1H, H-6), 6.34 (m, 1H, H-1'),
4.78-4.82 (m, 1H, H-3'), 4.25-4.33 (m, 1H, OCH), 4.11 (d, J=2.4 Hz,
1H, H-4'), 3.77-3.89 (m, 2H, H-5'), 3.49 (d, J=10.3 Hz, 3H,
CH.sub.3), 2.76 (m, 1H, NCH), 2.64 (m, 2H, NCH.sub.2), 2.39 (m, 1H,
H-2'), 2.05 (m, 1H, H-2'), 1.90 (s, 3H, C.dbd.C--CH.sub.3),
1.65-1.78 (m, 2H, OCCH.sub.2), 1.26 (d, J=6.4 Hz, 3H, OCHCH.sub.3),
1.04 (d, J=6.4 Hz, 6H, NCHMe.sub.2), 0.91 (s, 9H, SiCMe.sub.3),
0.10 (d, J=2.0 Hz, 6H, SiMe.sub.2); .sup.31P NMR (121 MHz,
CDCl.sub.3) .delta. 138.8
Example 15
Phophite Triester (32) From Phosphoramidite 13a With n-Butanol
[0207] 65
[0208] Diastereomerically pure phosphoramidite 13a (10 mg, fast
eluting component) in 0.5 ml CDCl.sub.3 was placed in 5 mm NMR
tube, and 50 .mu.l dry n-butanol (a large excess) was then added by
syringe, followed by 1 mg of dicyanoimidazole. The reactidn was
monitored by .sup.31P NMR until the reaction went to completion.
The phosphite triesters formed in a ratio of 7:1 (138.9 ppm:138.5
ppm) were purified on a silica gel column. .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 7.49 (d, J=1.5 Hz, 1H, H-6), 6.35 (dd, J=5.5,
8.8 Hz, 1H, H-1'), 4.83 (m, 1H, H-3'), 4.24-4.31 (m, 1H, OCH), 4.11
(d, J=2.0 Hz, 1H, H-4'), 3.71-3.89 (m, 4H, 2H-5', OCH.sub.2), 2.78
(m, 1H, NCH), 2.66 (m, 2H, NCH.sub.2), 2.39 (m, 1H, H-2'), 2.02 (m,
1H, H-2'), 1.89 (s, 3H, C.dbd.C--CH.sub.3), 1.63-1.77 (m, 2H,
OCMeCH.sub.2), 1.57 (m, 2H, CH.sub.2), 1.36 (m, 2H, CH.sub.2), 1.24
(d, J=6.3 Hz, 3H, OCHCH.sub.3), 1.04 (d, J=6.3 Hz, 6H,
NCHMe.sub.2), 0.91 (s, 9H, SiCMe.sub.3), 0.10 (d, J=2.0 Hz, 6H,
SiMe.sub.2); .sup.31P NMR (121 MHz, COCl.sub.3) .delta. 138.9.
Example 16
Phosphite Triester (33a) From Phosphoramidite 7a With Methanol
[0209] 66
[0210] Diastereomerically pure phosphoramidite 7a (10 mg, fast
eluting component) in 0.5 ml CDCl.sub.3 was put in 5 mm NMR tube,
and 50 .mu.l dry methanol (a large excess) was then added by
syringe, followed by 1 mg of 4,5-dicyano-2-bromoimidazole. The
reaction was monitored by .sup.31P NMR until the reation went to
completion. The phosphite triester consisted of only one
diastereomer (139.1 ppm) and was purified on a silica gel column.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.48 (s, 1H, H-6), 6.34
(dd, J=5.4, 8.8 Hz, 1H, H-1'), 4.78 (m, 1H, H-3'), 4.09 (m, 1H,
H-4'), 3.76-3.89 (m, 4H, 2H-5', OCH.sub.2), 3.50 (d, J=10.2 Hz, 3H,
OCH.sub.3), 2.80 (m, 1H, NCHMe.sub.2), 2.67 (t, J=6.8 Hz, 2H,
NHCH.sub.2), 2.39 (m, 1H, H-2'), 2.05 (m, 1H, H-2'), 1.90 (s, 3H,
C.dbd.C--CH.sub.3), 1.78 (m, 2H, OCHCH.sub.2), 1.03 (d, J=6.3 Hz,
6H, NCHMe.sub.2), 0.91 (s, 9H, SiCMe.sub.3), 0.10 (s, 6H,
SiMe.sub.2); .sup.31P NMR (121 MHz, CDCl.sub.3) .delta. 139.1; MS
(CI) m/e: 534 ([M+H.sup.+], 100%), 502 (14.4), 376 (13.6), 339
(74.6), 281 (20.4), 164 (59.8)
Example 17
Phophite Triester (33b) From Phosphoramidite 7b With Methanol
[0211] 67
[0212] Diastereomerically enriched (92%) phosphoramidite 7b (10 mg,
slow eluting component) in 0.5 ml CDCl.sub.3 was placed in 5 mm NMR
tube, and 50 .mu.l dry methanol (a large excess) was then added by
syringe, followed by 1 mg of dicyanobromoimidazole. The reaction
was monitored by .sup.31P NMR until the reaction went to
completion. Diastereomerically enriched phosphite triester 33b
(92%) in a ratio of 1:11 (139.2 ppm:138.8 ppm) was purified on a
silica gel column. .sup.31P NMR (121 MHz, CDCl.sub.3) .delta.
138.8
Example 18
Thiophosphonate (44)
[0213] 68
[0214] Diastereomerically pure 13a (100 mg, fast eluting component)
was dissolved in 5 ml CDCl.sub.3, and 0.5 ml MeOH was then added by
syringe, followed by 5 mg of 2-bromo-4,5-dicyanoimidazole. The
reaction was monitored by .sup.31P NMR until the reaction went to
completion with almost only one diastereomer of phosphite triester
20a (139.4 ppm) being found, and then 10 mg sulfur was added.
Within 5 minutes, the sulfurization went to completion. After
chromatography (ethyl acetate:triethylamine=1:1) sulfurized product
24 was obtained in an oily form. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 7.50 (d, J=1.4 Hz, 1H, H-6), 6.36 (dd, J=5.4, 9.3 Hz, 1H,
H-1'), 5.08 (m, 1H, H-3'), 4.63-4.72 (m, 1H, OCH), 4.25 (m, 1H,
H-4'), 3.88 (m, 2H, H-5'), 3.73 (d, J=13.7 Hz, 3H, OCH.sub.3), 2.77
(m, 1H, NCHMe.sub.2), 2.67 (t, J=6.4 Hz, 2H, NCH.sub.2), 2.48 (m,
1H, H-2'), 2.09 (m, 1H, H-2'), 1.90 (d, J=1.5 Hz, 3H,
C.dbd.C--CH.sub.3), 1.70-1.86 (m, 2H, OCCH.sub.2), 1.32 (d, J=5.9
Hz, 3H, OCHCH.sub.3), 1.04 (dd, J=2.9, 5.9 Hz, 6H, NCHMe.sub.2),
0.92 (s, 9H, SiCMe.sub.3), 0.12 (s, 6H, SiMe.sub.2); .sup.31P NMR
(121 MHz, CDCl.sub.3) .delta. 67.5
Example 19
2-Bromo-4,5-dicyanoimidazole (21)
[0215] 69
[0216] To 1.18 g (10 mmol) 4,5-dicyanoimidazole and 25 ml 0.1 M
NaOH was added 1.8 ml Br.sub.2 (35 mmol). The mixture was stirred
overnight at room temperature and then acidified with dilute HCl.
The solid was filtered, rinsed with water and recrystallized from
water to give 1.5 g of dicyanobromoimidazole 21 (yield 76.4%).:
m.p. 147-149.degree. C. (lit. 141-143.degree. C.); Rf=0.65 (ethyl
acetate:methanol=4:1); MS (EI): 198 ([M+2], 96%), 196 ([M.sup.+],
100%), 171 (28.5), 169 (29.2), 117 (27.4), 91 (19.0), 64 (20.6), 53
(22.4), 38 (18.8).
