U.S. patent application number 15/222910 was filed with the patent office on 2017-02-02 for methods for the synthesis of functionalized nucleic acids.
The applicant listed for this patent is WAVE LIFE SCIENCES LTD.. Invention is credited to David Charles Donnell Butler, Naoki Iwamoto, Meena, Gregory L. Verdine.
Application Number | 20170029457 15/222910 |
Document ID | / |
Family ID | 47558410 |
Filed Date | 2017-02-02 |
United States Patent
Application |
20170029457 |
Kind Code |
A1 |
Verdine; Gregory L. ; et
al. |
February 2, 2017 |
METHODS FOR THE SYNTHESIS OF FUNCTIONALIZED NUCLEIC ACIDS
Abstract
Described herein are methods for the synthesis of derivatives of
thiosulfonate reagents. Said reagents have utility for the
synthesis of phosphorothiotriesters from H-phosphonates in a
stereospecific fashion.
Inventors: |
Verdine; Gregory L.;
(Boston, MA) ; Meena;; (Belmont, MA) ;
Iwamoto; Naoki; (Brighton, MA) ; Butler; David
Charles Donnell; (Medford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WAVE LIFE SCIENCES LTD. |
Singapore |
|
SG |
|
|
Family ID: |
47558410 |
Appl. No.: |
15/222910 |
Filed: |
July 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14233579 |
Feb 27, 2014 |
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PCT/US12/46805 |
Jul 13, 2012 |
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15222910 |
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61509526 |
Jul 19, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/70 20130101;
C07C 309/66 20130101; C07D 295/108 20130101; C07H 21/04 20130101;
C07H 13/04 20130101; A61K 31/66 20130101 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Claims
1. A process for the preparation of phosphorothiotriesters of
structure IIIa comprising the steps of: i) reacting an
H-phosphonate of structure Ia with an silylating reagent to provide
a silyloxyphosphonate; and ii) reacting the silyloxyphosphonate
with a thiosulfonate reagent of structure IIa to provide a
phosphorothiotriester of structure IIIa; wherein, the H-phosphonate
of structure Ia has the following structure: ##STR00068## wherein,
W is independently selected from O, S, NH, or CH.sub.2; R.sup.3 is
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
Y.sup.1 is O, NR.sup.d, S, or Se; R.sup.a is a blocking group;
R.sup.c is a blocking group; each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; Y.sup.2 is O, NR.sup.d, or S; each instance of R.sup.4 is
independently hydrogen, --OH, --SH, --NR.sup.dR.sup.d, --N.sub.3,
halogen, alkyl, alkenyl, alkynyl, alkyl-Y.sup.1--,
alkenyl-Y.sup.1--, alkynyl-Y.sup.1--, aryl-Y.sup.1--,
heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c, and R.sup.b is a
blocking group; each instance of Ba is independently a blocked or
unblocked adenine, cytosine, guanine, thymine, uracil or modified
nucleobase; R.sup.5 is hydrogen, a blocking group, a linking moiety
connected to a solid support or a linking moiety connected to a
nucleic acid; and n is between 1 and about 200; and the
thiosulfonate reagent of structure IIa has the following structure:
##STR00069## wherein, X is alkyl, cycloalkyl, or heteroaryl; R is
alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, or R.sup.1-R.sup.2; R.sup.1 is selected from
--S-alkenylene-, --S-alkylene-, --S-alkylene-aryl-alkylene-,
--S--CO-aryl-alkylene-, or --S--CO-alkylene-aryl-alkylene-; R.sup.2
is selected from heterocyclo-alkylene-S--,
heterocyclo-alkenylene-S--, aminoalkyl-S--, or
(alkyl).sub.4N-alkylene-S--; and the phosphorothiotriester of
structure IIIa has the following structure: ##STR00070## wherein, W
is independently selected from O, S, NH, or CH.sub.2; R is alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, or R.sup.1-R.sup.2; R.sup.1 is selected from
--S-alkenylene-, --S-alkylene-, --S-alkylene-aryl-alkylene-,
--S--CO-aryl-alkylene-, or --S--CO-alkylene-aryl-alkylene-; R.sup.2
is selected from heterocyclo-alkylene-S--,
heterocyclo-alkenylene-S--, aminoalkyl-S--, or
(alkyl).sub.4N-alkylene-S--; R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; Y.sup.1 is O, NR.sup.d,
S, or Se; R.sup.a is a blocking group; R.sup.c is a blocking group;
each instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); each instance of
R.sup.e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; Y.sup.2 is O, NR.sup.d, or S;
each instance of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; R.sup.5 is hydrogen, a
blocking group, a linking moiety connected to a solid support or a
linking moiety connected to a nucleic acid; and n is between 1 and
about 200.
2. A process for the preparation of phosphorothiotriesters
comprising non-stereorandom phosphorous linkages of structure IIIb
comprising the steps of: i) reacting a H-phosphonate comprising
non-stereorandom phosphorous linkages of structure Ib with an
silylating reagent to provide a silyloxyphosphonate; and ii)
reacting the silyloxyphosphonate with a thiosulfonate reagent of
structure IIb to provide a phosphorothiotriester comprising
non-stereorandom phosphorous linkages of structure IIIb; wherein,
the H-phosphonate comprising non-stereorandom phosphorous linkages
of structure Ib has the following structure: ##STR00071## wherein,
W is independently selected from O, NH, or CH.sub.2; R.sup.3 is
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
Y.sup.1 is O, NR.sup.d, S, or Se; R.sup.a is a blocking group;
R.sup.c is a blocking group; each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; Y.sup.2 is O, NR.sup.d, or S; each instance of R.sup.4 is
independently hydrogen, --OH, --SH, --NR.sup.dR.sup.d, --N.sub.3,
halogen, alkyl, alkenyl, alkynyl, alkyl-Y.sup.1--,
alkenyl-Y.sup.1--, alkynyl-Y.sup.1--, aryl-Y.sup.1--,
heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c, and R.sup.b is a
blocking group; each instance of Ba is independently a blocked or
unblocked adenine, cytosine, guanine, thymine, uracil or modified
nucleobase; R.sup.5 is hydrogen, a blocking group, a linking moiety
connected to a solid support or a linking moiety connected to a
nucleic acid; and n is between 1 and about 200; and the
thiosulfonate reagent of structure IIb has the following structure:
##STR00072## wherein, X is alkyl, cycloalkyl, aryl, or heteroaryl;
R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, or R.sup.1-R.sup.2; R.sup.1 is selected
from --S-alkenylene-, --S-alkylene-, --S-alkylene-aryl-alkylene-,
--S--CO-aryl-alkylene-, or --S--CO-alkylene-aryl-alkylene-; R.sup.2
is selected from heterocyclo-alkylene-S--,
heterocyclo-alkenylene-S--, aminoalkyl-S--, or
(alkyl).sub.4N-alkylene-S--; and the chiral phosphorothiotriester
comprising non-stereorandom phosphorous linkages of structure IIIb
has the following structure: ##STR00073## wherein, W is
independently selected from O, NH, or CH.sub.2; R is alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl,
heteroaralkyl, or R.sup.1-R.sup.2; R.sup.1 is selected from
--S-alkenylene-, --S-alkylene-, --S-alkylene-aryl-alkylene-,
--S--CO-aryl-alkylene-, or --S--CO-alkylene-aryl-alkylene-; R.sup.2
is selected from heterocyclo-alkylene-S--,
heterocyclo-alkenylene-S--, aminoalkyl-S--, or
(alkyl).sub.4N-alkylene-S--; R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; Y.sup.1 is O, NR.sup.d,
S, or Se; R.sup.a is a blocking group; R.sup.c is a blocking group;
each instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); each instance of
R.sup.e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; Y.sup.2 is O, NR.sup.d, or S;
each instance of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; R.sup.5 is hydrogen, a
blocking group, a linking moiety connected to a solid support or a
linking moiety connected to a nucleic acid; and n is between 1 and
about 200.
3. The process of claim 1, wherein W is O.
4. The process of claim 1, wherein R.sup.1 is selected from:
##STR00074## and R.sup.2 is selected from: ##STR00075##
5. The process of claim 1, wherein the silylating reagent is
selected from 1,1,3,3-tetramethyl-1,3-diphenyldisilazane;
1,3-dimethyl-1,1,3,3-tetraphenyldisilazane;
1-(trimethylsilyl)imidazole; N-trimethylsilyl-N-methyl
trifluoroacetamide; bis(dimethylamino)dimethylsilane;
bromotrimethylsilane; chlorodimethyl(pentafluorophenyl)silane;
chlorotriethyl silane; chlorotriisopropylsilane;
chlorotrimethylsilane; dichlorodimethylsilane;
hexamethyldisilazane; N,N'-bis(trimethylsilyl)urea;
N,N-bis(trimethylsilyl)methylamine;
N,N-dimethyltrimethylsilylamine; N,O-bis(trimethylsilyl)acetamide;
N,O-bis(trimethylsilyl)carbamate;
N,O-bis(trimethylsilyl)trifluoroacetamide;
N-methyl-N-(trimethylsilyl)trifluoroacetamide;
N-methyl-N-trimethylsilylacetamide;
N-methyl-N-trimethylsilylheptafluorobutyramide;
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide;
N-methyl-N-trimethylsilylheptafluorobutyramide;
trimethylsilyltriflate; triethylsilyltriflate;
triisopropylsilyltriflate; or tert-butyldimethylsilyltriflate.
6. The process of claim 5, wherein the silylating reagent is
selected from N,O-bis(trimethylsilyl)trifluoroacetamide,
trimethylsilyltriflate, chlorotrimethylsilane, or 1-(trimethyl
silyl)imidazole.
7. The process of claim 6, wherein the silylating reagent is
selected from N,O-bis(trimethylsilyl)trifluoroacetamide.
8. The process of claim 1, wherein the H-phosphonate is covalently
linked to a solid phase.
9. A process for the preparation of phosphorothiotriesters of
structure IIIc comprising the steps of: i) reacting a H-phosphonate
of structure Ic with an silylating reagent to provide a
silyloxyphosphonate; ii) reacting the silyloxyphosphonate with a
bis(thiosulfonate) reagent of structure IVc to provide a
phosphorothiotriester comprising a thiosulfonate group of structure
Vc; iii) reacting the phosphorothiotriester comprising a
thiosulfonate group of structure Vc with a nucleophile of structure
VIc to provide the phosphorothiotriesters of structure IIIc;
wherein, the H-phosphonate of structure Ic has the following
structure: ##STR00076## wherein, W is independently selected from
O, S, NH, or CH.sub.2; R.sup.3 is --OH, --SH, --NR.sup.dR.sup.d,
--N.sub.3, halogen, hydrogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; Y.sup.1 is O, NR.sup.d,
S, or Se; R.sup.a is a blocking group; R.sup.c is a blocking group;
each instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); each instance of
R.sup.e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; Y.sup.2 is O, NR.sup.d, or S;
each instance of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; R.sup.5 is hydrogen, a
blocking group, a linking moiety connected to a solid support or a
linking moiety connected to a nucleic acid; and n is between 1 and
about 200; and the bis(thiosulfonate) reagent of structure IVc has
the following structure: ##STR00077## wherein, X is alkylene,
alkenylene, arylene, or heteroarylene; each R.sup.6 is
independently alkyl, cycloalkyl, aryl, or heteroaryl; the
nucleophile of structure VIc has the following structure:
R.sup.7--SH, wherein R.sup.7 is selected from alkyl, alkenyl, aryl,
heterocyclo, aminoalkyl, or (heterocyclo)alkyl; and
phosphorothiotriesters of structure IIIc has the following
structure: ##STR00078## wherein, W is independently selected from
O, S, NH, or CH.sub.2; R is R.sup.7--S--S--X-- R.sup.7 is alkyl,
alkenyl, aryl, heterocyclo, aminoalkyl, or (heterocyclo)alkyl; X is
alkylene, alkenylene, arylene, or heteroarylene; R.sup.3 is --OH,
--SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
Y.sup.1 is O, NR.sup.d, S, or Se; R.sup.a is a blocking group;
R.sup.c is a blocking group; each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; Y.sup.2 is O, NR.sup.d, or S; each instance of R.sup.4 is
independently hydrogen, --OH, --SH, --NR.sup.dR.sup.d, --N.sub.3,
halogen, alkyl, alkenyl, alkynyl, alkyl-Y.sup.1--,
alkenyl-Y.sup.1--, alkynyl-Y.sup.1--, aryl-Y.sup.1--,
heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c, and R.sup.b is a
blocking group; each instance of Ba is independently a blocked or
unblocked adenine, cytosine, guanine, thymine, uracil or modified
nucleobase; R.sup.5 is hydrogen, a blocking group, a linking moiety
connected to a solid support or a linking moiety connected to a
nucleic acid; n is between 1 and about 200; and wherein the
phosphorous linkages of the H-phosphonate of structure Ic, the
phosphorothiotriester comprising a thiosulfonate group of structure
Vc, and the phosphorothiotriesters of structure IIIc may optionally
comprise non-stereorandom phosphorous linkages.
10. The process of claim 8, wherein the phosphorothiotriesters of
structure IIIb comprise non-stereorandom phosphorous linkages and
the H-phosphonate of structure Ic comprise non-stereorandom
phosphorous linkages; and W is independently selected from O, NH,
or CH.sub.2.
11. The process of claim 9, wherein W is O.
12. The process of claim 9, wherein R.sup.6 is methyl.
13. The process of claim 9, wherein bis(thiosulfonate) reagent of
structure IVc is selected from: ##STR00079##
14. The process of claim 9, wherein the nucleophile of structure
VIc has the following structure: ##STR00080##
15. The process of claim 9, wherein the silylating reagent is
selected from 1,1,3,3-tetramethyl-1,3-diphenyldisilazane;
1,3-dimethyl-1,1,3,3-tetraphenyldisilazane;
1-(trimethylsilyl)imidazole; N-trimethylsilyl-N-methyl
trifluoroacetamide; bis(dimethylamino)dimethylsilane;
bromotrimethylsilane; chlorodimethyl(pentafluorophenyl)silane;
chlorotriethyl silane; chlorotriisopropylsilane;
chlorotrimethylsilane; dichlorodimethylsilane;
hexamethyldisilazane; N,N'-bis(trimethylsilyl)urea;
N,N-bis(trimethylsilyl)methylamine;
N,N-dimethyltrimethylsilylamine; N,O-bis(trimethylsilyl)acetamide;
N,O-bis(trimethylsilyl)carbamate;
N,O-bis(trimethylsilyl)trifluoroacetamide;
N-methyl-N-(trimethylsilyl)trifluoroacetamide;
N-methyl-N-trimethylsilylacetamide;
N-methyl-N-trimethylsilylheptafluorobutyramide;
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide;
N-methyl-N-trimethylsilylheptafluorobutyramide;
trimethylsilyltriflate; triethylsilyltriflate;
triisopropylsilyltriflate; or tert-butyldimethylsilyltriflate.
16. The process of claim 15, wherein the silylating reagent is
selected from N,O-bis(trimethylsilyl)trifluoroacetamide,
trimethylsilyltriflate, chlorotrimethylsilane, or 1-(trimethyl
silyl)imidazole.
17. The process of claim 16, wherein the silylating reagent is
selected from N,O-bis(trimethylsilyl)trifluoroacetamide.
18. The process of claim 17, wherein the H-phosphonate is
covalently linked to a solid phase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/509,526, filed Jul. 19, 2011, the entirety
of which is hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Oligonucleotides are useful in therapeutic, diagnostic,
research and nanomaterials applications. The use of natural
sequences of DNA or RNA for therapeutics is limited because of
their instability against extra and intracellular nucleases, poor
cell penetration and distribution. Additionally, in vitro studies
have shown that the properties of antisense nucleotides such as
binding affinity, sequence specific binding to the complementary
RNA (Cosstick and Eckstcin, 1985; LaPlanche et al., 1986; Latimer
et al., 1989; Hacia et al., 1994; Mesmaeker et al., 1995),
stability to nucleases are affected by the configurations of the
phosphorous atoms Therefore, there is a need for modified
oligonucleotides to impart stability towards ubiquitous nucleases,
increase binding affinity towards complementary RNA and increase
cell penetration and bio-distribution for a number of in-vitro and
in-vivo applications.
SUMMARY OF THE INVENTION
[0003] Described herein are methods for the synthesis of novel
functionalized nucleic acids and nucleic acid prodrugs. In some
embodiments, the nucleic acids comprise chiral phosphorous
moieties.