Example 20
Synthesis of (S)-Methyl-3-(5-imidazolyl)-2-hydroxypropionate (3)
Hydrochloride
[0217] 70
[0218] (L)-Histidine (3.103 g, 20 mmol) was first dissolved in 30
ml of 1 N hydrochloric acid solution. This solution was cooled down
to 0.degree. C., then a solution of sodium nitrite (2.070 g, 30
mmol) in 10 ml distilled water was added dropwise over a period of
1 hour. The solution was stirred overnight at 0.degree. C., then
evaporated to dryness in vacuo, with heating. 20 ml of distilled
water was added to the solid residue, the mixture was evaporated
once more with toluene in order to azeotropically remove the water
residue as much as possible. After drying the compound in high
vacuum overnight and without isolation of the intermediate acid,
the mixture was dissolved into 50 ml of dry methanol and stirred
under Ar. This solution was cooled down to 0.degree. C. and a
stream of gaseous hydrogen chloride was bubbled through the
mixture. After 1.5 hours, TLC indicated disappearance of the acid
and the reaction was stopped. The mixture was evaporated in vacuo
with heating to yield a sticky yellow solid that could be
crystallized from a mixture of ethanol and ether to yield 3.10 g of
(2). HCl, m.p. 139-142.degree. C., [.alpha.].sub.D-21.degree. (c
1.9, methanol, 25.degree. C.) (litt. -22.degree.) .sup.1HNMR (200
MHz, CD.sub.3OD) .delta. 8.90 (s, 1H, NCHN); 7.30 (s, 1H, NCHC);
4.40 (dd, ABX, .sup.3J.sub.Ha-Hx=5.40 Hz, .sup.3J.sub.Hb-Hx=5.00
Hz, CHOH); 3.72 (s, 3H, OCH.sub.3); 2.85-3.10 (ABX, 2H,
.sup.2J.sub.Ha-Hb=13.75 Hz, .sup.3J.sub.Hb-Hx=5.0 Hz,
.sup.3J.sub.Ha-Hx=5.4 Hz, CH.sub.2); .sup.13C NMR (non decoupled)
(125 MHz, D.sub.2O ) .delta. 175.3 (s, CO); 134.2 (d, NCHN); 129.7
(s, CH.sub.2CN); 117.6 (d, CCHN); 69.9 (d, CHOH); 52.6 (q,
CH.sub.3); 29.5 (t, CH.sub.2); M.S. (M+1).sup.+171
Example 21
Synthesis of Imidazo-oxazaphospholidine (4)
[0219] 71
[0220] In scrupulously dry glassware, compound (3) (0.236 g, 1.39
mmol) was added and dried in vacuo overnight, then put under an
atmosphere of Ar. This compound was suspended into 5 ml of dry
ether, then triethylamine (0.20 ml, 3.15 mmol) was added. The
suspension was cooled down to 0.degree. C. and stirred under Ar.
Then, methyl dichlorophosphite (0.15 ml, 1.58 mmol) was syringed
Unto the mixture quickly. As soon as the phosphite was introduced,
a thick white precipitate was observed, corresponding to the
formation of triethylammonium chloride. After 15 min, .sup.31P NMR
showed several signals between 176 and 120 ppm, as well as after 2
to 4 hours. After overnight stirring at room temperature, .sup.31P
NMR showed a single signal at 143.5 ppm. Compound decomposed upon
trying to handle it (dilution in dry ether, filtration in an Ar
atmosphere, concentration by evaporation of the ether) as indicated
by .sup.31P NMR by several peaks at 5-20 ppm, corresponding most
likely to H-phosphonates derivatives. Changing the reaction
conditions did not bring any improvement. Therefore, compound (4)
could not be further purified and analyzed.
Example 22
Synthesis of 1-Tritylimidazole (5)
[0221] 72
[0222] To a solution of trityl chloride (5.58 g, 20.0 mmol.) in dry
methylene chloride (100 ml) cooled down to 0.degree. C. and stirred
under Ar, was added dropwise over 1.5 hours a solution of imidazole
(1.36 g, 20.0 mmol.) and triethylamine (2.7 mml, 20 mmol.) in 50 ml
dry methylene chloride. At the end of the addition, the reaction
mixture was allowed to warm up to room temperature and stirred
under Ar at that temperature overnight. The reaction mixture was
then washed with 20 ml of a 10% solution of ammonium chloride, then
with 20 ml of distilled water. The organic phase was dried over
magnesium sulfate and evaporated in vacuo to yield quantitatively a
white solid. Recrystallization from methylene chloride/hexanes
yielded 5.60 g of (5) (yield=90% after recrystallization).
[0223] m.p. 214.degree. C.; .sup.1H NMR (200 MHz, CDCl.sub.3)
.delta. 7.43 (m, 1H, NCHN), 7.3-7.4 (m, 9H,
3.times.C.sub.6H.sub.5), 7.1-7.2 (m, 6H, 3.times.C.sub.6H.sub.5),
7.0 (m, 1H, Ph.sub.3CNCH.dbd.CH), 6.81 (m, 1H,
Ph.sub.3CNCH.dbd.CH); .sup.13 C NMR (50 MHz, CDCl.sub.3) .delta.
142.3, 139.0, 129.6, 128.2, 128.3, 121.6.
Example 23
Synthesis of (S)-1-(2-(1-Triphenylmethyl)-imidazolyl)-propan-2-ol
(S)-(6)
[0224] 73
[0225] To a solution of N-tritylimidazole (1.55 g, 5 mmol) in
freshly distilled TKF (50 ml) cooled down to -78.degree. C. and
stirred under dry Ar, was added a 2.5 M solution of n-butyllithium
in pentane (2.4 ml, 6 mmol). The addition lasted for 30 min, and
the deep red solution obtained was allowed to warm up to 0.degree.
C., stirred at room temperature for 1 hour, then cooled down again
to -78.degree. C. At that temperature, (S)-propylene oxide (0.35g,
6 mmol) was added dropwise. After 30 min, the solution was allowed
to warm up to 0.degree. C. and was stirred at that temperature for
12 hours until TLC indicated that the reaction did not further
proceed. The solution was poured into 50 ml of saturated NH.sub.4Cl
solution, and the resulting mixture was extracted with
CH.sub.2Cl.sub.2. After flash chromatography
(hexane:acetone:triethylamin- e 78:21:1), 1.44 g of the pure
product was collected in 78% yield: m.p. 201.degree. C.; .sup.1H
NMR (200 MHZ, CDCl.sub.3) .delta. 7.20-7.40 (m, 9H,
3.times.C.sub.6H.sub.5), 7.10-7.18 (m, 6H, 3.times.C.sub.6H.sub.5),
6.90 (d, 1H, .sup.3J.sub.H-H=1.2; CHNCPh.sub.3), 6.71 (d, 1H,
.sup.3J.sub.H-H=1.2; CH.dbd.CHNCPh.sub.3), 5.83 (b, 1H, OH),
3.40-3.60 (m, 1H, CHCH.sub.3), 1.78-2.05 (ABX, 2H,
.sup.3J.sub.Hb-Hx=3.2 Hz, .sup.3J.sub.Ha-Hx=8.5 Hz,
.sup.2J.sub.Ha-Hb=16.2 Hz, CH.sub.2), 0.81 (d, .sup.3J.sub.H-H=6.0
Hz, 3H, CH.sub.3); .sup.13C NMR (50 Mhz, CDCl.sub.3) .delta.
.sub.--149.2, 142.1, 129.6, 127.9, 127.7, 124.7, 121.0 (NCCN),
74.6, 65.0 (CHOH), 38.1 (CH.sub.2), 22.3 (CH.sub.3).
Example 24
Synthesis of (S)-1-(2-Imidazolyl)-propan-2-ol (S)-(7)
[0226] 74
[0227]
[0228] A solution of N-tritylimidazolylpropanol (S)-(6) (2.39 g,
6.51 mmol) in 80 ml methanol containing 4.3 ml glacial acetic acid
(5%) was refluxed for about 12 hours. After that time, TLC
indicated disappearance of the starting materials. The mixture was
concentrated in vacuo and a white precipitate appeared upon
addition of 50 ml of cold distilled water. The mixture was chilled,
then filtered, and the white precipitate was washed with cold
distilled water (10 ml). The filtrate was then evaporated twice,
and the residual yellow oil redissolved in 50 ml dry methanol and
passed through the weakly basic anion exchange resin (hydroxide
form) IRA-68. The solution was then evaporated to yield a solid
residue that could be recrystallized from a mixture of methanol and
ethyl acetate. yield: 0.80 g, 98% of pure (S)-(7). m.p.
119-121.degree. C.; .sup.1H NMR (200 Mhz, CD.sub.3OD) .delta. 6.96
(s, 2H, NCHCHN); 3.96 (m, 1H, CHCH.sub.3); 2.4-2.65 (ABX,
.sup.3J.sub.Ha-Hx=6.3 Hz, .sup.3J.sub.Hb-Hx=6.7 Hz,
.sup.3J.sub.Ha-Hb=14.5 Hz, CH.sub.2); 0.87 (d, 3H,
.sup.3J.sub.H-H=6.3 Hz, CH.sub.3); .sup.13C NMR (50 MHz,
CD.sub.3OD) .delta. 145.8 (s, CH2CNH); 121.2 (d, NCH.dbd.CHNH);
67.1 (d, CHOH); 38.4 (t, CH2); 23.1 (q, CH3); MS (CI): [M+1].sup.+.
127
Example 25
Synthesis of
1-Methoxy-3-methyl-imidazo-[2,1-e]-(3,4-dihydro)-oxazaphospho- rine
(8)
[0229] 75
[0230] In an NMR tube previously dried in vacuo and under Ar was
introduced 23.0 mg (0.20 mmol) of (S)-(2-imidazolyl)-propan-2-ol
(S)-(7), then the tube was sealed with a septum and flushed with
Ar. CDCl.sub.3 (0.7 ml) was then introduced, followed by 127 .mu.l
(1.0 mmol) of triethylamine. The alcohol did not dissolve, and this
suspension was cooled down to 0.degree. C. while shaking the tube.