[0004] One embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa comprising the steps of:
[0005] i) reacting an H-phosphonate of structure Ia with an
silylating reagent to provide a silyloxyphosphonate; and [0006] ii)
reacting the silyloxyphosphonate with a thiosulfonate reagent of
structure IIa to provide a phosphorothiotriester of structure IIIa;
[0007] wherein, [0008] the H-phosphonate of structure Ia has the
following structure:
[0008] ##STR00001## [0009] wherein, [0010] W is independently
selected from O, S, NH, or CH.sub.2; [0011] R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0012] Y.sup.1 is O,
NR.sup.d, S, or Se; [0013] R.sup.a is a blocking group; [0014]
R.sup.c is a blocking group; [0015] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0016] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; [0017] Y.sup.2 is O, NR.sup.d, or S; [0018] each instance
of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0019] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0020] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0021] n is
between 1 and about 200; and [0022] the thiosulfonate reagent of
structure IIa has the following structure:
##STR00002##
[0022] wherein, [0023] X is alkyl, cycloalkyl, or heteroaryl;
[0024] R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, or R.sup.1-R.sup.2; [0025] R.sup.1 is
selected from --S-alkenylene-, --S-alkylene-,
--S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-, or
--S--CO-alkylene-aryl-alkylene-; [0026] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0027] and the
phosphorothiotriester of structure IIIa has the following
structure:
[0027] ##STR00003## [0028] wherein, [0029] W is independently
selected from O, S, NH, or CH.sub.2; [0030] R is alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or
R.sup.1-R.sup.2; [0031] R.sup.1 is selected from --S-alkenylene-,
--S-alkylene-, --S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-,
or --S--CO-alkylene-aryl-alkylene-; [0032] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0033] R.sup.3 is
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
[0034] Y.sup.1 is O, NR.sup.d, S, or Se; [0035] R.sup.a is a
blocking group; [0036] R.sup.c is a blocking group; [0037] each
instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); [0038] each instance of
R.sup.e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; [0039] Y.sup.2 is O, NR.sup.d,
or S; [0040] each instance of R.sup.4 is independently hydrogen,
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0041] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0042] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0043] n is
between 1 and about 200.
[0044] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIa, wherein W is O.
[0045] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein R.sup.1 is
selected from:
##STR00004## [0046] R.sup.2 is selected from:
##STR00005##
[0047] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein the silylating
reagent is selected from [0048]
1,1,3,3-tetramethyl-1,3-diphenyldisilazane; [0049]
1,3-dimethyl-1,1,3,3-tetraphenyldisilazane; [0050]
1-(trimethylsilyl)imidazole; [0051] N-trimethylsilyl-N-methyl
trifluoroacetamide; [0052] bis(dimethylamino)dimethylsilane; [0053]
bromotrimethylsilane; [0054]
chlorodimethyl(pentafluorophenyl)silane; [0055]
chlorotriethylsilane; [0056] chlorotriisopropylsilane; [0057]
chlorotrimethylsilane; [0058] dichlorodimethylsilane; [0059]
hexamethyldisilazane; [0060] N,N'-bis(trimethylsilyl)urea; [0061]
N,N-bis(trimethylsilyl)methylamine; [0062]
N,N-dimethyltrimethylsilylamine; [0063]
N,O-bis(trimethylsilyl)acetamide; [0064]
N,O-bis(trimethylsilyl)carbamate; [0065]
N,O-bis(trimethylsilyl)trifluoroacetamide; [0066]
N-methyl-N-(trimethylsilyl)trifluoroacetamide; [0067]
N-methyl-N-trimethylsilylacetamide; [0068]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0069]
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; [0070]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0071]
trimethylsilyltriflate; [0072] triethylsilyltriflate; [0073]
triisopropylsilyltriflate; or [0074]
tert-butyldimethylsilyltriflate.
[0075] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate,
chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.
[0076] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide.
[0077] Another embodiment provides the process, wherein the
H-phosphonate is covalently linked to a solid phase.
[0078] One embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb comprising the steps of: [0079] i)
reacting a H-phosphonate comprising non-stereorandom phosphorous
linkages of structure Ib with an silylating reagent to provide a
silyloxyphosphonate; and [0080] ii) reacting the
silyloxyphosphonate with a thiosulfonate reagent of structure IIb
to provide a phosphorothiotriester comprising non-stereorandom
phosphorous linkages of structure IIIb; [0081] wherein, [0082] the
H-phosphonate comprising non-stereorandom phosphorous linkages of
structure Ib has the following structure:
[0082] ##STR00006## [0083] wherein, [0084] W is independently
selected from O, NH, or CH.sub.2; [0085] R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0086] Y.sup.1 is O,
NR.sup.d, S, or Se; [0087] R.sup.a is a blocking group; [0088]
R.sup.c is a blocking group; [0089] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0090] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; [0091] Y.sup.2 is O, NR.sup.d, or S; [0092] each instance
of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.e,
and R.sup.b is a blocking group; [0093] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0094] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0095] n is
between 1 and about 200; and [0096] the thiosulfonate reagent of
structure IIb has the following structure:
##STR00007##
[0096] wherein, [0097] X is alkyl, cycloalkyl, aryl, or heteroaryl;
[0098] R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, or R.sup.1-R.sup.2; [0099] R.sup.1 is
selected from --S-alkenylene-, --S-alkylene-,
--S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-, or
--S--CO-alkylene-aryl-alkylene-; [0100] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0101] and the
chiral phosphorothiotriester comprising non-stereorandom
phosphorous linkages of structure IIIb has the following
structure:
[0101] ##STR00008## [0102] wherein, [0103] W is independently
selected from O, NH, or CH.sub.2; [0104] R is alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or
R.sup.1-R.sup.2; [0105] R.sup.1 is selected from --S-alkenylene-,
--S-alkylene-, --S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-,
or --S--CO-alkylene-aryl-alkylene-; [0106] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0107] R.sup.3 is
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
[0108] Y.sup.1 is O, NR.sup.d, S, or Se; [0109] R.sup.a is a
blocking group; [0110] R.sup.c is a blocking group; [0111] each
instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); [0112] each instance of
R.sup.c is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; [0113] Y.sup.2 is O, NR.sup.d,
or S; [0114] each instance of R.sup.4 is independently hydrogen,
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0115] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0116] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0117] n is
between 1 and about 200.
[0118] Another embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb, wherein W is O.
[0119] Another embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb, wherein R.sup.1 is selected from:
##STR00009##
and [0120] R.sup.2 is selected from:
##STR00010##
[0121] Another embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb, wherein the silylating reagent is
selected from [0122] 1,1,3,3-tetramethyl-1,3-diphenyldisilazane;
[0123] 1,3-dimethyl-1,1,3,3-tetraphenyldisilazane; [0124]
1-(trimethylsilyl)imidazole; [0125] N-trimethylsilyl-N-methyl
trifluoroacetamide; [0126] bis(dimethylamino)dimethylsilane; [0127]
bromotrimethylsilane; [0128]
chlorodimethyl(pentafluorophenyl)silane; [0129]
chlorotriethylsilane; [0130] chlorotriisopropylsilane; [0131]
chlorotrimethylsilane; [0132] dichlorodimethylsilane; [0133]
hexamethyldisilazane; [0134] N,N'-bis(trimethylsilyl)urea; [0135]
N,N-bis(trimethylsilyl)methylamine; [0136]
N,N-dimethyltrimethylsilylamine; [0137]
N,O-bis(trimethylsilyl)acetamide; [0138]
N,O-bis(trimethylsilyl)carbamate; [0139]
N,O-bis(trimethylsilyl)trifluoroacetamide; [0140]
N-methyl-N-(trimethylsilyl)trifluoroacetamide; [0141]
N-methyl-N-trimethylsilylacetamide; [0142]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0143]
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; [0144]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0145]
trimethylsilyltriflate; [0146] triethylsilyltriflate; [0147]
triisopropylsilyltriflate; or [0148]
tert-butyldimethylsilyltriflate.
[0149] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate,
chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.
[0150] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide.
[0151] Another embodiment provides the process, wherein the
H-phosphonate is covalently linked to a solid phase.
[0152] One embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIc comprising the steps of:
[0153] i) reacting a H-phosphonate of structure Ic with an
silylating reagent to provide a silyloxyphosphonate; [0154] ii)
reacting the silyloxyphosphonate with a bis(thiosulfonate) reagent
of structure IVc to provide a phosphorothiotriester comprising a
thiosulfonate group of structure Vc; [0155] iii) reacting the
phosphorothiotriester comprising a thiosulfonate group of structure
Vc with a nucleophile of structure VIc to provide the
phosphorothiotriesters of structure IIIc; [0156] wherein, [0157]
the H-phosphonate of structure Ic has the following structure:
[0157] ##STR00011## [0158] wherein, [0159] W is independently
selected from O, S, NH, or CH.sub.2; [0160] R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0161] Y.sup.1 is O,
NR.sup.d, S, or Se; [0162] R.sup.a is a blocking group; [0163]
R.sup.c is a blocking group; [0164] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0165] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; [0166] Y.sup.2 is O, NR.sup.d, or S; [0167] each instance
of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0168] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0169] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0170] n is
between 1 and about 200; and [0171] the bis(thiosulfonate) reagent
of structure IVc has the following structure:
##STR00012##
[0171] wherein, [0172] X is alkylene, alkenylene, arylene, or
heteroarylene; [0173] each R.sup.6 is independently alkyl,
cycloalkyl, aryl, or heteroaryl; [0174] the nucleophile of
structure VIc has the following structure: [0175] R.sup.7--SH,
wherein R.sup.7 is selected from alkyl, alkenyl, aryl, heterocyclo,
aminoalkyl, or (heterocyclo)alkyl; [0176] and
phosphorothiotriesters of structure IIIc has the following
structure:
[0176] ##STR00013## [0177] wherein, [0178] W is independently
selected from O, S, NH, or CH.sub.2; [0179] R is R.sup.7--S--S--X--
[0180] R.sup.7 is alkyl, alkenyl, aryl, heterocyclo, aminoalkyl, or
(heterocyclo)alkyl; [0181] X is alkylene, alkenylene, arylene, or
heteroarylene; [0182] R.sup.3 is --OH, --SH, --NR.sup.dR.sup.d,
--N.sub.3, halogen, hydrogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0183] Y.sup.1 is O,
NR.sup.d, S, or Se; [0184] R.sup.a is a blocking group; [0185]
R.sup.c is a blocking group; [0186] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0187] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, L.sup.+1, or
K.sup.+1; Y.sup.2 is O, NR.sup.d, or S; [0188] each instance of
R.sup.4 is independently hydrogen, --OH, --SH, --NR.sup.dR.sup.d,
--N.sub.3, halogen, alkyl, alkenyl, alkynyl, alkyl-Y.sup.1--,
alkenyl-Y.sup.1--, alkynyl-Y.sup.1--, aryl-Y.sup.1--,
heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c, and R.sup.b is a
blocking group; [0189] each instance of Ba is independently a
blocked or unblocked adenine, cytosine, guanine, thymine, uracil or
modified nucleobase; [0190] R.sup.5 is hydrogen, a blocking group,
a linking moiety connected to a solid support or a linking moiety
connected to a nucleic acid; [0191] n is between 1 and about 200;
and [0192] wherein the phosphorous linkages of the H-phosphonate of
structure Ic, the phosphorothiotriester comprising a thiosulfonate
group of structure Vc, and the phosphorothiotriesters of structure
IIIc may optionally comprise non-stereorandom phosphorous
linkages.
[0193] Another embodiment provides the process wherein the
phosphorothiotriesters of structure IIIb comprise non-stereorandom
phosphorous linkages and the H-phosphonate of structure Ic comprise
non-stereorandom phosphorous linkages; and W is independently
selected from O, NH, or CH.sub.2. Another embodiment provides the
process wherein W is O.
[0194] Another embodiment provides the process wherein R.sup.6 is
methyl.
[0195] Another embodiment provides the process wherein
bis(thiosulfonate) reagent of structure IVc is selected from:
##STR00014##
[0196] Another embodiment provides the process wherein the
nucleophile of structure VIc has the following structure:
##STR00015##
[0197] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein the silylating
reagent is selected from [0198]
1,1,3,3-tetramethyl-1,3-diphenyldisilazane; [0199]
1,3-dimethyl-1,1,3,3-tetraphenyldisilazane; [0200]
1-(trimethylsilyl)imidazole; [0201] N-trimethylsilyl-N-methyl
trifluoroacetamide; [0202] bis(dimethylamino)dimethylsilane; [0203]
bromotrimethylsilane; [0204]
chlorodimethyl(pentafluorophenyl)silane; [0205]
chlorotriethylsilane; [0206] chlorotriisopropylsilane; [0207]
chlorotrimethylsilane; [0208] dichlorodimethylsilane; [0209]
hexamethyldisilazane; [0210] N,N'-bis(trimethylsilyl)urea; [0211]
N,N-bis(trimethylsilyl)methylamine; [0212]
N,N-dimethyltrimethylsilylamine; [0213]
N,O-bis(trimethylsilyl)acetamide; [0214]
N,O-bis(trimethylsilyl)carbamate; [0215]
N,O-bis(trimethylsilyl)trifluoroacetamide; [0216]
N-methyl-N-(trimethylsilyl)trifluoroacetamide; [0217]
N-methyl-N-trimethylsilylacetamide; [0218]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0219]
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; [0220]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0221]
trimethylsilyltriflate; [0222] triethylsilyltriflate; [0223]
triisopropylsilyltriflate; or [0224]
tert-butyldimethylsilyltriflate.
[0225] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate,
chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.
[0226] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide.
[0227] Another embodiment provides the process, wherein the
H-phosphonate is covalently linked to a solid phase.
INCORPORATION BY REFERENCE
[0228] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0229] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0230] FIG. 1 provides the .sup.31P NMR spectrum of Compound 100S
in CD.sub.3CN as described in Example 6;
[0231] FIG. 2 provides the .sup.31P NMR spectrum of Compound 100S
in CD.sub.3CN after adding BSTFA as described in Example 6;
[0232] FIG. 3 provides the .sup.31P NMR spectrum of Compound 100S
in CD.sub.3CN after adding BSTFA, TEA and MTS as described in
Example 6;
[0233] FIG. 4 provides the .sup.31P NMR spectrum of Compound 100R
in CD.sub.3CN as described in Example 6;
[0234] FIG. 5 provides the .sup.31P NMR spectrum of Compound 100R
in CD.sub.3CN as described in Example 6; and
[0235] FIG. 6 provides the .sup.31P NMR spectrum of Compound 100R
in CD.sub.3CN after adding BSTFA, TEA and MTS as described in
Example 6.
DETAILED DESCRIPTION OF THE INVENTION
[0236] Unless otherwise stated, the following terms used in this
application, including the specification and claims, have the
definitions given below. It must be noted that, as used in the
specification and the appended claims, the singular forms "a" "an"
and "the" include plural referents unless the context clearly
dictates otherwise. Unless otherwise indicated, conventional
methods of mass spectroscopy, NMR, HPLC, protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology are
employed. In this application, the use of "or" or "and" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes"
and "included" is not limiting.
Certain Chemical Terminology
[0237] Unless otherwise noted, the use of general chemical terms,
such as though not limited to "alkyl," "amine," "aryl," are
unsubstituted.
[0238] As used herein, C.sub.1-C.sub.x includes C.sub.1-C.sub.2,
C.sub.1-C.sub.3 . . . C.sub.1-C.sub.x. By way of example only, a
group designated as "C.sub.1-C.sub.4" indicates that there are one
to four carbon atoms in the moiety, i.e. groups containing 1 carbon
atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms, as well as
the ranges C.sub.1-C.sub.2 and C.sub.1-C.sub.3. Thus, by way of
example only, "C.sub.1-C.sub.4 alkyl" indicates that there are one
to four carbon atoms in the alkyl group, i.e., the alkyl group is
selected from among methyl, ethyl, propyl, iso-propyl, n-butyl,
iso-butyl, sec-butyl, and t-butyl. Whenever it appears herein, a
numerical range such as "1 to 10" refers to each integer in the
given range; e.g., "1 to 10 carbon atoms" means that the group may
have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms,
5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9
carbon atoms, or 10 carbon atoms.
[0239] The terms "heteroatom" or "hetero" as used herein, alone or
in combination, refer to an atom other than carbon or hydrogen.
Heteroatoms are may be independently selected from among oxygen,
nitrogen, sulfur, phosphorous, silicon, selenium and tin but are
not limited to these atoms. In embodiments in which two or more
heteroatoms are present, the two or more heteroatoms can be the
same as each another, or some or all of the two or more heteroatoms
can each be different from the others.