At that temperature, 18.9 .mu.l (0.20 mmol) of methyl
dichlorophosphite was syringed inside the tube. Upon shaking, the
alcohol dissolved instantaneously, and an exothermic reaction was
noticed. After about 1 hour, .sup.31P NMR revealed the presence of
several peaks around 120-140 ppm. After about 3 hours, a single
product (8) was observed by .sup.31P NMR, as evidenced by its
chemical shift at 118.8 ppm. There was no further characterization
of this compound, as all efforts to isolate it have been
unsuccessful and have led to the hydrolysis of this extremely water
sensitive bicyclic structure, most likely to the corresponding
H-phosphonate.
Example 26
Synthesis of
1-Ethoxy-3-methyl-imidazo-[2,1-e]-(3,4-dihydro)-oxazaphosphor- ine
(9)
[0231] 76
[0232] To an NMR tube previously dried in vacua and under Ar, was
introduced 18.9 mg (0.15 mmol) of (S)-(2-imidazolyl)-propan-2-ol
(S)-(7). The tube was then sealed with a septum and flushed with
Ar. Then, 0.7 ml CDCl.sub.3 was introduced, followed by 105 .mu.l
(0.75 mmol) of triethylamine. The alcohol did not dissolve, and
this suspension was cooled down to 0.degree. C. while shaking the
tube. At that temperature, 17.2 .mu.l (0.15 mmol) of ethyl
dichlarophosphite was syringed into the NMR tube. Upon shaking, the
alcohol dissolved instantaneously, and an exothermic reaction was
noticed. After about 2 hours, a single product was observed by
.sup.31P NMR as evidenced by its chemical shift at 118.2 ppm. After
about 1 hour, the presence of other products around 120-140 ppm was
noticed, one of which (121.1 ppm) is probably the other
diastereomer. There was no further characterization of this
compound.
Example 27
Synthesis of Thiophosphate Ethyl (S)-1-(2-Imidazolyl)-prop-2-yl
Isopropyl Ester (22)
[0233] 77
[0234] In an NMR tube, to a suspension of 18.9 mg (0.15 mmol)
(S)-1-(2-imidazolyl)-propan-2-ol (S)-(7) in 0.7 ml dry deuterated
chloroform and 0.21 ml triethylamine (1.5 mmol), shaken at room
temperature under Ar was introduced with a syringe 17.2 .mu.l (0.15
mmol) of ethyl dichlorophosphite. The reaction mixture was shaken
at room temperature and the chiral imidazolylpropanol dissolved
immediately in an exothermic process. At that point, .sup.31P NMR
indicated the formation of several products, among which was one
having a signal at 118.3 ppm and one having a signal at 120.5 ppm.
(suspected diastereomers). After one hour and regular shaking of
the NMR tube, .sup.31P NMR indicated only one peak at 119.3 ppm. At
that stage, 20 .mu.l (0.45 mmol) of iso propanol was introduced,
and the tube was shaken again. .sup.31P NMR indicated after 20 min
the presence of a single peak at 140.6 ppm, indicating that the
displacement of the imidazole moiety had given rise to a single
diastereomer (19). 32 mg sulfur (1 mmol) was then introduced and
the .sup.31P-NMR was again recorded. The spectrum indicated a
single peak at 64.8 ppm. The product (22) was then concentrated in
vacuo and purified by flash chromatography (ethyl
acetate/hexanes/triethylamine 79/20/1). .sup.1H NMR (200 MHz,
CDCl.sub.3) 6.96 (s, 2H, NCH.dbd.CH--N); 4.83-4.97 (m, 1H,
P--O--CH--CH.sub.3); 4.58-4.75 (dh, 1H, .sup.3J.sub.H-H=6.2 Hz,
.sup.3J.sub.H-P=9.6 Hz, OCH (CH.sub.3).sub.2); 3.96-4.12 (m, 2H,
P--O--CH.sub.2CH.sub.3); 2.98-3.20 (2.times.ABX, 2H,
.sup.2J.sub.Ha-H=15.5 Hz, .sup.3J.sub.Ha-Hx=6.23 Hz,
.sup.3J.sub.Hb-Hx=4.6 Hz, .sup.4J.sub.H-P=1.5 Hz,
CH.sub.2CHCH.sub.3); 1.21-1.34 (m, 12H,
CH(CH.sub.3)+CH(CH.sub.3).sub.2+CH.sub.2CH.sub.3) .sup.13C NMR (50
MHz, CDCl.sub.3) 144.00 (s, N--C.dbd.N); 121.70 (s, N--C.dbd.C--N);
74.90 (d, .sup.2J.sub.C-P=6.1 Hz, P--O--CH--(CH.sub.3)CH.- sub.2);
73.80 (d, .sup.2J.sub.C-P=5.7 Hz, P--O--CH.sub.2--CH.sub.3); 64.25
(d, .sup.2J.sub.C-P=5.8 Hz, P--O--CH(CH.sub.3).sub.2); 35.95 (s,
P--O--CH(CH.sub.3)CH.sub.2--); 23.40 (s, P--O--CH(CH.sub.3).sub.2);
21.06 (s, P--O--CH(CH.sub.3)CH.sub.2--); 15.83 (s,
P--O--CH.sub.2--CH.sub.3) .sup.31P NMR (81 MHz, CDCl.sub.3) .delta.
64.8 ppm M.S. (CI), [M+1].sup.+. 293.
Example 28
Synthesis of Thiophosphate Ethyl (S)-1-(2-Imidazolyl)-prop-2-yl 5
Terbutyldimethylsilylthysidinyl Ester (19)
[0235] 78
[0236] To a suspension of (S)-imidazolylpropanol (S)-(7) (0.30
mmol, 37.8 mq) in 2 ml dry dichloromethane and triethylamine (1.5
mmol, 0.21 ml) cooled down to 0.degree. C. and stirred under Ar,
was slowly added ethyl dichlorophosphite (0.30 mmol, 35 .mu.l). The
reaction mixture was then allowed to warm up to room temperature,
the solid starting materials dissolved, and after about 2 hours,
.sup.31P NMR indicated the presence of a single peak at 118.3 ppm.
At that time, the mixture was cooled down to 0.degree. C. again,
and at that temperature was added a mixture of 5'-tBDMS-thymidine
(1) (0.30 mmol, 106 mg) in 1.5 ml of dry methylene chloride. The
reaction mixture was allowed to warm up to room temperature again.
.sup.31P NMR indicated, after 30 min, a full conversion of the peak
at 118.3 ppm to a single peak at 141.2 ppm, assigned to (23).
Elemental sulfur S.sub.8 (0.9 mmol, 29 mg) was then added after
about 30 minutes. .sup.31P NMR indicated conversion of the
previously formed product to a single product (24) with a peak at
66.3 ppm. Evaporation of the reaction mixture followed by flash
chromatography (ethyl acetate/triethylamine 80/20) afforded thioate
(24) as a sticky solid (127 mg, 72%). .sup.1H NMR (300 MHz,
CD.sub.2Cl.sub.2) .delta. 7.48 (d, 1H, .sup.4J.sub.H-H=1.3 Hz,
C.dbd.CH); 6.94 (s, 2H, NCH.dbd.CHN); 6.25 (dd, 1H,
.sup.1J.sub.H-H=5.1 Hz, .sup.3J.sub.H-H=9.2 Hz, NCHO); 4.84-5.05
(m, 2H, POCH(CH.sub.3)CH.sub.2+POCHCH.sub.2O); 4.15-4.24 (m, 1H,
SiOCH.sub.2CHO); 3.96-4.14 (dq, 2H, .sup.3J.sub.H-P=9.4 Hz,
.sup.3J.sub.H-H=7.0 Hz, POCH.sub.2CH.sub.3); 3.76-3.92 (ABX, 2H,
.sup.2J.sub.Ha-Hb=11.5 Hz, .sup.3J.sub.Hb-Hx=2.5 Hz,
.sup.3J.sub.Ha-Hx=2.4 Hz, SiOCH.sub.2CHO); 2.98-3.08 (dd, 2H,
.sup.3J.sub.H-H=5.8 Hz, .sup.4J.sub.H-P=1.1 Hz,
POCH(CH.sub.3)CH.sub.2-Im- ); 1.98-2.08 (m, 2H, POCHCH.sub.2CHN);
1.88 (s, 3H, CH.dbd.CCH.sub.3); 1.39 (d, 3H, .sup.3J.sub.H-H=6.2
Hz, POCH(CH.sub.3)); 1.28 (dt, 3H, .sup.3J.sub.H-H=7.0 Hz,
.sup.4J.sub.H-P=0.9 Hz, POCH.sub.2CH.sub.3); 0.91 (s, 9H,
SiC(CH.sub.3).sub.3); 0.12 (s, 6H, Si(CH.sub.3).sub.2).
[0237] .sup.13C NMR (121 MHz, CD.sub.2Cl.sub.2, decoupled from
.sup.1H) .delta. 164.1 (s, NCOC(CH.sub.3)); 151.1 (s, NCON); 144.2
(s, NC.dbd.N); 135.5 (s, NC.dbd.C(CH.sub.3)C.dbd.O); 132.4 (s,
NC.dbd.CN); 186.1 (s, 11.5 (C(CH.sub.3)CO); 86.1 (s,
SiOCH.sub.2CHO); 86.0 (s, NCHO); 79.9 (d, .sup.2J.sub.C-P=4.4 Hz,
POCH(CH.sub.3)); 76.2 (d, .sup.2J.sub.C-P=5.7 Hz,
POCH.sub.2CH.sub.3); 65.0 (d, .sup.2J.sub.C-P=5.6 Hz, POCHCHO);
63.8 (s, SiOCH.sub.2); 39.4 (s, POCHCH.sub.2CHN); 36.6
(POCH(CH.sub.3)CH.sub.2); 26.1 (SiC(CH.sub.3).sub.3); 21.3
(POCH(CH.sub.3)); 18.6 (SiC(CH.sub.3).sub.3); 16.0 (s,
POCH.sub.2CH.sub.3); 12.7 (s, C.dbd.CCH.sub.3); -5.4 (d,
Si(CH.sub.3).sub.2); MS (CI) (M+1).sup.+. 589.
Example 29
1,2-O-3,5-O-Dicyclopentylidene-D-xylofuranose (25)
[0238] 79
[0239] To a solution of trimethyl orthoformate (5 mmol, 547 .mu.l)
and p-toluene sulfonic acid (0.2 mmol, 38 mg) in dioxane (10 ml)
under a nitrogen atmosphere at 0.degree. C., was added dropwise
cyclopentanone (40 mmol, 3.5 ml). This solution was stirred at room
temperature for 2 hours and D-xylose (2 mmol, 300 mg) was added
with stirring continued overnight. Then the reaction mixture was
neutralized with triethylamine. Evaporation of the solvent
furnished a yellow syrupy residue. A solution of the syrupy residue
in chloroform (20 ml) was washed with water (20 ml). The aqueous
layer was extracted with chloroform (3.times.15 ml). The combined
chloroform layers were dried (MgSO.sub.4). After removing the
solvent, the mixture was chromatographed on a silica gel column
(Hexane:Ethyl acetate=5:1) to give 340 mg white solid (60%).
.sup.1HNMR(200 MHz, CDCl3) .delta. 5.99 (.delta., J=3.80 Hz, 1H,
H-1), 4.45 (d, J=3.81 Hz, 1H, H-2), 4.24 (d, J=2.15 Hz, 1H, H-3),
4.14-3.46 (m, 3H, H-4, 2.times.H-5), 1.98-1.55 (m, 16H,
cyclopentylidene protons); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 121.23 (C-1OCOC-2), 109.24 (C-3OCOC-5), 105.08 (C-1), 84.49
(C-2), 74.25 (C-4), 71.83 (C-3), 61.50 (C-5), 39.52, 36.82, 36.19,
29.74, 24.09, 23.49, 22.80, 22.37 (cyclopentylidene carbons); MS
(CI) m/e: 283 (M+H.sup.+).
Example 30
1,2-O-Cyclopentylidene-D-xylofuranose (26)
[0240] 80
[0241] The 1,2-O-3,5-O-dicyclopentylidene-D-xylofuranose (25) (1
mmol, 282 mg) was dissolved in acetic acid-water (2:1) (14 ml) at
room temperature. The reaction mixture was stirred for 3 hours,
followed by TLC. Solvent was evaporated with high vacum, and
coevaporated with methanol three times and dried in vacuo overnight
to give 196 mg white solid (91%). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 5.94 (d, J=3.91 Hz, 1H, H-1), 4.44 (d, J=3.91 Hz, 1H, H-2),
4.32 (d, J=2.44 Hz, 1H, H-3), 4.18 (m, 1H, H-4), 4.10-4.02 (m, 2H,
2.times.H-5), 1.96 1.61 (m, 8H, cyclopentylidene protons); .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta. 121.41 (OCO), 104.53 (C-1), 85.59
(C-2), 78.76 (C-3), 76.83 (C-4), 61.07 (C-5), 36.84, 36.19
(CH.sub.2CCH.sub.2), 23.47, 22.80
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2); MS (CI): m/e 217
(M+H.sup.+).
Example 31
1,2-O-Cyclopentylidene-5'-O-tosyl-D-xylofuranose (27)
[0242] 81
[0243] To a solution of 1,2-O-cyclopentylidene-D-xylofuranose (26)
(0.81 mmol, 176 mg) in dry pyridine (6 ml) under nitrogen
atmosphere at 0.degree. C., p-toluenesulfonyl chloride (1.12 eq.,
173 mg) was added. The reaction mixture was stirred at 0.degree. C.
for 3 hours. Then 5 ml water was added to quench the reaction, and
the solvent was evaporated in high vacuum, and coevaporated with
toluene twice. The mixture was dissolved in chloroform, washed with
water three times and dried over MgSO.sub.4. Removal of the solvent
furnished 259 mg (27) as a white solid (86%). .sup.1H NMR (200 MHz,
CDCl.sub.3) .delta. 7.80-7.32 (AA'BB', 4H, Ph), 5.84 (d, J=3.63 Hz,
1H, H-1), 4.44 (d, J=3.7 Hz, 1H, H-2), 4.42-4.29 (m, 3H, H-3,
2.times.H-5), 4.27-4.09 (m, 1H, H-4), 2.44 (s, 3H, CH.sub.3),
1.89-1.61 (m, 8H, cyclopentylidene protons); .sup.13C NMR (270 MHz,
CDCl.sub.3) 145.34, 132.30, 130.06, 128.08 (aromatic), 121.86
(OCO), 104.80 (C-1), 85.15 (C-2), 77.73 (C-3), 74.41 (C-4), 66.21
(C-5), 37.02, 36.44 (CH.sub.2CCH.sub.2), 23.54, 22.98
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.- 2), 21.73 (CH.sub.3); MS (CI):
m/e 371 (M+H.sup.+).
Example 32
1,2-O-Dicyclopentylidene-5'-isopropylamine-D-xylofuranose (28)
[0244] 82
[0245] A solution of 1,2-O-cyclopentylidene-5'-tosyl-D-xylofuranose
(27) (7.1 mmol, 2.64 g) in isopropylamine (15 ml) was stirred at
55.degree. C. over night in a pressure bottle. The solvent was
removed by rotary evaporator and the remaining yellow syrup was
taken up with chloroform and washed with a saturated solution of
sodium bicarbonate, and then with brine. The organic phase was
dried over MgSO.sub.4, the solvent was evaporated and the residue
was flash chromatographed on silica gel (ethyl acetate-3%
triethylamine) to furnish 1.33 g (28) as a white solid (73%), m.p.
44-45.degree. C.; [.alpha.].sub.D.sup.20=31.06 (C-2, CHCl.sub.3);
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.0 (bs, NH) 5.90 (d,
J=3.91 Hz, 1H, H-1), 4.38 (d, J=3.91 Hz, 1H, H-2), 4.27 (d, J=2.44
Hz, 1H, H-3), 4.20 (d, J=2.93 Hz, 1H, H-4), 3.36-2.92 (ABX, 2H,
2.times.H-5), 2.74-2.70 (heptet, 1H, NCH), 1.95-1.63 (m, 8H,
cyclopentylidene protons), 1.04-1.03 (dd, 6H, Me.sub.2CH); .sup.13C
NMR (500 MHz, CDCl.sub.3) .delta. 121.06 (OCO), 104.82 (C-1) 86.14
(C-2), 78.30 (C-3), 77.07 (C-4), 48.64 (NCH), 45.90 (C-5), 36.85,
36.32 (CH.sub.2CCH.sub.2), 23.51, 22.84
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 22.64 (CH.sub.3CHN), 22.34
(CH.sub.3CHN); MS (CI): m/e 258 ([M+H.sup.+], 100%); HRMS (EI) m/e
calc'd for C.sub.13H.sub.23NO.sub.4) [M.sup.+]: 257.16270, found
257.1630.