[0240] The term "alkyl" as used herein, alone or in combination,
refers to a straight-chain or branched-chain saturated hydrocarbon
monoradical having from one to about ten carbon atoms, or one to
six carbon atoms. Examples include, but are not limited to methyl,
ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl,
2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl,
2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,
4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,
4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,
2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl,
isopentyl, neopentyl, tert-amyl and hexyl, and longer alkyl groups,
such as heptyl, octyl and the like. Whenever it appears herein, a
numerical range such as "C.sub.1-C.sub.6 alkyl" or "C.sub.1-6
alkyl", means that the alkyl group may consist of 1 carbon atom, 2
carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6
carbon atoms. In one embodiment, the "alkyl" is substituted. Unless
otherwise indicated, the "alkyl" is unsubstituted.
[0241] The term "alkenyl" as used herein, alone or in combination,
refers to a straight-chain or branched-chain hydrocarbon
monoradical having one or more carbon-carbon double-bonds and
having from two to about ten carbon atoms, or two to about six
carbon atoms. The group may be in either the cis or trans
conformation about the double bond(s), and should be understood to
include both isomers. Examples include, but are not limited to
ethenyl (--CH.dbd.CH.sub.2), 1-propenyl
(--CH.sub.2CH.dbd.CH.sub.2), isopropenyl
[--C(CH.sub.3).dbd.CH.sub.2], butenyl, 1,3-butadienyl and the like.
Whenever it appears herein, a numerical range such as
"C.sub.2-C.sub.6 alkenyl" or "C.sub.2-6 alkenyl", means that the
alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, 4
carbon atoms, 5 carbon atoms or 6 carbon atoms. In one embodiment,
the "alkenyl" is substituted. Unless otherwise indicated, the
"alkenyl" is unsubstituted.
[0242] The term "alkynyl" as used herein, alone or in combination,
refers to a straight-chain or branched-chain hydrocarbon
monoradical having one or more carbon-carbon triple-bonds and
having from two to about ten carbon atoms, or from two to about six
carbon atoms. Examples include, but are not limited to ethynyl,
2-propynyl, 2-butynyl, 1,3-butadiynyl and the like. Whenever it
appears herein, a numerical range such as "C.sub.2-C.sub.6 alkynyl"
or "C.sub.2-6 alkynyl", means that the alkynyl group may consist of
2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6
carbon atoms. In one embodiment, the "alkynyl" is substituted.
Unless otherwise indicated, the "alkynyl" is unsubstituted.
[0243] The terms "heteroalkyl", "heteroalkenyl" and "heteroalkynyl"
as used herein, alone or in combination, refer to alkyl, alkenyl
and alkynyl structures respectively, as described above, in which
one or more of the skeletal chain carbon atoms (and any associated
hydrogen atoms, as appropriate) are each independently replaced
with a heteroatom (i.e. an atom other than carbon, such as though
not limited to oxygen, nitrogen, sulfur, silicon, phosphorous, tin
or combinations thereof), or heteroatomic group such as though not
limited to --O--O--, --S--S--, --O--S--, --S--O--, .dbd.N--N.dbd.,
--N.dbd.N--, --N.dbd.N--NH--, --P(O).sub.2--, --O--P(O).sub.2--,
--P(O).sub.2--O--, --S(O)--, --S(O).sub.2--, --SnH.sub.2-- and the
like.
[0244] The terms "haloalkyl", "haloalkenyl" and "haloalkynyl" as
used herein, alone or in combination, refer to alkyl, alkenyl and
alkynyl groups respectively, as defined above, in which one or more
hydrogen atoms is replaced by fluorine, chlorine, bromine or iodine
atoms, or combinations thereof. In some embodiments two or more
hydrogen atoms may be replaced with halogen atoms that are the same
as each another (e.g. difluoromethyl); in other embodiments two or
more hydrogen atoms may be replaced with halogen atoms that are not
all the same as each other (e.g. 1-chloro-1-fluoro-1-iodoethyl).
Non-limiting examples of haloalkyl groups are fluoromethyl,
chloromethyl and bromoethyl. A non-limiting example of a
haloalkenyl group is bromoethenyl. A non-limiting example of a
haloalkynyl group is chloroethynyl.
[0245] The term "carbon chain" as used herein, alone or in
combination, refers to any alkyl, alkenyl, alkynyl, heteroalkyl,
heteroalkenyl or heteroalkynyl group, which is linear, cyclic, or
any combination thereof. If the chain is part of a linker and that
linker comprises one or more rings as part of the core backbone,
for purposes of calculating chain length, the "chain" only includes
those carbon atoms that compose the bottom or top of a given ring
and not both, and where the top and bottom of the ring(s) are not
equivalent in length, the shorter distance shall be used in
determining the chain length. If the chain contains heteroatoms as
part of the backbone, those atoms are not calculated as part of the
carbon chain length.
[0246] The term "cycloalkyl" as used herein, alone or in
combination, refers to a saturated, hydrocarbon monoradical ring,
containing from three to about fifteen ring carbon atoms or from
three to about ten ring carbon atoms, though may include
additional, non-ring carbon atoms as substituents (e.g.
methylcyclopropyl). Whenever it appears herein, a numerical range
such as "C.sub.3-C.sub.6 cycloalkyl" or "C.sub.3-6 cycloalkyl",
means that the cycloalkyl group may consist of 3 carbon atoms, 4
carbon atoms, 5 carbon atoms or 6 carbon atoms, i.e., is
cyclopropyl, cyclobutyl, cyclopentyl or cyclohepty, although the
present definition also covers the occurrence of the term
"cycloalkyl" where no numerical range is designated. The term
includes fused, non-fused, bridged and spiro radicals. A fused
cycloalkyl may contain from two to four fused rings where the ring
of attachment is a cycloalkyl ring, and the other individual rings
may be alicyclic, heterocyclic, aromatic, heteroaromatic or any
combination thereof. Examples include, but are not limited to
cyclopropyl, cyclopentyl, cyclohexyl, decalinyl, and bicyclo
[2.2.1] heptyl and adamantyl ring systems. Illustrative examples
include, but are not limited to the following moieties:
##STR00016##
and the like.
[0247] In one embodiment, the "cycloalkyl" is substituted. Unless
otherwise indicated, the "cycloalkyl" is unsubstituted.
[0248] The terms "non-aromatic heterocyclyl" and "heteroalicyclyl"
as used herein, alone or in combination, refer to a saturated,
partially unsaturated, or fully unsaturated nonaromatic ring
monoradicals containing from three to about twenty ring atoms,
where one or more of the ring atoms are an atom other than carbon,
independently selected from among oxygen, nitrogen, sulfur,
phosphorous, silicon, selenium and tin but are not limited to these
atoms. In embodiments in which two or more heteroatoms are present
in the ring, the two or more heteroatoms can be the same as each
another, or some or all of the two or more heteroatoms can each be
different from the others. The terms include fused, non-fused,
bridged and spiro radicals. A fused non-aromatic heterocyclic
radical may contain from two to four fused rings where the
attaching ring is a non-aromatic heterocycle, and the other
individual rings may be alicyclic, heterocyclic, aromatic,
heteroaromatic or any combination thereof. Fused ring systems may
be fused across a single bond or a double bond, as well as across
bonds that are carbon-carbon, carbon-hetero atom or hetero
atom-hetero atom. The terms also include radicals having from three
to about twelve skeletal ring atoms, as well as those having from
three to about ten skeletal ring atoms. Attachment of a
non-aromatic heterocyclic subunit to its parent molecule can be via
a heteroatom or a carbon atom. Likewise, additional substitution
can be via a heteroatom or a carbon atom. As a non-limiting
example, an imidazolidine non-aromatic heterocycle may be attached
to a parent molecule via either of its N atoms (imidazolidin-1-yl
or imidazolidin-3-yl) or any of its carbon atoms
(imidazolidin-2-yl, imidazolidin-4-yl or imidazolidin-5-yl). In
certain embodiments, non-aromatic heterocycles contain one or more
carbonyl or thiocarbonyl groups such as, for example, oxo- and
thio-containing groups. Examples include, but are not limited to
pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl,
piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl,
azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl,
thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl,
2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl,
dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,
dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,
3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl
and quinolizinyl. Illustrative examples of heterocycloalkyl groups,
also referred to as non-aromatic heterocycles, include:
##STR00017##
and the like.
[0249] The terms also include all ring forms of the carbohydrates,
including but not limited to the monosaccharides, the disaccharides
and the oligosaccharides. In one embodiment, the "non-aromatic
heterocyclyl" or "heteroalicyclyl" is substituted. Unless otherwise
indicated, the "non-aromatic heterocyclyl" or "heteroalicyclyl" is
unsubstituted.
[0250] The term "aryl" as used herein, alone or in combination,
refers to an aromatic hydrocarbon radical of six to about twenty
ring carbon atoms, and includes fused and non-fused aryl rings. A
fused aryl ring radical contains from two to four fused rings where
the ring of attachment is an aryl ring, and the other individual
rings may be alicyclic, heterocyclic, aromatic, heteroaromatic or
any combination thereof. Further, the term aryl includes fused and
non-fused rings containing from six to about twelve ring carbon
atoms, as well as those containing from six to about ten ring
carbon atoms. A non-limiting example of a single ring aryl group
includes phenyl; a fused ring aryl group includes naphthyl,
phenanthrenyl, anthracenyl, azulenyl; and a non-fused bi-aryl group
includes biphenyl. In one embodiment, the "aryl" is substituted.
Unless otherwise indicated, the "aryl" is unsubstituted.
[0251] The term "heteroaryl" as used herein, alone or in
combination, refers to an aromatic monoradicals containing from
about five to about twenty skeletal ring atoms, where one or more
of the ring atoms is a heteroatom independently selected from among
oxygen, nitrogen, sulfur, phosphorous, silicon, selenium and tin
but not limited to these atoms and with the proviso that the ring
of said group does not contain two adjacent O or S atoms. In
embodiments in which two or more heteroatoms are present in the
ring, the two or more heteroatoms can be the same as each another,
or some or all of the two or more heteroatoms can each be different
from the others. The term heteroaryl includes fused and non-fused
heteroaryl radicals having at least one heteroatom. The term
heteroaryl also includes fused and non-fused heteroaryls having
from five to about twelve skeletal ring atoms, as well as those
having from five to about ten skeletal ring atoms. Bonding to a
heteroaryl group can be via a carbon atom or a heteroatom. Thus, as
a non-limiting example, an imidazole group may be attached to a
parent molecule via any of its carbon atoms (imidazol-2-yl,
imidazol-4-yl or imidazol-5-yl), or its nitrogen atoms
(imidazol-1-yl or imidazol-3-yl). Likewise, a heteroaryl group may
be further substituted via any or all of its carbon atoms, and/or
any or all of its heteroatoms. A fused heteroaryl radical may
contain from two to four fused rings where the ring of attachment
is a heteroaromatic ring and the other individual rings may be
alicyclic, heterocyclic, aromatic, heteroaromatic or any
combination thereof. A non-limiting example of a single ring
heteroaryl group includes pyridyl; fused ring heteroaryl groups
include benzimidazolyl, quinolinyl, acridinyl; and a non-fused
bi-heteroaryl group includes bipyridinyl. Further examples of
heteroaryls include, without limitation, furanyl, thienyl,
oxazolyl, acridinyl, phenazinyl, benzimidazolyl, benzofuranyl,
benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzothiophenyl,
benzoxadiazolyl, benzotriazolyl, imidazolyl, indolyl, isoxazolyl,
isoquinolinyl, indolizinyl, isothiazolyl, isoindolyloxadiazolyl,
indazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazinyl, pyrrolyl,
pyrazinyl, pyrazolyl, purinyl, phthalazinyl, pteridinyl,
quinolinyl, quinazolinyl, quinoxalinyl, triazolyl, tetrazolyl,
thiazolyl, triazinyl, thiadiazolyl and the like, and their oxides,
such as for example pyridyl-N-oxide. Illustrative examples of
heteroaryl groups include the following moieties:
##STR00018##
and the like.
[0252] In one embodiment, the "heteroaryl" is substituted. Unless
otherwise indicated, the "heteroaryl" is unsubstituted.
[0253] The term "heterocyclyl" as used herein, alone or in
combination, refers collectively to heteroalicyclyl and heteroaryl
groups. Herein, whenever the number of carbon atoms in a
heterocycle is indicated (e.g., C.sub.1-C.sub.6 heterocycle), at
least one non-carbon atom (the heteroatom) must be present in the
ring. Designations such as "C.sub.1-C.sub.6 heterocycle" refer only
to the number of carbon atoms in the ring and do not refer to the
total number of atoms in the ring. Designations such as "4-6
membered heterocycle" refer to the total number of atoms that are
contained in the ring (i.e., a four, five, or six membered ring, in
which at least one atom is a carbon atom, at least one atom is a
heteroatom and the remaining two to four atoms are either carbon
atoms or heteroatoms). For heterocycles having two or more
heteroatoms, those two or more heteroatoms can be the same or
different from one another. Non-aromatic heterocyclic groups
include groups having only three atoms in the ring, while aromatic
heterocyclic groups must have at least five atoms in the ring.
Bonding (i.e. attachment to a parent molecule or further
substitution) to a heterocycle can be via a heteroatom or a carbon
atom. In one embodiment, the "heterocyclyl" is substituted. Unless
otherwise indicated, the "heterocycyl" is unsubstituted.
[0254] The terms "halogen", "halo" or "halide" as used herein,
alone or in combination refer to fluoro, chloro, bromo and/or
iodo.
[0255] The compounds, or their pharmaceutically acceptable salts
may contain one or more asymmetric centers and may thus give rise
to enantiomers, diastereomers, and other stereoisomeric forms that
may be defined, in terms of absolute stereochemistry, such as (R)-
or (S)-. When the compounds described herein contain olefinic
double bonds or other centers of geometric asymmetry, and unless
specified otherwise, it is intended that the compounds include both
Z and E geometric isomers (e.g., cis or trans). Likewise, all
possible isomers, as well as their racemic and optically pure
forms, and all tautomeric forms are also intended to be
included.
[0256] A "stereoisomer" refers to the relationship between two or
more compounds made up of the same atoms bonded by the same bonds
but having different three-dimensional structures, which are not
superimposable. The term "enantiomer" refers to two stereoisomers
that are nonsuperimposeable mirror images of one another. It is
contemplated that the various stereoisomers of the compounds
disclosed herein, and mixtures thereof, are within the scope of the
present disclosure and specifically includes enantiomers.
[0257] A "tautomer" refers to a compound wherein a proton shift
from one atom of a molecule to another atom of the same molecule is
possible. The compounds presented herein may exist as tautomers. In
solutions where tautomerization is possible, a chemical equilibrium
of the tautomers will exist. The exact ratio of the tautomers
depends on several factors, including temperature, solvent, and pH.
Some examples of tautomeric equilibrium are shown below.
##STR00019##
[0258] The term "non-stereorandom phosphorous linkage(s)" as used
herein refers to a chiral phosphorous atom in the phosphodiester,
or other isosteric linkage type, internucleotide linkage. For
embodiments comprising more than one phosphorous internucleotide
linkage, the handedness of chirality at phosphorous is
independently selected at each phosphorous atom. In one embodiment,
the oligonucleotide described herein is a pure diastereomer. In
another embodiment, the oligonucleotide is greater that 95%
diastereomeric purity. In another embodiment, the oligonucleotide
is greater that 90% diastereomeric purity.
[0259] "Optional" or "optionally" means that a subsequently
described event or circumstance may or may not occur and that the
description includes instances when the event or circumstance
occurs and instances in which it does not. For example, "optionally
substituted alkyl" means that the alkyl radical may or may not be
substituted and that the description includes both substituted aryl
radicals and aryl radicals having no substitution.
Certain Nucleic Acid Terminology
[0260] Natural nucleic acids have a phosphate backbone; artificial
nucleic acids may contain other types of backbones, but contain the
same bases.
[0261] The term "nucleotide" as used herein refers to a monomeric
unit of a polynucleotide that consists of a heterocyclic base, a
sugar, and one or more phosphate groups. The naturally occurring
bases, (guanine, (G), adenine (A), cytosine (C), thymine (T), and
uracil (U)) are derivatives of purine or pyrimidine, though it
should be understood that naturally and non-naturally occurring
base analogs are also included. The naturally occurring sugar is
the pentose (five-carbon sugar) deoxyribose (which forms DNA) or
ribose (which forms RNA), though it should be understood that
naturally and non-naturally occurring sugar analogs are also
included. Nucleic acids are linked via phosphate bonds to form
nucleic acids, or polynucleotides, though many other linkages are
known in the art (such as, though not limited to phosphorothioates,
boranophosphates and the like). Artificial nucleic acids include
PNAs (peptide nucleic acids), phosphothionates, and other variants
of the phosphate backbone of native nucleic acids.