Example 33
Chlorophosphoramidite (29)
[0246] 83
[0247] In a scrupulously dry NMR tube, 9.6 .mu.l (0.11 mmol)
phosphorus trichloride was placed via syringe, followed by 0.25 ml
CDCl.sub.3. The NMR tube was cooled at 0.degree. C., and a solution
of the 1,2-O-dicyclopentylidene-5'-isopropylamino-D-xylofuranose 28
(25.7 mg, 0.1 mmol) and triethylamine (27.8 ul, 0.22 mmol) in
CDCl.sub.3 (0.35 ml) were added under a nitrogen atmosphere with
shaking of the NMR tube. An exothermic reaction was noticed. The
NMR tube was then cooled to -78.degree. C., pumped and sealed. The
sealed NMR tube was heated to 40.degree. C. and the reaction was
followed by .sup.31P NMR until a single peak was found in the
.sup.31PNMR spectrum. The product was not isolated, and was used
directly in the following step. .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 5.75 (d, J=3.91 Hz, 1H, H-1), 4.51 (t, J=2.44 Hz, J=2.93
Hz, H-3), 4.37 (d, J=3.91 Hz, 1H, H-2), 4.19-4.17 (m, 1H, H-4),
3.42-3.34 (heptet, 1H, NCH), 3.33-3.00 (ARX, 7H, 2.times.H-5),
1.81-1.51 (m, 8H, cyclopentylidene protons), 1.05 (d, J=6.84 Hz,
6H, Me.sub.2CH); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 121.48
(OCO), 104.26 (C-1), 83.86, 83.83 (d, J=3.66 Hz, C-2), 73.04, 72.99
(d, J=6.41 Hz, C-3), 72.35 (C-4), 50.42, 50.13 (d, J=35.72 Hz,
C-5), 36.95, 36.91 (d, J=5.50 Hz, NCH), 36.62, 35.91
(CH.sub.2CCH.sub.2), 23.22, 22.50
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 20.89 20.79 (d, J=12.82 Hz,
CH.sub.3CHN), 19.64, 19.60 (d, J=5.50 Hz, CH.sub.3CHN); .sup.-P NMR
(202 MHz, CDCl.sub.3, 8 148.42.
Example 34
5'-O-(tert-Butyl Dimethyl Silyl)-thymidine-3'-O-phosphoramidite
(30)
[0248] 84
[0249] To the same NMR tube from the example 29, a solution of
5'-O-(tert-butyldimethylsilyl)-thymidine (35.6 mg, 100.1 mmol) in
0.45 ml CDCl.sub.3 and triethylamine (14 .mu.l, 0.11 mmol) was
added slowly at 0.degree. C. under nitrogen atmosphere. Then the
NMR tube was cooled to -78.degree. C., pumped and sealed. The
sealed NMR tube was heated to 50.degree. C. and the reaction was
followed by .sup.31P NMR until a single peak corresponding to a new
product was found in the .sup.31PNMR spectrum. The solution was
poured into flask and taken up with ethyl acetate (prewashed with a
saturated solution of sodium bicarbonate) and washed with saturated
solution of sodium bicarbonate. The organic layer was dried over
MgSO.sub.4, the solvent was removed to furnish a white foam in a
quantatitive yield. The crude pruduct was flash chromatographed on
a silica gel column (hexane-ethyl acetate-triethylamine=5:3:2);
m.p. 68-70.degree. C.; [.alpha.].sub.D.sup.20=62.9.degree. (c=0.5,
CHCl.sub.3) .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.98 (bs, 1H,
NH), 7.46 (d, J=1.00 Hz, 1H, H-6), 6.34-6.31 (dd, J=5.86 Hz, J=8.30
Hz, 1H, H-1'), 5.88 (d, J=3.91 Hz, 1H, H-1"), 4.56-4.53 (m, 1H,
H-3'), 4.41 (d, J=3.91 Hz, 1H, H-2"), 4.35 (m, 1H, H-3"), 4.17 (d,
J=1.95 Hz, 1H, H-4"), 4.05 (d, J=1.95 Hz, 1H, H-4'), 3.89-3.76
(ABX, 2H, 2.times.H-5'), 3.45-3.39 (m, 2H, H-5", NCH), 3.03-2.99
(m, 1H, H-5"), 2.37-2.34 (m, 1H, H-2'), 2.09-2.05 (m, 1H, H-2'),
1.88 (d, J=1.47 Hz, 3H, MeC.dbd.C), 1.95-1.61 (m, 8H,
cyclopentylidene protons), 1.11-1.00 (m, 6H, Me.sub.2CH), 0.92 (s,
9H, t-BuSi), 0.09 (d, J=1.47 Hz, 6H, Me.sub.2Si); .sup.13C NMR (125
MHz, CDCl.sub.3) .delta. 163.77 (C-4), 150.35 (C-2), 135.17 (C-6),
121.42 (OCO), 110.96 (C-5), 104.59 (C-1"), 86.38, 86.36 (d, J=2.75
Hz, C-4'), 84.76 (C-1'), 84.74 (C-2"), 73.78, 73.63 (d, J=19.23 Hz,
C-3'), 73.07, 73.05 (d, J=1.83, C-4"), 71.83, 71.80 (d, J=3.66 Hz,
C-3"), 63.17 (C-5'), 50.09, 49.80 (d, J-36.63 Hz, NCH), 40.27,
40.23 (d, J=4.58 Hz, C-2'), 36.89, 36.20 (CH.sub.2CCH.sub.2),
36.11, 36.08 (d, J=3.21H.sub.8, C-5"), 25.91 (SiCMe3), 23.44, 22.76
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 22.02, 21.95 (d, J=9.16 Hz,
CH.sub.3CHN), 21.69, 21.64 (d, J=6.41 Hz, CH.sub.3CHN), 18.31
(SiCMe3), 12.51 (CH.sub.3C.dbd.C), -5.41, -5.48 (d, J=9.16 Hz,
Me.sub.2Si); .sup.31P NMR (81 MHz, CDCl.sub.3) .delta. 130.14; MS
(CI): m/e 642 ([M+H.sup.+], 78%); EI m/e [M.sup.+] 641, HRMS (EI)
m/e calc'd for C.sub.28H.sub.45N.sub.3O.sub.9PSi [M.sup.+-CH.sub.3]
626.26625, found 626.26670, and calc'd
C.sub.23H.sub.39N.sub.3O.sub.9PSi [M.sup.+-C.sub.4H.sub.9]:
584.21930, found 584.2190. HRMS FAB (glycerol) m/e calcd. for
C.sub.29H.sub.49N.sub.3O.sub.9PSi [MH.sup.+] 642, 2975; found
642.2973.
Example 35
Protected Phosphorothioate Dinucleotide (34)
[0250] 85
[0251] In a dried NMR tube, phosphoramidite 30 (15 mg, 0.0234
mmol), 3'-O-(tert-butyldimethylsilyl)thymidine (10 mg, 1.2 eq.) and
4,5-dicyano-2-bromo-imidazole 21 (9.17 mg, 2.0 eq.) were added.
Then the NMR tube was dried in vacuum overnight. Dry acetonitile
(0.6 ml) was injected into the NMR tube at 0.degree. C. under
nitrogen atmosphere. The solid dissolved instantly. The reaction
was moved to room temperature and was followed by .sup.31P NMR.
Within 5 minutes, the peak corresponding to the phosphoramidite
disappeared and two new peaks (143.76 ppm, 142.55 ppm=6:1) were
formed. The clean .sup.31P NMR spectrum suggested use of this
product directly in the following sulphurization without isolation.
To this solution, Beaucage's reagent (5.6 mg, 1.2 eq.) in 140 ul
acetonitrile (0.2M) was added. Instantaneously the .sup.31P NMR
showed another two peaks (68.43 ppm, 68.23 ppm=1:6), while the
peaks corresponding to the starting material disappeared. The
solution in NMR tube was transfered to a flask. Then the mixture
was redisolved in ethyl acetate, washed with saturated sodium
bicarbonate and water, and dried over MgSO.sub.4. Removal of the
solvent gave 22 mg of white solid 34 (91%). The product was then
purified by chromatography on a sllica gel column (ethyl
acetate:methanol=95:5). .sup.1H NMR (500 MHz, DMSO-d6) .delta. 7.45
(s, 1H, .sup.3H-6), 7.42 (s, 1H, .sup.5H-6), 6.19-6.15 (m, 2H,
.sup.5H-1', .sup.3H-1'), 5.86, 5.85 (d, J=3.91 Hz, 1H, H-1"),
5.05-5.04 (m, 1H, .sup.5H-3'), 4.78-4.77 (m, 1H, H-3"), 4.60, 4.59
(d, J=3.42 Hz, 1H, H-2"), 4.38-4.36 ((m, 1H, H-4"), 4.26-4.13 (m,
4H, .sup.1H-3', 2.times..sup.3H-5', .sup.1H-4'), 3.93, 3.91 (m, 1H,
.sup.3H-4'), 3.81-3.73 (m, 2H, 2.times..sup.5H-5'), 2.66-2.52 (m,
3H, NCH, 2.times.H-5"), 2.43-2.40 (m, 1H, .sup.5H-2'), 2.31-2.22
(m, 2H, 2.times..sup.3H-2'), 2.09-2.07 (m, 1H, .sup.5H-2'), 1.78
(s, 3H, .sup.3CH.sub.3C.dbd.C), 1.76 (s, 3H,
.sup.5CH.sub.3C.dbd.C), 1.62-1.56 (m, 8H, cyclopentylidene
protons), 0.94-0.89 (m, 6H, Me.sub.2CHN), 0.36 (s, 9H,
.sup.3t-BuSi), 0.85 (s, 9H, .sup.5t-BuSi), 0.069 (s, 6H,
Me.sub.2Si), 0.065 (s, 6H, Me.sub.2Si); .sup.13CNMR (125 MHz,
CDCl.sub.3) .delta. 163.97, 163.66 (.sup.5C-4, .sup.3C-4), 150.39,
150.04 (.sup.5C-2, .sup.3C-2), 135.58, 134.49 (.sup.5C-6,
.sup.3C-6), 121.77 (OCO), 111.26, 111.04 (.sup.5C-5, .sup.3C-5),
104.00 (C-1"), 85.59, 85.56 (d, J=6.41 Hz, .sup.5C-4'), 85.48
(.sup.3C-1'), 84.69 84.61, 84.39 (.sup.5C-1', .sup.3C-4'), 83.47
(C-2"), 80.85, 80.64 (d, C-3"), 80.69, 80.66 (d, .sup.5C-3'),
79.03, 78.97 (d, .sup.3C-3'), 71.49 (C-4"), 67.66, 67.52 (d, J=4.58
Hz, .sup.3C-5'), 63.47 (.sup.5C-5'), 48.93 (NCH), 45.23 (C-5"),
40.37 (.sup.3C-2'), 39.14, 39.09 (d, J=6.41 Hz, .sup.5C-2'), 37.06,
36.16 (CH.sub.2CCH.sub.2), 25.88, 25.60 (.sup.5SiCMe.sub.3,
.sup.3SiCMe.sub.3), 23.52, 22.85
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 22.65 22.39 (NCMe.sub.2),
18.28, 17.81 (.sup.5SiCMe.sub.3, 3SiCMe.sub.3), 12.51, 12.46
(.sup.5C.dbd.CCH.sub.3, .sup.3C.dbd.CCH.sub.3), -4.66, -4.83,
-5.40, -5.46 (.sup.5SiMe2, .sup.3SiMe.sub.2); .sup.31P NMR (202
MHz, CDCl.sub.3) .delta. 68.43, 68.23 (1:6) MS (FAB): m/e 1030
(M+H.sup.+).