[0262] The term "nucleoside" refers to a moiety wherein a
nucleobase or a modified nucleobase is covalently bound to a sugar
or modified sugar.
[0263] The term "sugar" refers to a monosaccharide in closed and/or
open form. Sugars include, but are not limited to, ribose,
deoxyribose, pentofuranose, pentopyranose, and hexopyranose
moieties.
[0264] The term "modified sugar" refers to a moiety that can
replace a sugar. The modified sugar mimics the spatial arrangement,
electronic properties, or some other physicochemical property of a
sugar.
[0265] The terms "nucleic acid" and "polynucleotide" as used herein
refer to a polymeric form of nucleotides of any length, either
ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms
refer to the primary structure of the molecules and, thus, include
double- and single-stranded DNA, and double- and single-stranded
RNA. These terms include, as equivalents, analogs of either RNA or
DNA made from nucleotide analogs and modified polynucleotides such
as, though not limited to, methylated and/or capped
polynucleotides. The terms encompass poly- or oligo-ribonucleotides
(RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA
derived from N-glycosides or C-glycosides of nucleobases and/or
modified nucleobases; nucleic acids derived from sugars and/or
modified sugars; and nucleic acids derived from phosphate bridges
and/or modified phosphorous-atom bridges. The term encompasses
nucleic acids containing any combinations of nucleobases, modified
nucleobases, sugars, modified sugars, phosphate bridges or modified
phosphorous atom bridges. Examples include, and are not limited to,
nucleic acids containing ribose moieties, the nucleic acids
containing deoxy-ribose moieties, nucleic acids containing both
ribose and deoxyribose moieties, nucleic acids containing ribose
and modified ribose moieties. The prefix poly- refers to a nucleic
acid containing about 1 to about 10,000 nucleotide monomer units
and wherein the prefix oligo- refers to a nucleic acid containing
about 1 to about 200 nucleotide monomer units.
[0266] The term "nucleobase" refers to the parts of nucleic acids
that are involved in the hydrogen-bonding that binds one nucleic
acid strand to another complementary strand in a sequence specific
manner. The most common naturally-occurring nucleobases are adenine
(A), guanine (G), uracil (U), cytosine (C), and thymine (T).
[0267] The term "modified nucleobase" refers to a moiety that can
replace a nucleobase. The modified nucleobase mimics the spatial
arrangement, electronic properties, or some other physicochemical
property of the nucleobase and retains the property of
hydrogen-bonding that binds one nucleic acid strand to another in a
sequence specific manner. A modified nucleobase can pair with all
of the five naturally occurring bases (uracil, thymine, adenine,
cytosine, or guanine) without substantially affecting the melting
behavior, recognition by intracellular enzymes or activity of the
oligonucleotide duplex.
[0268] The term "chiral reagent" refers to a compound that is
chiral or enantiopure and can be used for asymmetric induction in
nucleic acid synthesis.
[0269] The term "chiral ligand" or "chiral auxiliary" refers to a
moiety that is chiral or enantiopure and controls the
stereochemical outcome of a reaction.
[0270] In a condensation reaction, the term "condensing reagent"
refers to a reagent that activates a less reactive site and renders
it more susceptible to attack by a nucleophile.
[0271] The term "blocking group" refers to a group that transiently
masks the reactivity of a functional group. The functional group
can be subsequently unmasked by removal of the blocking group.
[0272] The term "moiety" refers to a specific segment or functional
group of a molecule. Chemical moieties are often recognized
chemical entities embedded in or appended to a molecule.
[0273] The term "solid support" refers to any support which enables
synthetic mass production of nucleic acids and can be reutilized at
need. As used herein, the term refers to a polymer, that is
insoluble in the media employed in the reaction steps performed to
synthesize nucleic acids, and is derivatized to comprise reactive
groups.
[0274] The term "linking moiety" refers to any moiety optionally
positioned between the terminal nucleoside and the solid support or
between the terminal nucleoside and another nucleoside, nucleotide,
or nucleic acid.
[0275] A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in
its either single stranded form or a double-stranded helix. This
term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure of
particular double-stranded DNA molecules, sequences can be
described herein according to the normal convention of giving only
the sequence in the 5' to 3' direction along the non-transcribed
strand of DNA (i.e., the strand having a sequence homologous to the
mRNA).
[0276] As used herein, an "antisense" nucleic acid molecule
comprises a nucleotide sequence which is complementary to a "sense"
nucleic acid encoding a protein, e.g., complementary to the coding
strand of a double-stranded cDNA molecule, complementary to an mRNA
sequence or complementary to the coding strand of a gene.
Accordingly, an antisense nucleic acid molecule can hydrogen bond
to a sense nucleic acid molecule.
[0277] As used herein, a "complementary DNA" or "cDNA" includes
recombinant polynucleotides synthesized by reverse transcription of
mRNA and from which intervening sequences (introns) have been
removed.
Synthetic Methods for the Preparation Novel Functionalized Nucleic
Acids and Nucleic Acid Prodrugs
[0278] Described herein are methods for the synthesis of novel
functionalized nucleic acids and nucleic acid prodrugs. In some
embodiments, the nucleic acids comprise chiral phosphorous
moieties.
[0279] One embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa comprising the steps of:
[0280] i) reacting an H-phosphonate of structure Ia with an
silylating reagent to provide a silyloxyphosphonate; and [0281] ii)
reacting the silyloxyphosphonate with a thiosulfonate reagent of
structure IIa to provide a phosphorothiotriester of structure IIIa;
[0282] wherein, [0283] the H-phosphonate of structure Ia has the
following structure:
[0283] ##STR00020## [0284] wherein, [0285] W is independently
selected from O, S, NH, or CH.sub.2; [0286] R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0287] Y.sup.1 is O,
NR.sup.d, S, or Se; [0288] R.sup.a is a blocking group; [0289]
R.sup.c is a blocking group; [0290] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0291] each instance of R.sup.c is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; [0292] Y.sup.2 is O, NR.sup.d, or S; [0293] each instance
of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0294] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0295] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0296] n is
between 1 and about 200; and [0297] the thiosulfonate reagent of
structure IIa has the following structure:
##STR00021##
[0297] wherein, [0298] X is alkyl, cycloalkyl, or heteroaryl;
[0299] R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, or R.sup.1-R.sup.2; [0300] R.sup.1 is
selected from --S-alkenylene-, --S-alkylene-,
--S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-, or
--S--CO-alkylene-aryl-alkylene-; [0301] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0302] and the
phosphorothiotriester of structure IIIa has the following
structure:
[0302] ##STR00022## [0303] wherein, [0304] W is independently
selected from O, S, NH, or CH.sub.2; [0305] R is alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or
R.sup.1-R.sup.2; [0306] R.sup.1 is selected from --S-alkenylene-,
--S-alkylene-, --S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-,
or --S--CO-alkylene-aryl-alkylene-; [0307] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0308] R.sup.3 is
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
[0309] Y.sup.1 is O, NR.sup.d, S, or Se; [0310] R.sup.a is a
blocking group; [0311] R.sup.c is a blocking group; [0312] each
instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); [0313] each instance of
R.sup.e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; [0314] Y.sup.2 is O, NR.sup.d,
or S; [0315] each instance of R.sup.4 is independently hydrogen,
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0316] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0317] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0318] n is
between 1 and about 200.
[0319] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein W is O.
[0320] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein R.sup.1 is
selected from:
##STR00023##
and [0321] R.sup.2 is selected from:
##STR00024##
[0322] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein the silylating
reagent is selected from [0323]
1,1,3,3-tetramethyl-1,3-diphenyldisilazane; [0324]
1,3-dimethyl-1,1,3,3-tetraphenyldisilazane; [0325]
1-(trimethylsilyl)imidazole; [0326] N-trimethylsilyl-N-methyl
trifluoroacetamide; [0327] bis(dimethylamino)dimethylsilane; [0328]
bromotrimethylsilane; [0329]
chlorodimethyl(pentafluorophenyl)silane; [0330]
chlorotriethylsilane; [0331] chlorotriisopropylsilane; [0332]
chlorotrimethylsilane; [0333] dichlorodimethylsilane; [0334]
hexamethyldisilazane; [0335] N,N'-bis(trimethylsilyl)urea; [0336]
N,N-bis(trimethylsilyl)methylamine; [0337]
N,N-dimethyltrimethylsilylamine; [0338]
N,O-bis(trimethylsilyl)acetamide; [0339]
N,O-bis(trimethylsilyl)carbamate; [0340]
N,O-bis(trimethylsilyl)trifluoroacetamide; [0341]
N-methyl-N-(trimethylsilyl)trifluoroacetamide; [0342]
N-methyl-N-trimethylsilylacetamide; [0343]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0344]
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; [0345]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0346]
trimethylsilyltriflate; [0347] triethylsilyltriflate; [0348]
triisopropylsilyltriflate; or [0349]
tert-butyldimethylsilyltriflate.
[0350] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate,
chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.
[0351] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide.
[0352] Another embodiment provides the process, wherein the
H-phosphonate is covalently linked to a solid phase.
[0353] One embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb comprising the steps of: [0354] i)
reacting a H-phosphonate comprising non-stereorandom phosphorous
linkages of structure Ib with an silylating reagent to provide a
silyloxyphosphonate; and [0355] ii) reacting the
silyloxyphosphonate with a thiosulfonate reagent of structure IIb
to provide a phosphorothiotriester comprising non-stereorandom
phosphorous linkages of structure IIb; [0356] wherein, [0357] the
H-phosphonate comprising non-stereorandom phosphorous linkages of
structure Ib has the following structure:
[0357] ##STR00025## [0358] wherein, [0359] W is independently
selected from O, NH, or CH.sub.2; [0360] R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0361] Y.sup.1 is O,
NR.sup.d, S, or Se; [0362] R.sup.a is a blocking group; [0363]
R.sup.c is a blocking group; [0364] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0365] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K+; Y.sup.2 is O, NR.sup.d, or S; [0366] each instance of R.sup.4
is independently hydrogen, --OH, --SH, --NR.sup.dR.sup.d,
--N.sub.3, halogen, alkyl, alkenyl, alkynyl, alkyl-Y.sup.1--,
alkenyl-Y.sup.1--, alkynyl-Y.sup.1--, aryl-Y.sup.1--,
heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c, and R.sup.b is a
blocking group; [0367] each instance of Ba is independently a
blocked or unblocked adenine, cytosine, guanine, thymine, uracil or
modified nucleobase; [0368] R.sup.5 is hydrogen, a blocking group,
a linking moiety connected to a solid support or a linking moiety
connected to a nucleic acid; and [0369] n is between 1 and about
200; and [0370] the thiosulfonate reagent of structure IIb has the
following structure:
##STR00026##
[0370] wherein, [0371] X is alkyl, cycloalkyl, aryl, or heteroaryl;
[0372] R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,
heteroaryl, heteroaralkyl, or R.sup.1-R.sup.2; [0373] R.sup.1 is
selected from --S-alkenylene-, --S-alkylene-,
--S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-, or
--S--CO-alkylene-aryl-alkylene-; R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0374] and the
chiral phosphorothiotriester comprising non-stereorandom
phosphorous linkages of structure IIIb has the following
structure:
[0374] ##STR00027## [0375] wherein, [0376] W is independently
selected from O, NH, or CH.sub.2; [0377] R is alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, or
R.sup.1-R.sup.2; [0378] R.sup.1 is selected from --S-alkenylene-,
--S-alkylene-, --S-alkylene-aryl-alkylene-, --S--CO-aryl-alkylene-,
or --S--CO-alkylene-aryl-alkylene-; [0379] R.sup.2 is selected from
heterocyclo-alkylene-S--, heterocyclo-alkenylene-S--,
aminoalkyl-S--, or (alkyl).sub.4N-alkylene-S--; [0380] R.sup.3 is
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl,
alkenyl, alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--,
alkynyl-Y.sup.1--, aryl-Y.sup.1--, heteroaryl-Y.sup.1--,
--P(O)(R.sup.e).sub.2, --HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c;
[0381] Y.sup.1 is O, NR.sup.d, S, or Se; [0382] R.sup.a is a
blocking group; [0383] R.sup.c is a blocking group; [0384] each
instance of R.sup.d is independently hydrogen, alkyl, alkenyl,
alkynyl, aryl, acyl, substituted silyl, carbamate,
--P(O)(R.sup.e).sub.2, or --HP(O)(R.sup.e); [0385] each instance of
R.sup.e is independently hydrogen, alkyl, aryl, alkenyl, alkynyl,
alkyl-Y.sup.2--, alkenyl-Y.sup.2--, alkynyl-Y.sup.2--,
aryl-Y.sup.2--, or heteroaryl-Y.sup.2--, or a cation which is
Na.sup.+1, Li.sup.+1, or K.sup.+1; [0386] Y.sup.2 is O, NR.sup.d,
or S; [0387] each instance of R.sup.4 is independently hydrogen,
--OH, --SH, --NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0388] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0389] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0390] n is
between 1 and about 200.
[0391] Another embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb, wherein W is O.
[0392] Another embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb, wherein R.sup.1 is selected from:
##STR00028## [0393] R.sup.2 is selected from:
##STR00029##
[0394] Another embodiment provides a process for the preparation of
phosphorothiotriesters comprising non-stereorandom phosphorous
linkages of structure IIIb, wherein the silylating reagent is
selected from [0395] 1,1,3,3-tetramethyl-1,3-diphenyldisilazane;
[0396] 1,3-dimethyl-1,1,3,3-tetraphenyldisilazane; [0397]
1-(trimethylsilyl)imidazole; [0398] N-trimethylsilyl-N-methyl
trifluoroacetamide; [0399] bis(dimethylamino)dimethylsilane; [0400]
bromotrimethylsilane; [0401]
chlorodimethyl(pentafluorophenyl)silane; [0402]
chlorotriethylsilane; [0403] chlorotriisopropylsilane; [0404]
chlorotrimethylsilane; [0405] dichlorodimethylsilane; [0406]
hexamethyldisilazane; [0407] N,N'-bis(trimethylsilyl)urea; [0408]
N,N-bis(trimethylsilyl)methylamine; [0409]
N,N-dimethyltrimethylsilylamine; [0410]
N,O-bis(trimethylsilyl)acetamide; [0411]
N,O-bis(trimethylsilyl)carbamate; [0412]
N,O-bis(trimethylsilyl)trifluoroacetamide; [0413]
N-methyl-N-(trimethylsilyl)trifluoroacetamide; [0414]
N-methyl-N-trimethylsilylacetamide; [0415]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0416]
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; [0417]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0418]
trimethylsilyltriflate; [0419] triethylsilyltriflate; [0420]
triisopropylsilyltriflate; or [0421]
tert-butyldimethylsilyltriflate.
[0422] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate,
chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.
[0423] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide.
[0424] Another embodiment provides the process, wherein the
H-phosphonate is covalently linked to a solid phase.
[0425] One embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIc comprising the steps of:
[0426] i) reacting a H-phosphonate of structure Ic with an
silylating reagent to provide a silyloxyphosphonate; [0427] ii)
reacting the silyloxyphosphonate with a bis(thiosulfonate) reagent
of structure IVc to provide a phosphorothiotriester comprising a
thiosulfonate group of structure Vc; [0428] iii) reacting the
phosphorothiotriester comprising a thiosulfonate group of structure
Vc with a nucleophile of structure VIc to provide the
phosphorothiotriesters of structure IIIc; [0429] wherein, [0430]
the H-phosphonate of structure Ic has the following structure:
[0430] ##STR00030## [0431] wherein, [0432] W is independently
selected from O, S, NH, or CH.sub.2; [0433] R.sup.3 is --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, hydrogen, alkyl, alkenyl,
alkynyl, alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0434] Y.sup.1 is O,
NR.sup.d, S, or Se; [0435] R.sup.a is a blocking group; [0436]
R.sup.c is a blocking group; [0437] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0438] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; [0439] Y.sup.2 is O, NR.sup.d, or S; [0440] each instance
of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0441] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0442] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; and [0443] n is
between 1 and about 200; and [0444] the bis(thiosulfonate) reagent
of structure IVc has the following structure:
##STR00031##
[0444] wherein, [0445] X is alkylene, alkenylene, arylene, or
heteroarylene; [0446] each R.sup.6 is independently alkyl,
cycloalkyl, aryl, or heteroaryl; [0447] the nucleophile of
structure VIc has the following structure: [0448] R.sup.7--SH,
wherein R.sup.7 is selected from alkyl, alkenyl, aryl, heterocyclo,
aminoalkyl, or (heterocyclo)alkyl; [0449] and
phosphorothiotriesters of structure IIIc has the following
structure:
[0449] ##STR00032## [0450] wherein, [0451] W is independently
selected from O, S, NH, or CH.sub.2; [0452] R is R.sup.7--S--S--X--
[0453] R.sup.7 is alkyl, alkenyl, aryl, heterocyclo, aminoalkyl, or
(heterocyclo)alkyl; [0454] X is alkylene, alkenylene, arylene, or
heteroarylene; [0455] R.sup.3 is --OH, --SH, --NR.sup.dR.sup.d,
--N.sub.3, halogen, hydrogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --P(O)(R.sup.e).sub.2,
--HP(O)(R.sup.e), --OR.sup.a or --SR.sup.c; [0456] Y.sup.1 is O,
NR.sup.d, S, or Se; [0457] R.sup.a is a blocking group; [0458]
R.sup.c is a blocking group; [0459] each instance of R.sup.d is
independently hydrogen, alkyl, alkenyl, alkynyl, aryl, acyl,
substituted silyl, carbamate, --P(O)(R.sup.e).sub.2, or
--HP(O)(R.sup.e); [0460] each instance of R.sup.e is independently
hydrogen, alkyl, aryl, alkenyl, alkynyl, alkyl-Y.sup.2--,
alkenyl-Y.sup.2--, alkynyl-Y.sup.2--, aryl-Y.sup.2--, or
heteroaryl-Y.sup.2--, or a cation which is Na.sup.+1, Li.sup.+1, or
K.sup.+1; [0461] Y.sup.2 is O, NR.sup.d, or S; [0462] each instance
of R.sup.4 is independently hydrogen, --OH, --SH,
--NR.sup.dR.sup.d, --N.sub.3, halogen, alkyl, alkenyl, alkynyl,
alkyl-Y.sup.1--, alkenyl-Y.sup.1--, alkynyl-Y.sup.1--,
aryl-Y.sup.1--, heteroaryl-Y.sup.1--, --OR.sup.b, or --SR.sup.c,
and R.sup.b is a blocking group; [0463] each instance of Ba is
independently a blocked or unblocked adenine, cytosine, guanine,
thymine, uracil or modified nucleobase; [0464] R.sup.5 is hydrogen,
a blocking group, a linking moiety connected to a solid support or
a linking moiety connected to a nucleic acid; [0465] n is between 1
and about 200; and [0466] wherein the phosphorous linkages of the
H-phosphonate of structure Ic, the phosphorothiotriester comprising
a thiosulfonate group of structure Vc, and the
phosphorothiotriesters of structure IIIc may optionally comprise
non-stereorandom phosphorous linkages.
[0467] Another embodiment provides the process wherein the
phosphorothiotriesters of structure IIIb comprise non-stereorandom
phosphorous linkages and the H-phosphonate of structure Ic comprise
non-stereorandom phosphorous linkages; and W is independently
selected from O, NH, or CH.sub.2. Another embodiment provides the
process wherein W is O.
[0468] Another embodiment provides the process wherein R.sup.6 is
methyl.
[0469] Another embodiment provides the process wherein
bis(thiosulfonate) reagent of structure IVc is selected from:
##STR00033##
[0470] Another embodiment provides the process wherein the
nucleophile of structure VIc has the following structure:
##STR00034##
[0471] Another embodiment provides a process for the preparation of
phosphorothiotriesters of structure IIIa, wherein the silylating
reagent is selected from [0472]
1,1,3,3-tetramethyl-1,3-diphenyldisilazane; [0473] 1,3-dimethyl-1,
1,3,3-tetraphenyldisilazane; [0474] 1-(trimethylsilyl)imidazole;
[0475] N-trimethylsilyl-N-methyl trifluoroacetamide; [0476]
bis(dimethylamino)dimethylsilane; [0477] bromotrimethylsilane;
[0478] chlorodimethyl(pentafluorophenyl)silane; [0479]
chlorotriethylsilane; [0480] chlorotriisopropylsilane; [0481]
chlorotrimethylsilane; [0482] dichlorodimethylsilane; [0483]
hexamethyldisilazane; [0484] N,N'-bis(trimethylsilyl)urea; [0485]
N,N-bis(trimethylsilyl)methylamine; [0486]
N,N-dimethyltrimethylsilylamine; [0487]
N,O-bis(trimethylsilyl)acetamide; [0488]
N,O-bis(trimethylsilyl)carbamate; [0489]
N,O-bis(trimethylsilyl)trifluoroacetamide; [0490]
N-methyl-N-(trimethylsilyl)trifluoroacetamide; [0491]
N-methyl-N-trimethylsilylacetamide; [0492]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0493]
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide; [0494]
N-methyl-N-trimethylsilylheptafluorobutyramide; [0495]
trimethylsilyltriflate; [0496] triethylsilyltriflate; [0497]
triisopropylsilyltriflate; or [0498]
tert-butyldimethylsilyltriflate.
[0499] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide, trimethylsilyltriflate,
chlorotrimethylsilane, or 1-(trimethylsilyl)imidazole.
[0500] Another embodiment provides the process, wherein the
silylating reagent is selected from
N,O-bis(trimethylsilyl)trifluoroacetamide.
[0501] Another embodiment provides the process, wherein the
H-phosphonate is covalently linked to a solid phase.
Modified Oligonucleotides
[0502] Oligonucleotides have several pharmaceutical properties
which can be improved through the application of prodrug
strategies. In particular, oligonucleotides are rapidly degraded by
nucleases and exhibit poor cellular uptake through the cytoplasmic
cell membrane (Poijarvi-Virta et al., Curr. Med. Chem. (2006),
13(28); 3441-65; Wagner et al., Med. Res. Rev. (2000),
20(6):417-51; Peyrottes et al., Mini Rev. Med. Chem. (2004),
4(4):395-408; Gosselin et al., (1996), 43(1):196-208; Bologna et
al., (2002), Antisense & Nucleic Acid Drug Development
12:33-41). In one example, Vives et al., (Nucleic Acids Research
(1999), 27(20):4071-76) found that tert-butyl SATE
pro-oligonucleotides displayed markedly increased cellular
penetration compared to the parent oligonucleotide. Described
herein are methods for the synthesis of modified oligonucleotides
or pronucleotides.
Reaction Conditions and Reagents Used in the Methods of the
Invention.
Conditions
[0503] The steps of reacting a molecule comprising an achiral
H-phosphonate moiety and a nucleoside comprising a 5'-OH moiety to
form a condensed intermediate can occur without isolating any
intermediates. In some embodiments, the steps of reacting a
molecule comprising an achiral H-phosphonate moiety and a
nucleoside comprising a 5'-OH moiety to form a condensed
intermediate occurs is a one-pot reaction. In an embodiment, a
molecule comprising an achiral H-phosphonate moiety, condensing
reagent, chiral reagent, and compound comprising a free
nucleophilic moiety are added to the reaction mixture at different
times. In another embodiment, a molecule comprising an achiral
H-phosphonate moiety, condensing reagent, and chiral reagent are
present in the same reaction vessel or same pot. In another
embodiment, a molecule comprising an achiral H-phosphonate moiety,
condensing reagent, chiral reagent, and compound comprising a free
nucleophilic moiety are present in the same reaction or same pot.
This allows the reaction to be performed without isolation of
intermediates and eliminates time-consuming steps, resulting in an
economical and efficient synthesis. In specific embodiments, the
achiral H-phosphonate, condensing reagent, chiral amino alcohol,
5'-OH nucleoside are present at the same time in a reaction. In a
further embodiment, the formation of the chiral intermediate for
condensation is formed in situ and is not isolated prior to the
condensation reaction. In another embodiment, a molecule comprising
an achiral H-phosphonate moiety has been activated by reaction with
a condensing reagent, chiral reagent in a different reaction vessel
from that used when reacting the chiral intermediate with the
compound comprising a free 5'-OH moiety.
Synthesis on Solid Support
[0504] In some embodiments, the synthesis of the nucleic acid is
performed in solution. In other embodiments, the synthesis of the
nucleic acid is performed on solid phase. The reactive groups of a
solid support may be unprotected or protected. During
oligonucleotide synthesis a solid support is treated with various
reagents in several synthesis cycles to achieve the stepwise
elongation of a growing oligonucleotide chain with individual
nucleotide units. The nucleoside unit at the end of the chain which
is directly linked to the solid support is termed "the first
nucleoside" as used herein. The first nucleoside is bound to the
solid support via a linker moiety, i.e. a diradical with covalent
bonds to both the polymer of the solid support and the nucleoside.
The linker stays intact during the synthesis cycles performed to
assemble the oligonucleotide chain and is cleaved after the chain
assembly to liberate the oligonucleotide from the support.
[0505] Solid supports for solid-phase nucleic acid synthesis
include the supports described in, e.g., U.S. Pat. Nos. 4,659,774,
5,141,813, 4,458,066; Caruthers U.S. Pat. Nos. 4,415,732,
4,458,066, 4,500,707, 4,668,777, 4,973,679, and 5,132,418; Andrus
et al. U.S. Pat. Nos. 5,047,524, 5,262,530; and Koster U.S. Pat.
No. 4,725,677 (reissued as Re34,069). In some embodiments, the
solid phase is an organic polymer support. In other embodiments,
the solid phase is an inorganic polymer support. In some
embodiments, the organic polymer support is polystyrene,
aminomethyl polystyrene, a polyethylene glycol-polystyrene graft
copolymer, polyacrylamide, polymethacrylate, polyvinylalcohol,
highly cross-linked polymer (HCP), or other synthetic polymers,
carbohydrates such as cellulose and starch or other polymeric
carbohydrates, or other organic polymers and any copolymers,
composite materials or combination of the above inorganic or
organic materials. In other embodiments, the inorganic polymer
support is silica, alumina, controlled poreglass (CPG), which is a
silica-gel support, or aminopropyl CPG. Other useful solid supports
include fluorous solid supports (see e.g., WO/2005/070859), long
chain alkylamine (LCAA) controlled pore glass (CPG) solid supports
(sce e.g., S. P. Adams, K. S. Kavka, E. J. Wykes, S. B. Holder and
G. R. Galluppi, J. Am. Chem. Soc., 1983, 105, 661-663; G. R. Gough,
M. J. Bruden and P. T. Gilham, Tetrahedron Lett., 1981, 22,
4177-4180). Membrane supports and polymeric membranes (see e.g.
Innovation and Perspectives in Solid Phase Synthesis, Peptides,
Proteins and Nucleic Acids, ch 21 pp 157-162, 1994, Ed. Roger Epton
and U.S. Pat. No. 4,923,901) are also useful for the synthesis of
nucleic acids. Once formed, a membrane can be chemically
functionalized for use in nucleic acid synthesis. In addition to
the attachment of a functional group to the membrane, the use of a
linker or spacer group attached to the membrane may be used to
minimize steric hindrance between the membrane and the synthesized
chain.
[0506] Other suitable solid supports include those generally known
in the art to be suitable for use in solid phase methodologies,
including, for example, glass sold as Primer.TM. 200 support,
controlled pore glass (CPG), oxalyl-controlled pore glass (see,
e.g., Alul, et al., Nucleic Acids Research, 1991, 19, 1527),
TentaGel Support--an aminopolyethyleneglycol derivatized support
(see, e.g., Wright, et al., Tetrahedron Lett., 1993, 34, 3373), and
Poros-a copolymer of polystyrene/divinylbenzene.
[0507] Surface activated polymers have been demonstrated for use in
synthesis of natural and modified nucleic acids and proteins on
several solid supports mediums. The solid support material can be
any polymer suitably uniform in porosity, has sufficient amine
content, and sufficiently flexible to undergo any attendant
manipulations without losing integrity. Examples of suitable
selected materials include nylon, polypropylene, polyester,
polytetrafluoroethylene, polystyrene, polycarbonate, and
nitrocellulose. Other materials can serve as the solid support,
depending on the design of the investigator. In consideration of
some designs, for example, a coated metal, in particular gold or
platinum can be selected (see e.g., US publication No.
20010055761). In one embodiment of oligonucleotide synthesis, for
example, a nucleoside is anchored to a solid support which is
functionalized with hydroxyl or amino residues. Alternatively, the
solid support is derivatized to provide an acid labile
trialkoxytrityl group, such as a trimethoxytrityl group (TMT).
Without being bound by theory, it is expected that the presence of
the trialkoxytrityl protecting group will permit initial
detritylation under conditions commonly used on DNA synthesizers.
For a faster release of oligonucleotide material in solution with
aqueous ammonia, a diglycoate linker is optionally introduced onto
the support.
Linking Moiety
[0508] A linking moiety or linker is optionally used to connect the
solid support to the compound comprising a free nucleophilic
moiety. Suitable linkers are known such as short molecules which
serve to connect a solid support to functional groups (e.g.,
hydroxyl groups) of initial nucleosides molecules in solid phase
synthetic techniques. In some embodiments, the linking moiety is a
succinamic acid linker, or a succinate linker
(--CO--CH.sub.2--CH.sub.2--CO--), or an oxalyl linker (--CO--CO--).
In other embodiments, the linking moiety and the nucleoside are
bonded together through an ester bond. In other embodiments, the
linking moiety and the nucleoside are bonded together through an
amide bond. In further embodiments, the linking moiety connects the
nucleoside to another nucleotide or nucleic acid. Suitable linkers
are disclosed in, for example, Oligonucleotides And Analogues A
Practical Approach, Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter
1.
[0509] A linker moiety is used to connect the compound comprising a
free nucleophilic moiety to another nucleoside, nucleotide, or
nucleic acid. In some embodiments, the linking moiety is a
phosphodiester linkage. In other embodiments, the linking moiety is
an H-phosphonate moiety. In yet other embodiments, the linking
moiety is an X-phosphonate moiety.
Solvents for Synthesis
[0510] Synthesis of the nucleic acids is performed in an aprotic
organic solvent. In some embodiments, the solvent is acetonitrile,
pyridine, or NMP. In some embodiments, the solvent is acetone,
acetontrile, NMP, ethyl acetate, THF, dioxane, DMF, DMSO, DCM,
chloroform, pyridine, 2,6-lutidine, HMPA, HMPT, DMA, glyme,
diglyme, sulfone, methyl tert-butyl ether, or combinations thereof.
In some embodiments, the solvent is a polar, aprotic organic
solvent. In some embodiments, the solvent is anhydrous.
Acidification Conditions to Remove Blocking Groups.
[0511] Acidification to remove blocking groups is accomplished by a
Bronsted acid or Lewis acid. In some embodiments, acidification is
used to remove R.sup.1 blocking groups. Useful Bronsted acids are
carboxylic acids, alkylsulfonic acids, arylsulfonic acids,
phosphoric acid and its derivatives, phosphonic acid and its
derivatives, alkylphosphonic acids and their derivatives,
arylphosphonic acids and their derivatives, phosphinic acid,
dialkylphosphinic acids, and diarylphosphinic acids which have a
pKa (25.degree. C. in water) value of -0.6 (trifluoroacetic acid)
to 4.76 (acetic acid) in an organic solvent or water (in the case
of 80% acetic acid). The concentration of the acid (1 to 80%) used
in the acidification step depends on the acidity of the acid.
Consideration to the acid strength must be taken into account as
strong acid conditions will result in
depurination/depyrimidination, wherein purinyl or pyrimidinyl bases
are cleaved from ribose ring.
##STR00035##
[0512] In some embodiments, acidification is accomplished by a
Lewis acid in an organic solvent. Useful Lewis acids are ZnX.sub.2
wherein X is Cl, Br, I, or CF.sub.3SO.sub.3.
[0513] In some embodiments, the acidifying comprises adding an
amount of a Bronsted or Lewis acid effective to convert the
condensed intermediate into the compound of Formula 4 without
removing purine moieties from the condensed intermediate.
[0514] Acids that are useful in the acidifying step also include,
but are not limited to 10% phosphoric acid in an organic solvent,
10% hydrochloric acid in an organic solvent, 1% trifluoroacetic
acid in an organic solvent, 3% dichloroacetic acid in an organic
solvent or 80% acetic acid in water. The concentration of any
Bronsted or Lewis acid used in the process is selected such that
the concentration of the acid does not exceed a concentration that
causes cleavage of the nucleobase from the sugar moiety.