[0252] (II). 4,5-dicyano-2-bromo-imidazole 21 (7.6 mg, 2.5 eq.),
phosphoramidite 30 (10 mg, 0.0156 mmol) and
3'-O-(tert-butyldimethylsilyl- )thymidine (6.7 mg, 1.2 eq.) were
added to a dried NMR tube. The the NMR tube was dried under vacuum
overnight, and dry CDCl.sub.3 (0.5 ml) was injected into the NMR
tube at 0.degree. C. under an argon atmosphere. The reaction was
followed by .sup.31P NMR until the reaction went to completion. The
.sup.31P NMR spectrum showed that the peak corresponding to the
phosphoramidite at 130 ppm had disappeared, and that two new peaks
appeared at 142.634, 141.880 ppm in a ratio of 40:1. MS (FAB)
[M+H+]: calc'd for C.sub.45H.sub.76N.sub.5PO.sub.14Si.sub.2
phosphite triester 998, found 998.4.
[0253] Beaucage's reagent (3.8 mg, 1.2 eq.) was added directly to
the solution. The .sup.31PNMR(202 MHz, CDCl.sub.3, 0.degree. C.)
instantaeously showed another two peaks at 67.831 and 67.514 ppm in
the same ratio, while the peaks corresponding to the phosphite
triester disappeared. The solution in the NMR tube was transfered
to a flask, the solvent was evaporated, and the product was then
purified by chromatography on a silica gel column (ethyl
acetate:hexane:triethylamine- =60:35:5) to give only one isomer.
.sup.31PNMR (202 MHz, CDCl.sub.3, room temperature) .delta.
68.291.
[0254] (III) Virtually identical results were obtained when the
reaction was carried out at -15.degree. C. for 7 hours in
CDCl.sub.3, except that the ratio of isomers was .sup..about.68:1.
MS FAB (nitrobenzyl alcohol): m/e [MH.sup.+] 1030 HRMS FAB CsI m/e
calcd. for C.sub.45H.sub.77N.sup.5O.- sub.14PSSi.sub.2 [MH.sup.+],
1030.4464; found 1030.4460.
Example 36
Phosphorothioate Dinucleotide (35)
[0255] 86
[0256] Protected phosphorothioate dinucleotide 34 (14 mg, 0.0136
mmol) from Example 35 was dissolved in 1 ml 70% TFA-H.sub.2O at
0.degree. C. with stirring, and then the reaction was allowed to
proceed at room temperature. The reaction was followed by TLC until
the spot corresponding to the starting material disappeared.
Evaporation of the solvent and coevaporation with methanol three
times furnished a white solid. The crude product was purified with
preparative TLC plate (0.5 mm) (CH.sub.2Cl.sub.2:MeOH=5:1) to give
7.2 mg of a white solid (35) (94%); .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta. 7.87 (s, 1H, .sup.3H-6), 7.85 (s, 1H,
.sup.5H-6), 6.36-6.33 (dd, J=6.35 Hz, J=7.81 Hz, 1H, 5H-1'),
6.30-6.27 (dd, J=6.35 Hz, J=7.33 Hz .sup.3H-1'), 5.06-5.03 (m, 1H,
.sup.5H-3') 4.53-5.52 (m, 1H, .sup.1H-3'), 4.21-4.06 (m, 4H,
.sup.1H-4', 2.times..sup.3H-5', .sup.3H-4'), 3.84-3.80 (m, 2H,
2.times..sup.5H-5'), 2.50-2.46 (m, 1H, .sup.5H-2'), 2.31-2.24 (m,
2H, 2.times..sup.3H-2'), 2.21-2.17 (m, 1H, .sup.5H-2'), 1.96 (s,
3H, .sup.3CH.sub.3C.dbd.C), 1.87 (s, 3H, .sup.5CH.sub.3C.dbd.C);
.sup.31P NMR (202 MHz, CD.sub.3OD) .delta. 58.64: 58.57 (6:1) MS
(FAB): m/e, 563 (M+H.sup.+).
Example 37
1,2-O-3,5-O-Dicyclopentylidene-L-xylofuranose (36)
[0257] 87
[0258] To a solution of trimethyl orthoformate (68 mmol, 7.5 ml)
and p-toluene sulfonic acid (2 mmol, 380 mg) in dioxane (45 ml)
under a nitrogen atmosphere at 0.degree. C., was added dropwise
cyclopentanone (500 mmol, 45 ml). This solution was stirred at room
temperature for 2 hours and L-xylose (20 mmol, 3.0 g) was added
with stirring continued overnight. Then the reaction mixture was
neutralized with triethylamine. Evaporation of the solvent
furnished a yellow syrupy residue. A solution of the syrupy residue
in chloroform (50 ml) was washed with water (50 ml). The aqueous
layer was extracted with chloroform (3.times.20 ml), and the
combined chloroform layers were dried over (MgSO.sub.4). After
removing the solvent, the mixture was chromatographed on a silica
gel column (hexane:ethyl acetate=5:1) to give 3.5 g white solid 36
(62%). .sup.1HNMR (500 MHz, CDCl.sub.3) .delta. 5.96 (d, J=3.91 Hz,
1H, H-1), 4.42 (d, J=3.91 Hz, 1H, H-2), 4.21 (d, J=1.95 Hz, 1H,
H-3), 4.09-3.98 (m, 3H, H-4, 2.times.H-5), 1.97-1.57 (m, 16H,
cyclopentylidene protons); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 121.20 (C-1OCOC-2), 109.20 (C-3OCOC-5), 105.05 (C-1), 84.46
(C-2), 74.22 (C-4), 71.80 (C-3), 61.47 (C-5), 39.49, 36.80, 36.16,
29.71, 24.07, 23.47, 22.77, 22.34 (cyclopentylidene carbons); MS
(CI) m/e: 283 (M+H.sup.+).
Example 38
1,2-O-Cyclopentylidene-L-xylofuranose (37)
[0259] 88
[0260] 1,2-O-3,5-O-dicyclopentylidene-L-xylofuranose (36) (10 mmol,
2.82 g) was dissolved in acetic acid-water (2:1) (60 ml) at room
temperature. The reaction mixture was stirred for 7 hours. The
reaction was followed by TLC. The solvent was evaporated with high
vacum, coevaporated with methanol three times, and dried in vacuo
overnight to give 2.16 g white solid 37 (100%). .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 5.92 (d, J=3.91 Hz, 1H, H-1), 4.42 (d,
J=3.91 Hz, 1H, H-2), 4.30 (d, J=2.93 Hz, 1H, H-3), 4.17-4.14 (m,
1H, H-4), 4.07-3.97 (m, 2H, 2.times.H-5), 1.96-1.60 (m, 8H,
cyclopentylidene protons); .sup.13C NMR (125 MHz, CDCl.sub.3)
.delta. 121.42 (OCO), 104.52 (C-1), 85.56 (C-2), 78.81 (C-3), 76.77
(C-4), 61.01 (C-5), 36.84, 36.19 (CH.sub.2CCH.sub.2), 23.47,
2522.79 (CH.sub.2CH.sub.2CCH.sub.2CH.sub.2); MS (CI): m/e 217
(M+H.sup.+).