[0515] In some embodiments, acidification comprises adding 1%
trifluoroacetic acid in an organic solvent. In some embodiments,
acidification comprises adding about 0.1% to about 8%
trifluoroacetic acid in an organic solvent. In other embodiments,
acidification comprises adding 3% dichloroacetic acid in an organic
solvent. In other embodiments, acidification comprises adding about
0.1% to about 10% dichloroacetic acid in an organic solvent. In yet
other embodiments, acidification comprises adding 3%
trichloroacetic acid in an organic solvent. In yet other
embodiments, acidification comprises adding about 0.1% to about 10%
trichloroacetic acid in an organic solvent. In some embodiments,
acidification comprises adding 80% acetic acid in water. In some
embodiments, acidification comprises adding about 50% to about 90%,
or about 50% to about 80%, about 50% to about 70%, about 50% to
about 60%, about 70% to about 90% acetic acid in water. In some
embodiments, the acidification comprises the further addition of
cation scavengers to the acidic solvent. In specific embodiments,
the cation scavengers can be triethylsilane or triisopropylsilane.
In some embodiments, R.sup.1 is deblocked prior to the step of
acidifying the condensed intermediate. In some embodiments, R.sup.1
is deblocked by acidification, which comprises adding 1%
trifluoroacetic acid in an organic solvent. In some embodiments,
R.sup.1 is deblocked by acidification, which comprises adding 3%
dichloroacetic acid in an organic solvent. In some embodiments,
R.sup.1 is deblocked by acidification, which comprises adding 3%
trichloroacetic acid in an organic solvent.
Removal of Blocking Moieties or Groups
[0516] Functional groups such as hydroxyl or amino moieties which
are located on nucleobases or sugar moieties are routinely blocked
with blocking (protecting) groups (moieties) during synthesis and
subsequently deblocked. In general, a blocking group renders a
chemical functionality of a molecule inert to specific reaction
conditions and can later be removed from such functionality in a
molecule without substantially damaging the remainder of the
molecule (see e.g., Green and Wuts, Protective Groups in Organic
Synthesis, 2.sup.nd Ed., John Wiley & Sons, New York, 1991).
For example, amino groups can be blocked with nitrogen blocking
groups such as phthalimido, 9-fludrenylmethoxycarbonyl (FMOC),
triphenylmethylsulfenyl, t-BOC, 4,4'-dimethoxytrityl (DMTr),
4-methoxytrityl (MMTr), 9-phenylxanthin-9-yl (Pixyl), trityl (Tr),
or 9-(p-methoxyphenyl)xanthin-9-yl (MOX). Carboxyl groups can be
protected as acetyl groups. Hydroxy groups can be protected such as
tetrahydropyranyl (THP), t-butyldimethylsilyl (TBDMS),
1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (Ctmp),
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp),
1-(2-chloroethoxy)ethyl, 3-methoxy-1,5-dicarbomethoxypentan-3-yl
(MDP), bis(2-acetoxyethoxy)methyl (ACE), triisopropylsilyloxymethyl
(TOM), 1-(2-cyanoethoxy)ethyl (CEE), 2-cyanoethoxymethyl (CEM),
[4-(N-dichloroacetyl-N-methylamino)benzyloxy]methyl, 2-cyanoethyl
(CN), pivaloyloxymethyl (PivOM), levunyloxymethyl (ALE). Other
representative hydroxyl blocking groups have been described (see
e.g., Beaucage et al., Tetrahedron, 1992, 46, 2223). In some
embodiments, hydroxyl blocking groups are acid-labile groups, such
as the trityl, monomethoxytrityl, dimethoxytrityl,
trimethoxytrityl, 9-phenylxanthin-9-yl (Pixyl) and
9-(p-methoxyphenyl)xanthin-9-yl (MOX). Chemical functional groups
can also be blocked by including them in a precursor form. Thus an
azido group can be considered a blocked form of an amine as the
azido group is easily converted to the amine. Further
representative protecting groups utilized in nucleic acid synthesis
are known (see e.g. Agrawal et al., Protocols for Oligonucleotide
Conjugates, Eds., Humana Press, New Jersey, 1994, Vol. 26, pp.
1-72).
[0517] Various methods are known and used for removal of blocking
groups from the nucleic acids. In some embodiments, all blocking
groups are removed. In other embodiments, the blocking groups are
partially removed. In yet other embodiments, reaction conditions
can be adjusted to remove blocking groups on certain moieties. In
certain embodiments where R.sup.2 is a blocking group, removal of
the blocking group at R.sup.2 is orthogonal to the removal of the
blocking group at R.sup.1. The blocking groups at R.sup.1 and
R.sup.2 remain intact during the synthesis steps and are
collectively removed after the chain assembly. In some embodiments,
the R.sup.2 blocking group are removed simultaneously with the
cleavage of the nucleic acids from the solid support and with the
removal of the nucleobase blocking groups. In specific embodiments,
the blocking group at R.sup.1 is removed while the blocking groups
at R.sup.2 and nucleobases remain intact. Blocking groups at
R.sup.1 are cleavable on solid supports with an organic base such
as a primary amine, a secondary amine, or a mixture thereof.
Deblocking of the R.sup.1 position is commonly referred to as front
end deprotection.
[0518] In an embodiment, the nucleobase blocking groups, if
present, are cleavable after the assembly of the respective nucleic
acid with an acidic reagent. In another embodiment, one or more of
the nucleobase blocking groups is cleavable under neither acidic
nor basic conditions, e.g. cleavable with fluoride salts or
hydrofluoric acid complexes. In yet another embodiment, one or more
of the nucleobase blocking groups are cleavable after the assembly
of the respective nucleic acid in the presence of base or a basic
solvent, and wherein the nucleobase blocking group is stable to the
conditions of the front end deprotection step with amines.
[0519] In some embodiments, blocking groups for nucleobases are not
required. In other embodiments, blocking groups for nucleobases are
required. In yet other embodiments, certain nucleobases require
blocking group while other nucleobases do not require blocking
groups. In embodiments where the nucleobases are blocked, the
blocking groups are either completely or partially removed under
conditions appropriate to remove the blocking group at the front
end. For example, R.sup.1 can denote OR.sup.a, wherein R.sup.a is
acyl, and Ba denotes guanine blocked with an acyl group including,
but not limited to isobutyryl, acetyl or
4-(tert-butylphenoxy)acetyl. The acyl groups at R.sup.1 and Ba will
be removed or partially removed during the same deblocking
step.
Stereochemistry of Oligonucleoside Phosphorothioate Linkages
[0520] Oligonucleoside phosphorothioates have shown therapeutic
potential (Stein et al., Science (1993), 261:1004-12; Agrawal et
al., Antisence Res. and Dev. (1992), 2:261-66; Bayever et al.,
Antisense Res. and Dev. (1993), 3:383-390). Oligonucleoside
phosphorothioates prepared without regard to the stereochemistry of
the phosphorothioate exist as a mixture of 2.sup.n diastereomers,
where n is the number of internucleotide phosphorothioates
linkages. The chemical and biological properties of these
diastereomeric phosphorothioates can be distinct. For example, Wada
et al (Nucleic Acids Symposium Series No. 51 p. 119-120;
doi:10.1093/nass/nrm060) found that
stereodefined-(Rp)-(Ups).sub.9U/(Ap).sub.9A duplex showed a higher
Tm value than that of natural-(Up).sub.9U/(Ap).sub.9A and
stereodefined-(Sp)-(Ups).sub.9U did not form a duplex. In another
example, in a study by Tang et al., (Nucleosides Nucleotides
(1995), 14:985-990) stereopure Rp-oligodeoxyribonucleoside
phosphorothioates were found to possess lower stability to
nucleases endogenous to human serum that the parent
oligodeoxyribonucleoside phosphorothioates with undefined
phosphorous chirality.
Nucleobases and Modified Nucleobases
[0521] The nucleobase Ba utilized in the compounds and methods
described herein is a natural nucleobase or a modified nucleobase
derived from natural nucleobases. Examples include, but are not
limited to, uracil, thymine, adenine, cytosine, and guanine having
their respective amino groups protected by acyl protecting groups,
2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil,
2,6-diaminopurine, azacytosine, pyrimidine analogs such as
pseudoisocytosine and pseudouracil and other modified nucleobases
such as 8-substituted purines, xanthine, or hypoxanthine (the
latter two being the natural degradation products). The modified
nucleobases disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048,
Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and
Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7,
313, are also contemplated as Ba moieties of the compounds and
methods described herein.
[0522] Compounds represented by the following general formulae are
also contemplated as modified nucleobases:
##STR00036##
[0523] In the formulae above, R.sup.8 is a linear or branched
alkyl, aryl, aralkyl, or aryloxylalkyl group having 1 to 15 carbon
atoms, including, by way of example only, a methyl, isopropyl,
phenyl, benzyl, or phenoxymethyl group; and each of R.sup.9 and
R.sup.10 represents a linear or branched alkyl group having 1 to 4
carbon atoms.
[0524] Modified nucleobases also include expanded-size nucleobases
in which one or more benzene rings has been added. Nucleic base
replacements described in the Glen Research catalog
(www.glenresearch.com); Krueger A T et al, Acc. Chem. Res., 2007,
40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner
S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F.
E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I.,
Curr. Opin. Chem. Biol., 2006, 10, 622-627, are contemplated as
useful for the synthesis of the nucleic acids described herein.
Some examples of these expanded-size nucleobases are shown
below:
##STR00037##
[0525] Herein, modified nucleobases also encompass structures that
are not considered nucleobases but are other moieties such as, but
not limited to, corrin- or porphyrin-derived rings.
Porphyrin-derived base replacements have been described in
Morales-Rojas, H and Kool, E T, Org. Lett., 2002, 4, 4377-4380.
Shown below is an example of a porphyrin-derived ring which can be
used as a base replacement:
##STR00038##
[0526] Other modified nucleobases also include base replacements
such as those shown below:
##STR00039##
[0527] Modified nucleobases which are fluorescent are also
contemplated. Non-limiting examples of these base replacements
include phenanthrene, pyrene, stillbene, isoxanthine,
isozanthopterin, terphenyl, terthiophene, benzoterthiophene,
coumarin, lumazine, tethered stillbene, benzo-uracil, and
naphtho-uracil, as shown below:
##STR00040##
[0528] The modified nucleobases can be unsubstituted or contain
further substitutions such as heteroatoms, alkyl groups, or linking
moieties connected to fluorescent moieties, biotin or avidin
moieties, or other protein or peptides. Modified nucleobases also
include certain `universal bases` that are not nucleobases in the
most classical sense, but function similarly to nucleobases. One
representative example of such a universal base is
3-nitropyrrole.
[0529] Other nucleosides can also be used in the process disclosed
herein and include nucleosides that incorporate modified
nucleobases, or nucleobases covalently bound to modified sugars.
Some examples of nucleosides that incorporate modified nucleobases
include 4-acetylcytidine; 5-(carboxyhydroxylmethyl)uridine;
2'-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyluridine; dihydrouridine;
2'-O-methylpseudouridine; beta,D-galactosylqueosine;
2'-O-methylguanosine; N.sup.6-isopentenyladenosine;
1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine;
1-methylinosine; 2,2-dimethylguanosine; 2-methyladenosine;
2-methylguanosine; N.sup.7-methylguanosine; 3-methyl-cytidine;
5-methylcytidine; N.sup.6-methyladenosine; 7-methylguanosine;
5-methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine;
beta,D-mannosylqueosine; 5-methoxycarbonylmethyluridine;
5-methoxyuridine; 2-methylthio-N.sup.6-isopentenyladenosine;
N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;
N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;
uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);
pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;
2-thiouridine; 4-thiouridine; 5-methyluridine;
2'-O-methyl-5-methyluridine; and 2'-O-methyluridine.
[0530] In some embodiments, nucleosides include 6'-modified
bicyclic nucleoside analogs that have either (R) or (S)-chirality
at the 6'-position and include the analogs described in U.S. Pat.
No. 7,399,845. In other embodiments, nucleosides include
5'-modified bicyclic nucleoside analogs that have either (R) or
(S)-chirality at the 5'-position and include the analogs described
in US Patent Application Publication No. 20070287831.
[0531] In some embodiments, the nucleobases or modified nucleobases
comprises biomolecule binding moieties such as antibodies, antibody
fragments, biotin, avidin, streptavidin, receptor ligands, or
chelating moieties. In other embodiments, Ba is 5-bromouracil,
5-iodouracil, or 2,6-diaminopurine. In yet other embodiments, Ba is
modified by substitution with a fluorescent or biomolecule binding
moiety. In some embodiments, the substituent on Ba is a fluorescent
moiety. In other embodiments, the substituent on Ba is biotin or
avidin.
Modified Sugars of the Nucleotide/Nucleoside.
[0532] The most common naturally occurring nucleotides are ribose
sugars linked to the nucleobases adenosine (A), cytosine (C),
guanine (G), and thymine (T) or uracil (U). Also contemplated are
modified nucleotides wherein the phosphate group or the modified
phosphorous atom moieties in the nucleotides can be linked to
various positions of the sugar or modified sugar. As non-limiting
examples, the phosphate group or the modified phosphorous-atom
moiety can be linked to the 2', 3', 4' or 5' hydroxyl moiety of a
sugar or modified sugar. Nucleotides that incorporate the modified
nucleobases described above can also be used in the process
disclosed herein. In some embodiments, nucleotides or modified
nucleotides comprising an unprotected --OH moiety are used in the
process disclosed herein.
[0533] In addition to the ribose moiety described in Schemes 1-4b,
other modified sugars can also be incorporated in the nucleic acids
disclosed herein. In some embodiments, the modified sugars contain
one or more substituents at the 2' position including one of the
following: F; CF.sub.3, CN, N.sub.3, NO, NO.sub.2, O-, S-, or
N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or
O-alkyl-O-alkyl, O-alkyl-N-alkyl or N-alkyl-O-alkyl wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1-C.sub.10 alkyl or C.sub.2-C.sub.10 alkenyl and alkynyl.
Examples of substituents include, and are not limited to,
O(CH.sub.2).sub.nOCH.sub.3, and O(CH.sub.2).sub.nNH.sub.2, wherein
n is from 1 to about 10, MOE, DMAOE, DMAEOE. Also contemplated
herein are modified sugars described in WO 2001/088198; and Martin
et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments,
modified sugars comprise substituted silyl groups, an RNA cleaving
group, a reporter group, a fluorescent label, an intercalator, a
group for improving the pharmacokinetic properties of a nucleic
acid, or a group for improving the pharmacodynamic properties of a
nucleic acid, and other substituents having similar properties. The
modifications may be made at the at the 2', 3', 4', 5', or 6'
positions of the sugar or modified sugar, including the 3' position
of the sugar on the 3'-terminal nucleotide or in the 5' position of
the 5'-terminal nucleotide.
[0534] Modified sugars also include sugar mimetics such as
cyclobutyl or cyclopentyl moieties in place of the pentofuranosyl
sugar. Representative United States patents that teach the
preparation of such modified sugar structures include, but are not
limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; and
5,359,044. Some modified sugars that are contemplated include:
##STR00041##
[0535] Other non-limiting examples of modified sugars include
glycerol, which form glycerol nucleic acid (GNA) analogues. One
example of a GNA analogue is shown below and is described in Zhang,
R et al., J. Am. Chem. Soc., 2008, 130, 5846-5847; Zhang L, et al.,
J. Am. Chem. Soc., 2005, 127, 4174-4175 and Tsai C H et al., PNAS,
2007, 14598-14603:
##STR00042## [0536] wherein X is as defined herein. Another example
of a GNA derived analogue, flexible nucleic acid (FNA) based on the
mixed acetal aminal of formyl glycerol, is described in Joyce G F
et al., PNAS, 1987, 84, 4398-4402 and Heuberger B D and Switzer C,
J. Am. Chem. Soc., 2008, 130, 412-413, and is shown below:
##STR00043##
[0537] Other non-limiting examples of modified sugars include
hexopyranosyl (6' to 4'), pentopyranosyl (4' to 2'), pentopyranosyl
(4' to 3'), or tetrofuranosyl (3' to 2') sugars.
[0538] Hexopyranosyl (6' to 4') sugars contemplated include:
##STR00044##
[0539] Pentopyranosyl (4' to 2') sugars contemplated include:
##STR00045##
[0540] Pentopyranosyl (4' to 3') sugars contemplated include:
##STR00046##
[0541] Tetrofuranosyl (3' to 2') sugars contemplated include:
##STR00047##
[0542] Other modified sugars contemplated include:
##STR00048##
[0543] Further contemplated are the sugar mimetics illustrated
below wherein X is selected from S, Se, CH.sub.2, N-Me, N-Et or
N-iPr.
##STR00049## ##STR00050##
[0544] The modified sugars and sugar mimetics can be prepared by
methods known in the art, including, but not limited to: A.
Eschenmoser, Science (1999), 284:2118; M. Bohringer et al, Helv.
Chim. Acta (1992), 75:1416-1477; M. Egli et al, J. Am. Chem. Soc.
(2006), 128(33):10847-56; A. Eschenmoser in Chemical Synthesis:
Gnosis to Prognosis, C. Chatgilialoglu and V. Sniekus, Ed., (Kluwer
Academic, Netherlands, 1996), p. 293; K.-U. Schoning et al, Science
(2000), 290:1347-1351; A. Eschenmoser et al, Helv. Chim. Acta
(1992), 75:218; J. Hunziker et al, Helv. Chim. Acta (1993), 76:259;
G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K. Groebke et
al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser, Science
(1999), 284:2118.
Blocking Groups
[0545] In the reactions described, it is necessary in certain
embodiments to protect reactive functional groups, for example
hydroxy, amino, thiol or carboxy groups, where these are desired in
the final product, to avoid their unwanted participation in the
reactions. Protecting groups are used to block some or all reactive
moieties and prevent such groups from participating in chemical
reactions until the protective group is removed. In one embodiment,
each protective group is removable by a different means. Protective
groups that are cleaved under totally disparate reaction conditions
fulfill the requirement of differential removal. In some
embodiments, protective groups are removed by acid, base, and/or
hydrogenolysis. Groups such as trityl, dimethoxytrityl, acetal and
t-butyldimethylsilyl are acid labile and are used in certain
embodiments to protect carboxy and hydroxy reactive moieties in the
presence of amino groups protected with Cbz groups, which are
removable by hydrogenolysis, and/or Fmoc groups, which are base
labile. In other embodiments, carboxylic acid and hydroxy reactive
moieties are blocked with base labile groups such as, but not
limited to, methyl, ethyl, and acetyl in the presence of amines
blocked with acid labile groups such as t-butylcarbamate or with
carbamates that are both acid and base stable but hydrolytically
removable.
[0546] In another embodiment, hydroxy reactive moieties are blocked
with hydrolytically removable protective groups such as the benzyl
group, while amine groups capable of hydrogen bonding with acids
are blocked with base labile groups such as Fmoc. In another
embodiment, carboxylic acid reactive moieties are protected by
conversion to simple ester compounds, or they are, in yet another
embodiment, blocked with oxidatively-removable protective groups
such as 2,4-dimethoxybenzyl, while co-existing amino groups are
blocked with fluoride labile silyl or carbamate blocking
groups.
[0547] Allyl blocking groups are useful in the presence of acid-
and base-protecting groups since the former are stable and can be
subsequently removed by metal or pi-acid catalysts. For example, an
allyl-blocked hydroxy groups can be deprotected with a
Pd(O)-catalyzed reaction in the presence of acid labile
t-butylcarbamate or base-labile acetate amine protecting groups.
Yet another form of protecting group is a resin to which a compound
or intermediate is attached. As long as the residue is attached to
the resin, that functional group is blocked and cannot react. Once
released from the resin, the functional group is available to
react.
[0548] Typically blocking/protecting groups useful in the synthesis
of the compounds described herein are, by way of example only:
##STR00051##
[0549] Representative protecting groups useful to protect
nucleotides during synthesis include base labile protecting groups
and acid labile protecting groups. Base labile protecting groups
are used to protect the exocyclic amino groups of the heterocyclic
nucleobases. This type of protection is generally achieved by
acylation. Three commonly used acylating groups for this purpose
are benzoyl chloride, phenoxyacetic anhydride, and isobutyryl
chloride. These protecting groups are stable to the reaction
conditions used during nucleic acid synthesis and are cleaved at
approximately equal rates during the base treatment at the end of
synthesis.
[0550] In some embodiments, the 5'-protecting group is trityl,
monomethoxy trityl, dimethoxytrityl, trimethoxytrityl,
2-chlorotrityl, DATE, TBTr, 9-phenylxanthine-9-yl (Pixyl), or
9-(p-methoxyphenyl)xanthine-9-yl (MOX).
[0551] In some embodiments, thiol moieties are incorporated in the
compounds described herein and are protected. In some embodiments,
the protecting groups include, but are not limited to, pixyl,
trityl, benzyl, p-methoxybenzyl (PMB), or tert-butyl (t-Bu).
[0552] Other protecting groups, plus a detailed description of
techniques applicable to the creation of protecting groups and
their removal are described in Greene and Wuts, Protective Groups
in Organic Synthesis, 3.sup.rd Ed., John Wiley & Sons, New
York, N.Y., 1999, and Kocienski, Protective Groups, Thieme Verlag,
New York, N.Y., 1994, which are incorporated herein by reference
for such disclosure.
[0553] The examples provided below further illustrate and exemplify
the compounds of the present invention and methods of preparing
such compounds. It is to be understood that the scope of the
present invention is not limited in any way by the scope of the
following examples and preparations.
EXAMPLES
Example 1
Synthesis of Methanethiosulfonate Reagents
##STR00052## ##STR00053## ##STR00054## ##STR00055##
[0555] Compound 2:
[0556] A solution of (Z)-but-2-ene-1,4-diol (0.93 ml, 11.3 mmol)
and triethylamine (3.3 ml, 24 mmol) in DCM (50 mL) was added in a
dropwise fashion to a stirring ice cold solution of methanesulfonyl
chloride (1.9 ml, 24 mmol) in DCM (50 mL). After stirring for 0.5 h
at r.t. the mixture was poured onto ice and extracted. The organic
layer was collected, dried (MgSO.sub.4), filtered and reduced to
2.66 g, 96% of compound 2, which was judged by NMR to be
sufficiently pure for direct use in the next step of the
reaction.
[0557] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 5.94 (ddd, J=5.4,
4.1, 1.3 Hz, 2H), 4.83 (dd, J=4.1, 1.3 Hz, 4H), 3.04 (s, 6H);
.sup.13C NMR 128.34, 64.38, 38.27; MS (ESI+ve): calc (M+NH.sub.4):
262.04. found: 262.05. R.sub.f=0.3 (1:1 EtOAc/hexane).
[0558] Compound 3:
[0559] A solution of sodium methanesulfonothioate (1.51 g, 11.3
mmol) in MeOH (20 ml) was treated with neat (Z)-but-2-ene-1,4-diyl
dimethanesulfonate (1.25 g, 5.12 mmol) at r.t. After 5 min,
precipitation was observed to occur. After 36 h, the mixture was
partitioned between water and DCM. The organic layer was separated,
dried (MgSO.sub.4), filtered and reduced to afford a colorless oil.
Column chromatography (ISCO) gave the pure product as a pale
colorless oil. Column chromatography gave pure compound 3 (0.89 g,
63%) as a colorless oil.
[0560] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 5.84 (ddd, J=6.6,
5.1, 1.5 Hz, 2H), 3.92 (dd, J=5.1, 1.5 HZ, 4H), 3.33 (s, 6H);
.sup.13C NMR 128.1, 51.47, 33.13; MS (ESI+ve): calc (M+NH.sub.4):
294.00. found: 294.04. R.sub.f=0.4 (1:1 EtOAc/hexane).
[0561] Compound 4:
[0562] Under argon atmosphere, morpholine (10 g, 115 mmol) was
added to ethylene sulfide (15 g, 250 mmol) in a round bottom flask.
The reaction was stirred for 7 hrs and was directly loaded on to a
silica gel column. The column was washed with DCM first and then 2%
MeOH/DCM was used to obtain compound 4 (15.3 g, 91%) as colorless
oil.
[0563] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 3.67-3.59 (m, 4H),
2.63-2.52 (m, 2H), 2.51-2.45 (m, 2H), 2.44-2.34 (m, 4H); MS
(ESI+ve): calc (M+H)+=148.07. found: 148.1.
[0564] Compound 5:
[0565] A DCM solution (1 mL) of 2-morpholinoethanethiol (0.21 g,
1.44 mmol) was added dropwise via syringe to a stirring solution
compound 3 (0.40 g, 1.44 mmol) in DCM (10 mL) at r.t. Immediately
after addition, the TLC was checked, to reveal rapid formation of
product and some quantity of dimer. After 0.5 h, the mixture was
partitioned by addition of water. Upon extraction, the organic
layer was separated then dried (MgSO.sub.4), filtered and reduced
in vacuo. Column chromatography gave compound 5 (0.29 g, 58%) as
colorless oil.
[0566] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 5.78 (m, 2H), 3.92
(d, J=7.3 Hz, 2H), 3.70 (t, J=4.7 Hz, 4H), 3.46 (d, J=5.5 Hz, 2H),
3.31 (s, 3H), 2.84 (dd, J=7.8, 6.7 Hz, 2H), 2.66 (dd, J=7.8, 6.7,
2H), 2.48 (t, J=4.6 Hz, 4H); .sup.13C NMR 130.35, 126.27, 66.97,
58.20, 53.67, 51.52, 36.22, 35.16, 33.67; MS (ESI+ve): calc (M+H):
344.05. found: 344.06. R.sub.f=0.3 (EtOAc).
[0567] Compound 5b:
[0568] A DCM solution (1 mL) of compound 4b (395 mg, 1.085 mmol)
was added dropwise via syringe to a stirring DCM (15 mL) solution
compound 3 (300 mg, 1.085 mmol) at r.t. After 1 h, the resulting
solution was partitioned by addition of water. Upon extraction, the
organic layer was separated then dried (MgSO.sub.4), filtered and
reduced in vacuo. Column chromatography gave compound 5b as a
colorless oil (0.35 g, 58%). .sup.1H NMR (399 MHz, CDCl.sub.3)
.delta. 5.83-5.70 (m, 2H), 5.35-5.21 (dt, J=26.0, 9.3 Hz, 2H),
5.16-5.07 (m, 1H), 4.59-4.54 (d, J=9.5 Hz, 1H), 4.29-4.23 (m, 1H),
4.23-4.18 (m, 1H), 3.99-3.88 (dd, J=6.7, 1.2 Hz, 2H), 3.80-3.72
(ddd, J=10.1, 4.6, 2.6 Hz, 1H), 3.64-3.56 (m, 1H), 3.50-3.43 (m,
1H), 3.31 (s, 3H), 2.09 (s, 3H), 2.03 (s, 6H), 2.00 (s, 3H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 170.68, 170.30, 169.51,
169.30, 129.43, 127.14, 87.73, 76.49, 73.89, 69.16, 67.99, 61.99,
51.64, 35.89, 33.58, 20.95, 20.80, 20.74, 20.71; MS (ESI+ve): calc
(M+NH.sub.4.sup.+): 578.07. found: 577.96. R.sub.f=0.5 (1:1
EtOAc/hexane).
[0569] Compound 6:
[0570] An ice cold solution of (Z)-but-2-ene-1,4-diol (0.93 ml,
11.3 mmol) and triethylamine (1.6 mL, 11.5 mmol) in DCM (50 ml) was
treated dropwise via syringe with pivaloyl chloride (1.4 ml, 11.4
mmol) over 2 min. After 1 h, TLC showed good reaction.
[0571] The resulting mixture was partitioned by addition of water.
Upon extraction, the organic layer was separated then dried
(MgSO.sub.4), filtered and reduced in vacuo. This crude compound
was found: by TLC (Rf=0.6, 1:1 EtOAc/hexane) to contain no starting
diol and was used crude to prepare the mesylate. The crude material
was taken up in DCM (50 ml) containing triethylamine (1.7 mL, 12
mmol) and cooled on an ice bath. Methanesulfonyl chloride (0.98 ml,
12.66 mmol) was added dropwise via syringe over 2 min. TLC
immediately after addition indicated complete consumption of
starting material. The resulting mixture was partioned by addition
of water. Upon extraction, the organic layer was separated then
dried (MgSO.sub.4), filtered and reduced in vacuo. Column
chromatography gave pure compound 6, 1.48 g, 52%, as a colorless
oil.
[0572] 1H NMR (399 MHz, CDCl3) .delta. 5.89-5.75 (m, 2H), 4.89-4.84
(d, J=5.7 Hz, 2H), 4.68-4.63 (d, J=5.9 Hz, 2H), 3.03 (s, 3H), 1.19
(s, 9H); 13C NMR (100 MHz, CDCl3) .delta. 178.28, 130.61, 126.11,
65.08, 59.65, 38.84, 38.21, 27.25; MS (ESI+ve): calc (M+NH4):
268.12. found: 268.20. Rf=0.3 (20% EtOAc/hexane).
[0573] Compound 7:
[0574] A MeOH (10 ml) solution of sodium methanesulfonothioate
(0.63 g, 4.70 mmol) and (Z)-4-(methylsulfonyloxy)but-2-enyl
pivalate (1.00 g, 4.00 mmol) was stirred at r.t. for 18 h with
formation of a white precipitate (after 10 min). The resulting
mixture was partitioned by addition of water and DCM. Upon
extraction into DCM, the organic layer was separated then dried
(MgSO.sub.4), filtered and reduced in vacuo. Column chromatography
gave compound 7, 0.83 g, 78% as a colorless oil.
[0575] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 5.82-5.73 (m, 2H),
4.73-4.66 (m, 2H), 3.95-3.87 (m, 2H), 3.32 (s, 3H), 1.19 (s, 9H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 178.35, 129.37, 127.32,
59.50, 51.44, 38.84, 33.61, 27.28; MS (ESI+ve): calc (M+NH.sub.4):
284.10. found: 284.19. R.sub.f=0.4 (20% EtOAc/hexane).
[0576] Compound 9:
[0577] Pivaloyl chloride (0.60 g, 5.0 mmol) was added in a dropwise
fashion to a stirring solution of S-2-hydroxyethyl
methanesulfonothioate (0.65 g, 4.16 mmol) in DCM (20 ml). After 2 h
at r.t. the resulting mixture with white precipitate was
partitioned with water. The organic layer was separated, dried
(Ns.sub.2SO.sub.4), filtered and reduced to an oil. Column gave
compound 9 as a colorless oil (0.45 g, 45%). .sup.1H NMR (399 MHz,
CDCl.sub.3) .delta. 4.39-4.34 (t, J=6.3 Hz, 2H), 3.44-3.39 (t,
J=6.3 Hz, 2H), 3.36 (s, 3H), 1.20 (s, 9H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 62.10, 51.11, 38.96, 35.19, 27.24; MS (ESI+ve):
calc (M+NH.sub.4): 158.08. found: 158.04. R.sub.f=0.3 (20%
EtOAc/hexane).
[0578] Compound 11:
[0579] Pivaloyl chloride (4.96 ml, 40.3 mmol) was added dropwise
via syringe to an ice cold DCM solution (50 mL) of
2-(hydroxymethyl)phenol (5 g, 40.3 mmol) and triethylamine (5.61
ml, 40.3 mmol). An ice-cold solution of the crude pivalate ester
was treated with triethylamine (6.74 ml, 48.4 mmol) and 50 mL DCM.
Methanesulfonyl chloride (3.43 ml, 44.3 mmol) was then added slowly
(5 min) via syringe and the resulting mixture was warmed to r.t.
The mixture was poured onto ice and the organic layer was separated
then washed with sat NaHCO.sub.3 (aq), dried (MgSO.sub.4), filtered
and reduced to 10.5 g crude pale yellow oil.
[0580] Column (ISCO) gave pure 11 5.45 g, 47%.
[0581] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 7.53-7.46 (dd,
7.7, 1.8 Hz, 1H), 7.46-7.40 (dt, 7.7, 1.8 Hz, 1H), 7.32-7.24 (t,
7.7 Hz, 1H), 7.13-7.06 (d, 7.7 Hz, 1H), 5.21 (s, 2H), 2.79 (s, 3H),
1.40 (s, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 177.05,
150.06, 131.18, 131.07, 126.35, 125.94, 123.21, 66.88, 39.48,
38.82, 27.30, 27.26. MS (ESI+ve): calc (M+NH.sub.4): 304.12. found:
303.99. R.sub.f=0.4 (20% EtOAc/hexane).
[0582] Compound 12:
[0583] A MeOH (20 mL) solution of sodium methanesulfonothioate
(0.825 g, 6.15 mmol) was treated with
2-((methylsulfonyloxy)methyl)phenyl pivalate (1.76 g, 6.15 mmol) at
r.t. and left to stir for 18 h. The mixture was partitioned between
water and DCM. The organic layer was separated, dried (MgSO.sub.4),
filtered and reduced to afford a colorless oil. Column
chromatography gave pure compound 12 as a pale colorless oil, 0.754
g, 41%.