Example 39
1,2-O-cyclopentylidene-5'-O-tosyl-L-xylofuranose (38)
[0261] 89
[0262] To a solution of 1,2-O-cyclopentylidene-L-xylofuranose (37)
(9.4 mmol, 2.03 g) in dry pyridine (25 ml) under nitrogen
atmosphere at 0.degree. C., p-toluenesulfonyl chloride (1.2 eq.,
2.15 g) was added. The reaction mixture was stirred at 0.degree. C.
for 12 hours. Then 10 ml water was added to quench the reaction,
and the solvent evaporated in high vacum, and coevaporated with
toluene twice. The mixture was dissolved in chloroform, washed with
water three times and dried over MgSO.sub.4. Removal of the solvent
furnished 2.96 g of 38 as a white solid (85%). .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 7.78-7.32 (AA'BB', 4H, Ph), 5.83 (d,
J=3.42 Hz, 1H, H-1), 4.42 (d, J=3.91 Hz, 1H, H-2), 4.42-4.24 (m,
3H, H-3, 2.times.H-5), 4.16-4.08 (m, 1H, H-4), 2.42 (s, 3H,
CH.sub.3), 1.90-1.63 (m, 8H, cyclopentylidene protons); .sup.13C
NMR (270 MHz, CDCl.sub.3) .delta. 145.25 , 130.26, 130.09, 130.01,
129.96 (aromatic), 121.71 (OCO), 104.69 (C-1), 85.04 (C-2), 77.67
(C-4), 74.25 (C-3), 66.39 (C-5), 36.8, 36.30 (CH.sub.2CCH.sub.2),
23.42, 22.85 (CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 21.63 (CH.sub.3);
MS (CI): m/e 371 (M+H.sup.+).
Example 40
1,2-O-Dicyclopentylidene-5'-isopropylamine-L-xylofuranose (39)
[0263] 90
[0264] A solution of 1,2-O-cyclopentylidene-5'-tosyl-L-xylofuranose
(38) (7.0 mmol, 2.6 g) in isopropylamine (15 ml) was stirred at
55.degree. C. overnight in a pressure bottle. The solvent was
removed by rotary evaporator and the remaining yellow syrup was
taken up with chloroform and washed with a saturated solution of
sodium bicarbonate and with brine. The organic phase was dried over
MgSO.sub.4, the solvent was evaporated and the residue was flash
chromatographed on silica gel (ethyl acetate-3% triethylamine) to
furnish 1.30 g white solid 39 (72%). m.p. 39-41.degree. C.;
[.alpha.].sub.D.sup.20=-31.37 (c-2, CHCl.sub.3); .sup.1H NMR (500
MHz, CDCl.sub.3) .delta. 8.0 (bs, NH), 5.88 (d, J=3.91 Hz, 1H,
H-1), 4.37 (d, J=3.91 Hz, 1H, H-2), 4.25 (d, J=2.93 Hz, 1H, H-3),
4.19 (m, 1H, H-4), 3.34-2.91 (ABX, 2H, 2.times.H-5), 2.73-2.71
(heptet, 1H, NCH), 1.92-1.61 (m, 8H, cyclopentylidene protons),
1.03-1.01 (dd, J=2.44 Hz, J=6.35 Hz, 6H, Me.sub.2CH); .sup.13C NMR
(500 MHz, CDCl.sub.3) .delta. 121.00 (OCO), 104.75 (C-1), 86.06
(C-2), 78.20 (C-3), 77.00 (C-4), 48.62 (NCH), 45.82 (C-5), 36.78,
36.25 (CH.sub.2CCH.sub.2), 23.46, 22.78
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 22.57 (CH.sub.3CHN), 22.27
(CH.sub.3CHN); MS (CI): m/e 258 ([M+H.sup.+]; 100%); HRMS (EI) m/e
calc'd for C.sub.13H.sub.23NO.sub.4 [M.sup.+]: 257.16270, found
257.16250.
Example 41
Chlorophosphoramidite (40)
[0265] 91
[0266] In a scrupulously dry NMR tube, 9.6 .mu.l (0.11 mmol)
phorsphorus trichloride was added via syringe, then 0.25 ml
CDCl.sub.3 was simlarly added. This NMR tube was cooled to
0.degree. C., and a solution of the
1,2-O-dicyclopentylidene-5'-isopropylamine-L-xylofuranose (17)
(25.7 mg, 0.1 mmol) and triethylamine (27.8 ul, 0.22 mmol) in
CDCl.sub.3 (0.35 ml) was added under a nitrogen atmosphere with
shaking of the NMR tube. An exothermic reaction was noticed. The
NMR tube was then cooled to -78.degree. C., pumped and sealed. The
sealed NMR tube was heated to 40.degree. C. and the reaction was
followed by .sup.31PNMR until a single peak was found in the
.sup.31PNMR spectrum. The product was not isolated, and was
directly used in the following step. .sup.31P NMR (300 MHz, CDCl3)
.delta. 148.75
Example 42
5'-O-(tert-Butyl Dimethyl Silyl)-thymidine-3'-O-Phosphoramidite
(41)
[0267] 92
[0268] To the same NMR tube from previous Example 41, a solution of
5'-O-(tert-butyl dimethyl silyl)-thymidine (35.6 mg, 0.1 mmol) in
0.45 ml CDCl.sub.3 and triethylamine (14 .mu.l, 0.11 mmol) was
added slowly at 0.degree. C. under a nitrogen atmosphere. Then the
NMR tube was cooled to -78.degree. C., pumped and sealed. The
sealed NMR tube was heated to 50.degree. C. and the reaction was
followed by .sup.31PNMR until a single peak corresponding to a new
product was found in the .sup.31PNMR spectrum. The solution was
poured into a flask, and taken up with ethyl acetate (prewashed
with a saturated solution of sodium bicarbonate) and washed with
saturated solution of sodium bicarbonate. The organic layer was
dried over MgSO.sub.4, and the solvent was removed to furnish a
white foam in a quantatitive yield. The crude pruduct was flash
chromatographyed on a silica gel column (hexane-ethyl
acetate-triethylamine=5:3:2) to furnish white crystals (41); m.p.
99-101.degree. C.; [.alpha.].sub.D.sup.20 -72.0.degree. (c=0.5,
CHCl.sub.3) .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.77 (bs, 1H,
NH), 7.46 (s, 1H, H-6), 6.33-6.30 (dd, J=5.86 Hz, J=7.81 Hz, 1H,
H-1'), 5.88 (d, J=3.91 Hz, 1H, H-1"), 4.56-4.53 (m, 1H, H-3'), 4.43
(d, J=3.42 Hz, 1H, H-2"), 4.35 (m, 1H, H-3"), 4.18 (d, J=1.95 Hz,
1H, H-4"), 4.05 (m, 1H, H-4'), 3.90-3.76 (ABX, 2H, 2.times.H-5'),
3.45-3.42 (m, 2H, H-5", NCH), 3.03-2.99 (m, 1H, H-5"), 2.38-2.35
(m, 1H, H-2'), 2.12-2.06 (m, 1H, H-2'), 1.89 (s, 3H, MeC.dbd.C),
1.96-1.62 (m, 8H, cyclopentylidene protons), 1.11-1.08 (m, 6H,
Me.sub.2CH), 0.90 (s, 9H, t-BuSi), 0.09, (d, J=1.95 Hz, 6H,
Me.sub.2Si); .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 163.74
(C-4), 150.31 (C-2), 135.18 (C-6), 121.44 (OCO), 110.93 (C-5),
104.59 (C-1"), 86.61, 86.57 (d, J=5.49 Hz, C-4'), 84.75, 84.73 (d,
J=2.75 Hz, C-1'), 84.70 (C-2"), 73.07, 73.05 (d, J=1.83 Hz, C-3'),
73.03, 72.89 (d, J=17.4, C-4"), 71.82, 71.78 (d, J=4.58 Hz, C-3"),
62.98 (C-5'), 50.05, 49.76 (d, J=36.63 Hz, NCH), 39.94, 39.92 (d,
J=2.75 Hz, C-2'), 36.86, 36.19 (CH.sub.2CCH.sub.2), 36.11, 36.08
(d, J=3.06H.sub.8, C-5 "), 25.92 (SiCMe3), 23.45, 22.76
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 21.99, 21.91 (d, J=9.16 Hz,
CH.sub.3CHN), 21.69, 21.64 (d, J=6.41 Hz, CH.sub.3CHN), 18.35
(SiCMe3), 12.52(CH.sub.3C.dbd.C), -5.39, -5.45 (d, J=9.16 Hz,
Me.sub.2Si); .sup.31P NMR (81 MHz, CDCl.sub.3) .delta. 129.34; MS
(CI): m/e 642 (M+H.sup.+). EI: m/e 641 [M+]. MS FAB (nitrobenzyl
alchol): m/e [MH.sup.+] 642, HRMS FAB (glcerol) m/e calcd. for
C.sub.29H.sub.49N.sub.3O.sub.9 PSi [MH.sup.+] 642.2975; found
642.2973.
Example 43
Protected Phosphorothioate Dinucleotide (42)
[0269] 93
[0270] I: In a dried NMR tube, phosophoramidite 41 (15 mg, 0.0234
mmol), 3'-O-(tert-butyldimethylsilyl)thymidine (10 mg, 1.2 eq.) and
4,5-dicyano-2-bromo-imidazole 21 (9.17 mg, 2.0 eq.) were added.
Then the NMR tube was dried in vacuum overnight. Dry acetonitile
(0.6 ml) was injected into the NMR tube at room temperature under a
nitrogen atmosphere. The solid dissolved instantly. The reaction
was followed by .sup.31PNMR. Within 5 minutes, the peak
corresponding to the phosphoramidite disappeared. To this solution,
Beaucage's reagent (5.6 mg, 1.2 eq.) in 140 .mu.l acetonitrile
(0.2M) was added. The peaks corresponding to the starting material
disappeared. The solution in NMR tube was transfered to a flask,
and the solvent evaporated. Then the mixture was redisolved in
ethyl acetate, washed with saturated sodium bicarbonate and water,
and dried over MgSO.sub.4. After removal of the solvent, the
product was purified by chromatography on a silica gel column
(ethyl acetate:methanol=95:5) to furnish 42 in a diastereomeric
ratio of 7:1 (69.12, 68.91 ppm). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta. 7.46 (s, 1H, .sup.3H-6), 7.27 (s, 1H, .sup.5H-6), 6.34-610
(m, 2H, .sup.5H-1', .sup.3H-1'), 5.86, 5.87 (d, J=3.42 Hz, 1H,
H-1"), 5.17-5.14 (m, 1H, 5H-3'), 4.83-4.81 (d, J=10.25 Hz, 1H,
H-3"), 4.56, 4.55 (d, J=3.91 Hz, 1H, H-2"), 4.38 (m, 2H,
.sup.3H-3', H-4"), 4.25-4.21 (m, 3H, 2.times..sup.3H-5',
.sup.5H-4'), 3.98 (m, 1H, .sup.3H-4'), 3.91-3.85 (m, 2H,
2.times..sup.5H-5'), 2.85, 2.84 (d, J=6.35 Hz, 1H, 2.times.H-5"),
2.81 (m, 1H, NCH), 2.52-2.47 (dd, 2H, .sup.5H-2'), 2.25-2.23 (m,
2H, 2.times..sup.3H-2'), 2.08-2.02 (m, 1H, .sup.5H-2'), 1.91 (s,
3H, .sup.5CH.sub.3C.dbd.C), 1.89 (s, 3H, .sup.3CH.sub.3C.dbd.C),
1.92-1.65 (m, 8H, cyclopentylidene protons), 1.05, 1.04 (d, J=5.86
Hz, 6H, Me.sub.2CHN), 0.90 (s, 9H, .sup.3t-BuSi), 0.86 (s, 9H,
.sup.5t-BuSi), 0.11 (s, 6H, Me.sub.2Si), 0.05 (s, 6H, Me.sub.2Si);
.sup.13CNMR (125 MHz, CDCl.sub.3) .delta. 163.80, 163.59
(.sup.5C-4, .sup.3C-4), 150.33, 150.15 (.sup.5C-2, .sup.3C-2),
136.13, 134.64 (.sup.5C-6, .sup.3C-6), 122.03 (OCO), 111.43, 111.11
(.sup.5C-5, .sup.3C-5), 104.28 (C-1"), 86.29 (.sup.3C-1'), 85.95,
85.90 (d, J=6.41 Hz, .sup.5C-4'), 84.80 84.72, 84.69 (.sup.5C-1',
.sup.3C-4'), 83.59 (C-2"), 80.85, 80.81 (d, J=4.58 Hz, C-3"),
80.69, 80.66 (d, J=4.58 Hz, .sup.5C-3'), 79.03, 78.97 (d, J=8.24
Hz, .sup.3C-3'), 71.30 (C-4"), 67.38, 67.34 (d, J=5.50 Hz,
.sup.3C-5'), 63.37 (.sup.5C-5'), 48.91 (NCH), 45.18 (C-5"), 40.35
(.sup.3C-2'), 39.12, 39.09 (d, J=3.66 Hz, .sup.5C-2'), 37.14, 36.23
(CH.sub.2CCH.sub.2), 25.88, 25.65 (.sup.5SiCM.sub.3,
.sup.3SiCe.sub.3), 23.55, 22.89
(CH.sub.2CH.sub.2CCH.sub.2CH.sub.2), 22.80, 22.55 (NCHMe.sub.2),
18.28, 17.86 (.sup.5SiCMe.sub.3, 3SiCMe.sub.3), 12.52, 12.49
(.sup.5C.dbd.CCH.sub.3, .sup.3C.dbd.CCH), -4.66, -4.63, -5.40,
-5.46 (.sup.5SiMe2, .sup.3SiMe.sub.2); .sup.31P NMR (121 MHz,
CDCl.sub.3) .delta. 69.12, 68.91 (7:1); MS (FAB): m/e
(M+H.sup.+).
Example 44
Phosphorothioate Dinucleotide (43)
[0271] 94
[0272] The protected phosphorothioate dinucleotide 42 obtained from
example 43-I (15 mg, 0.014 mmol) was dissolved in 1 ml 701
TFA-H.sub.2O at 0.degree. C. with stirring and, the reaction was
allowed to proceed at room temperature. The reaction was followed
by TLC until the spot corresponding to the starting material
disappeared. Evaporation of the solvent and coevaporation with
methanol three times furnished a white solid. The crude product was
purified with preparative TLC plate (0.5 mm) (CH.sub.2Cl.sub.2:
MeOH-5:1) to give 43 as a white solid: .sup.1H NMR (500 MHz,
CD.sub.3OD) .delta. 7.91 (8, 1H, .sup.3H-6), 7.86 (s, 1H,
.sup.5H-6), 6.36-6.33 (dd, J=6.35 Hz, J=7.81 Hz, 1H, .sup.5H-1'),
6.29-6.26 (dd, J=5.86 Hz, J=7.81 Hz .sup.3H-1'), 5.08-5.05 (m, 1H,
.sup.5H-3') 4.52-3.51 (m, 1H, .sup.3H-3'), 4.21 (m, 1H,
.sup.5H-4'), 4.14-4.11 (m, 2H, 2.times..sup.3H-5'), 4.04 (m, 1H,
.sup.3H-4'), 3.86-3.79 (m, 2H, 2.times..sup.5H-5'), 2.48-2.44 (m,
1H, .sup.3H-2'), 2.31-2.24 (m, 2H, 2.times..sup.5H-2'), 2.23-2.16
(m, 1H, .sup.3H-2'), 1.97 (s, 3H, .sup.3CH.sub.3C.dbd.C), 1.87 (s,
3H, .sup.5CH.sub.3C.dbd.C); .sup.31P NMR (121 MHz, CDCl.sub.3)
.delta. 59.14: 59.08 (1:7); MS (FAEB): m/e (M+H.sup.+)
Example 45
Synthesis of .gamma.-Aminoalcohol 44 (Scheme 1)
[0273] The synthesis of amino-alcohol 44 is shown in Scheme 1
below: 95
[0274] Starting from L-mannonic-.gamma.-lactone 51, both diols are
protected by conversion to their acetonides according to standard
procedures, using acetone with para-toluenesulfonic acid as a
catalyst, to yield bis-acetonide lactone 52.
[0275] The bis-acetonide is then reacted in presence of samarium
iodide in a mixture of THF and ethylene glycol at room temperature
according to the procedure described by Christian Girard, Ph.D.
Thesis, Universite de Montreal, 1995. This reaction proceeds with
high yield and high regioselectivity and affects the acetonide
located on the a-position to the ester, yielding
.beta.-hydroxyester 53.
[0276] The next step is a classical acid-catalyzed deprotection of
the acetonide to give triol product 54. The diol function of the
triol is then cleaved by sodium periodate on alumina in methanol.
The intermediate aldehyde undergoes a reductive amination in
presence of isopropylamine and sodium cyanoborohydride to yield
.gamma.-aminoalcohol 55, which is isolated and stabilized as the
hydrochloride salt. See Robert Hambalek, Ph.D. Thesis, McGill
University, 1992.
[0277] .gamma.-aminoalcohol 55 is then employed as a chiral
precursor to the stereocontrolled synthesis of a P-chiral
phosphorothioate dimer that can be deprotected by a base-catalyzed
.beta.-elimination. An example of this type of elimination is found
in Takahata et al., J. Org. Chem. 1995, 60, 5628-5633.
[0278] Those skilled in the art will appreciate that numerous
changes and modifications may be made to the preferred embodiments
of the invention and that such changes and modifications may be
made without departing from the spirit of the invention. It is
therefore intended that the appended claims cover all equivalent
variations as fall within the true spirit and scope of the
invention.
[0279] It is intended that each of the patents, publications, and
other published documents mentioned on referred to in this
specification be herein incorporated by reference in their
entirety.
* * * * *