[0584] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 7.48-7.44 (dd,
J=7.7, 1.7 Hz, 1H), 7.39-7.34 (td, J=7.8, 1.7 Hz, 1H), 7.25-7.20
(td, J=7.6, 1.2 Hz, 1H), 7.10-7.06 (dd, J=8.2, 1.2 Hz, 1H), 4.29
(s, 2H), 2.90 (s, 3H), 1.39 (s, 9H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 176.69, 149.59, 131.17, 129.85, 127.41, 126.18,
123.40, 51.43, 39.47, 36.01, 27.30; MS (ESI+ve): calc (M+NH.sub.4):
320.10. found: 320.09. R.sub.f=0.4 (20% EtOAc/hexane).
[0585] Compound 14:
[0586] Chloromethyl pivalate (0.478 ml, 3.32 mmol) was added to a
stirring mixture of sodium iodide (0.050 g, 0.33 mmol) and sodium
methanesulfonothioate (0.445 g, 3.32 mmol) in acetone (7 ml) at
r.t. After 24 h, TLC showed good conversion to product. The solvent
was removed, and the residue was partitioned between water and DCM.
The organic layer was separated and dried (MgSO.sub.4), filtered
and reduced to afford a colorless oil. Column chromatography gave
pure 14 as a slightly pink solid, 0.41 g, 55%.
[0587] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 5.67 (s, 2H), 3.39
(s, 3H), 1.24 (s, 9H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
177.35, 67.84, 52.20, 38.93, 27.05. R.sub.f=0.5 (20%
EtOAc/hexane).
[0588] Compound 16:
[0589] Prepared from 15 and NaMTS as described previously: U.S.
Pat. No. 3,484,473 .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 4.86
(s, 2H), 3.45 (s, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
52.15, 41.50.
[0590] Compound 18:
[0591] Prepared from 17 and NaMTS as described previously: Chem.
Pharm. Bull. Vol. 12(11) p. 1271, 1964.
[0592] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 3.55 (s, 4H), 3.40
(s, 6H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 50.67,
35.96.
[0593] Compound 19:
[0594] A DCM solution (1 mL) of 2-morpholinoethanethiol (0.17 g,
1.2 mmol) was added dropwise via syringe to a stirring solution of
compound 18 (300 mg, 1.2 mmol) in DCM (10 mL) at r.t. Immediately
after addition, the TLC was checked, to reveal rapid formation of
product and some dimer. After 0.5 h, the mixture was partitioned by
addition of NaHCO.sub.3. Upon extraction, the organic layer was
separated then dried (MgSO.sub.4), filtered and reduced in vacuo.
Column chromatography gave pure 19 (0.20 g, 53%) as a colorless
oil. .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 3.73-3.67 (t, J=4.7
Hz, 4H), 3.51-3.46 (m, 2H), 3.35 (s, 3H), 3.07-3.01 (m, 2H),
2.88-2.83 (m, 2H), 2.69-2.63 (m, 2H), 2.52-2.43 (t, J=4.6 Hz, 4H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 66.96, 57.91, 53.58,
50.79, 37.66, 36.10, 35.52; MS (ESI+ve): calc (M+H): 318.03. found:
318.04. Rf=0.3 (EtOAc).
[0595] Compound 21:
[0596] Compound 20 is converted to compound 21 by a procedure
analogous to that described for compound 11.
[0597] Compound 22:
[0598] Compound 21 is converted to compound 22 by a procedure
analogous to that described for compound 12.
[0599] Compound 23:
[0600] Compound 23 is prepared according to a literature method
(Journal of Medicinal Chemistry, 50(23), 5568-5570; 2007.)
[0601] Compound 24:
[0602] An ice-cold pyridine solution (10 mL) of compound 23 (1
mmol) is treated successively, in a dropwise fashion with acetyl
chloride (1 mmol), then after 5 min with MsCl (1.1 mmol). The
solution is warmed to room temperature then the solvent is removed.
The residue is dissolved in EtOAc, washed with water, dried
(MgSO.sub.4), filtered and reduced in vacuo. Purification by column
chromatography affords pure compound 24.
[0603] Compound 25:
[0604] Compound 24 is converted to compound 25 by a procedure
analogous to that described for compound 12.
[0605] Compound 27:
[0606] Compound 26 is converted to compound 27 by a procedure
analogous to that described for compound 14.
[0607] Compound 29:
[0608] Compound 28 is converted to compound 29 by a procedure
analogous to that described for compound 14.
[0609] Compound 30:
[0610] Compound 30 is prepared according to a literature method
(Tetrahedron, 42(2), 601-7; 1986.)
[0611] Compound 31:
[0612] Compound 31 is prepared from compound 30 according to a
patent procedure (US 20090181444)
[0613] Compound 33:
[0614] Compound 33 is prepared from compound 32 according to a
patent procedure (US 20090181444)
[0615] Compound 36:
[0616] An ice-cold DCM (20 mL) solution of compound 34 (1 mmol) is
treated with NEt.sub.3 (1 mmol) followed by the dropwise addition
of TMS-Cl (1.1 mmol). After 1 h, the solution is washed with water,
dried (MgSO.sub.4), filtered and reduced in vacuo. The crude TMS
protected material is redissolved in THF (10 mL), whereon PPh.sub.3
(1.2 mmol), compound 35 (1.2 mmol), then DEAD (1.2 mmol, dropwise)
are added in succession. After stirring at r.t. for 18 h, the
solvent is removed under vacuum, the residue is redissolved in DCM,
the solution of which is washed with water, dried (MgSO.sub.4),
filtered and reduced in vacuo. Purification by column
chromatography affords pure compound 36.
[0617] Compound 37:
[0618] A THF (10 mL) solution of compound 36 (0.5 mmol) is treated
with TBAF (1 mmol of a 1M solution in THF), with monitoring by TLC.
On completion of TMS cleavage, the solvent is removed under vacuum,
the residue is redissolved in DCM, the solution of which is washed
with water, dried (MgSO.sub.4), filtered and reduced in vacuo. The
crude alcohol is redissolved in pyridine (5 mL), and TsCl (0.55
mmol) is added. After 18 h at r.t., the solvent is removed, the
residue is redissolved in DCM, the solution of which is washed with
water, dried (MgSO.sub.4), filtered and reduced in vacuo.
Purification by column chromatography affords pure compound 37.
[0619] Compound 38:
[0620] Compound 37 is converted to compound 38 by a procedure
analogous to that described for compound 12.
[0621] Compound 40:
[0622] An ice-cold DCM (20 mL) solution of compound 39 (1 mmol) is
treated with NEt.sub.3 (1 mmol) followed by the dropwise addition
of TMS-Cl (1.1 mmol). After 1 h, the solution is washed with water,
dried (MgSO.sub.4), filtered and reduced in vacuo. The crude TMS
protected material is redissolved in THF (10 mL), whereon PPh.sub.3
(1.2 mmol), potassium p-toluenethiosulfonate (KTTS, 1.2 mmol),
anhydrous ZnCl.sub.2 (1 mmol) then DEAD (1.2 mmol, dropwise) are
added in succession. After stirring at r.t. for 18 h, the solvent
is removed under vacuum, the residue is redissolved in DCM, the
solution of which is washed with water, dried (MgSO.sub.4),
filtered and reduced in vacuo. Purification by column
chromatography affords pure compound 40.
[0623] Compound 41:
[0624] A THF (10 mL) solution of compound 40 (0.5 mmol) is treated
with TBAF (1 mmol of a 1M solution in THF), with monitoring by TLC.
On completion of TMS cleavage, the solvent is removed under vacuum,
the residue is redissolved in DCM, the solution of which is washed
with water, dried (MgSO.sub.4), filtered and reduced in vacuo. The
crude alcohol is redissolved in THF (10 mL), whereon PPh.sub.3 (1.2
mmol), compound 35 (1.2 mmol), then DEAD (1.2 mmol, dropwise) are
added in succession. After stirring at r.t. for 18 h, the solvent
is removed under vacuum, the residue is redissolved in DCM, the
solution of which is washed with water, dried (MgSO.sub.4),
filtered and reduced in vacuo. Purification by column
chromatography affords pure compound 40.
[0625] Compound 42:
[0626] Compound 41 is converted to compound 42 by a procedure
analogous to that described for compound 14.
Example 2
Thioalkylation of H-Phosphonates to Provide Phosphorothiotriesters
in Solution Phase
##STR00056## ##STR00057## ##STR00058##
[0628] Compound 100:
[0629] The synthetic procedure for Di-DMTr H-phosphonate TT dimer
(100) has been previously described (Froehler, Brian C.; Ng, Peter
G.; Matteucci, Mark D., Nucleic Acids Research (1986), 14(13),
5399-5407; Garegg, Per J.; Lindh, Ingvar; Regberg, Tor; Stawinski,
Jacek; Stroemberg, Roger; Henrichson, Christina Tetrahedron Letters
(1986), 27(34), 4051-4054).
[0630] Compound 101:
[0631] Compound 100, mixture of diastereomers (200 mg, 0.176 mmol)
was dissolved in ACN (6 mL) then trimethylsilyl
2,2,2-trifluoro-N-(trimethylsilyl)acetamidate (227 mg, 0.882 mmol)
was added. A solution of
(Z)--S-4-((2-morpholinoethyl)disulfanyl)but-2-enyl
methanesulfonothioate (121 mg, 0.353 mmol) in ACN (2 mL) was then
added, over the course of 1 h in 3 approximately equal portions,
with monitoring by TLC and HPLC/MS. After 3 h, the resulting
solution was partitioned by addition of water. Upon extraction, the
organic layer was separated then dried (MgSO.sub.4), filtered and
reduced in vacuo. Column chromatography gave compound 101 as a
white foam, 225 mg, 91%.
[0632] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 9.72 (d, br, 1H),
9.27, (d, br, 1H), 7.53 (dd, J=25.0, 1 Hz, 1H), 7.42, (t, J=7.0 Hz,
2H), 7.37-7.16 (m, 17H), 6.83 (m, 8H), 6.43-6.28 (m, 2H), 5.63-5.42
(m, 2H), 5.21 (q, J=7.1 Hz, 1H), 4.27 (m, br, 1H), 3.94 (m, br,
2H), 3.77 (m, 12H), 3.74-3.60 (m, 6H), 3.51-3.22 (m, 5H), 2.82-2.76
(m, 2H), 2.68-2.60 (m, 2H), 2.59-2.46 (m, 5H), 2.44-2.33 (m, 2H),
2.03-1.88 (m, 1H), 1.84 (m, 3H), 1.75-1.66 (m, 1H), 1.48-1.32 (dd,
J=11.8, 1.2 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
164.10, 164.07, 164.00, 163.94, 159.14, 159.10, 150.80, 150.78,
150.75, 150.63, 145.09, 144.30, 144.27, 136.31, 136.27, 136.22,
136.18, 135.95, 135.82, 135.43, 135.35, 135.33, 135.24, 135.22,
130.52, 130.43, 130.40, 129.49, 129.30, 128.54, 128.43, 128.39,
127.64, 127.57, 113.78, 113.76, 113.73, 113.67, 112.05, 111.56,
87.77, 87.66, 87.58, 85.77, 85.59, 84.63, 84.51, 74.42, 74.33,
67.02, 66.95, 63.63, 63.49, 58.27, 58.23, 55.60, 55.58, 53.69,
53.62, 39.48, 39.26, 39.18, 35.88, 35.61, 35.43, 35.36, 28.18,
12.83, 12.79, 12.02, 11.95.; .sup.31P NMR (162 MHz, CDCl.sub.3)
.delta. 29.25, 29.12; MS (ESI+ve): calc (M+H): 1398.46. found:
1398.64. R.sub.f=0.4 (5% MeOH/DCM).
[0633] Compound 201:
[0634] Compound 101 (0.150 g, 0.107 mmol) was stirred with 3%
TCA/DCM (10 mL) over 10 min. TLC and HPLC/MS showed that the
reaction was complete. 10 mL of MeOH was added and stirring
continued for 2 min. Solvents were evaporated and the residue was
purified by column chromatography to give compound 201 (85 mg,
100%) as a white solid.
[0635] .sup.1H NMR (399 MHz, CD.sub.3OD) .delta. 7.78 (dd, J=7.2,
1.3 Hz, 1H), 7.53 (d, J=1.3 Hz, 1H), 6.33-6.27 (m, 2H), 5.83-5.70
(m, 2H), 5.25-5.19 (m, 1H), 4.47-4.30 (m, 3H), 4.27-4.22 (m, 1H),
4.11-4.05 (m, 1H), 3.89-3.82 (t, J=4.8 Hz, 4H), 3.85 (m, 2H),
3.76-3.70 (ddd, J=15.5, 7.2, 1.7 Hz, 2H), 3.52 (dd, J=7.3, 3.7 Hz,
2H), 3.28-3.19 (br, 2H), 3.16-3.05 (br, 4H), 3.05-2.98 (ddd, J=9.8,
5.5, 2.0 Hz, 2H), 2.62-2.52 (tdd, J=11.5, 5.7, 1.9 Hz, 1H),
2.47-2.36 (m, 1H), 2.33-2.28 (m, 2H), 1.92-1.87 (m, 6H); .sup.31P
NMR (162 MHz, CD.sub.3OD) .delta. 30.22, 30.19; MS (ESI+ve): calc
(M+H): 794.20. found: 794.18. R.sub.f=0.3 (10% MeOH/DCM).
[0636] Compound 102:
[0637] Compound 100 (400 mg, 0.352 mmol) was converted to compound
102 by a procedure analogous to that described for compound 101
(417 mg, 90%).
[0638] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 9.17 (d, J=6.0 Hz,
1H), 9.13-9.00 (d, J=25.7 Hz, 1H), 7.58-7.49 (dd, J=26.3, 1.5 Hz,
1H), 7.45-7.40 (ddd, J=8.0, 5.2, 1.3 Hz, 2H), 7.40-7.18 (m, 17H),
6.87-6.81 (m, 8H), 6.44-6.30 (m, 2H), 5.65-5.53 (m, 1H), 5.53-5.44
(m, 1H), 5.26-5.16 (quintet, J=6.4 Hz, 1H), 4.61-4.54 (m, 2H),
4.30-4.24 (m, 1H), 4.19-4.13 (m, 1H), 3.97-3.88 (m, 2H), 3.80-3.72
(m, 12H), 3.69-3.57 (m, 1H), 3.54-3.30 (m, 5H), 2.61-2.49 (dt,
J=14.4, 5.4 Hz, 1H), 2.44-2.32 (m, 1H), 2.02-1.91 (dt, J=12.5, 5.4
Hz, 1H), 1.85-1.80 (dd, J=5.0, 1.3 Hz, 3H), 1.76-1.63 (m, 1H),
1.43-1.36 (dd, J=10.2, 1.2 Hz, 3H), 1.19-1.14 (d, J=2.0 Hz, 8H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 178.22, 178.17, 163.82,
163.80, 163.75, 158.92, 158.88, 150.52, 150.43, 144.90, 144.88,
144.10, 144.05, 136.11, 136.08, 136.05, 136.01, 135.59, 135.28,
135.16, 135.03, 135.01, 130.30, 130.23, 130.19, 130.16, 128.69,
128.64, 128.59, 128.39, 128.34, 128.23, 128.21, 128.17, 127.42,
127.34, 113.54, 113.45, 111.85, 111.82, 111.41, 111.36, 87.59,
87.43, 87.37, 85.47, 85.33, 84.43, 84.29, 84.08, 84.00, 83.92,
74.24, 67.36, 63.38, 63.26, 59.42, 55.37, 39.22, 38.77, 27.94,
27.24, 12.57, 11.80, 11.74; .sup.31P NMR (162 MHz, CDCl.sub.3)
.delta. 29.23, 28.97; MS (ESI+ve): calc (M+H): 1338.51. found:
1338.84. R.sub.f=0.5 (5% MeOH/DCM).
[0639] Compound 202:
[0640] Compound 102 (200 mg, 0.151 mmol) was converted to compound
202 by a procedure analogous to that described for compound 101
(105 mg, 97%).
[0641] .sup.1H NMR (399 MHz, CD.sub.3OD) .delta. 7.81-7.75 (dd,
J=8.2, 1.3 Hz, 1H), 7.57-7.51 (dd, J=8.2, 1.3 Hz, 1H), 6.33-6.23
(m, 2H), 5.85-5.75 (m, 1H), 5.75-5.66 (m, 1H), 5.26-5.19 (m, 1H),
4.72-4.66 (m, 2H), 4.47-4.30 (m, 3H), 4.27-4.20 (m, 1H), 4.11-4.04
(m, 1H), 3.83-3.76 (m, 2H), 3.74-3.64 (m, 2H), 2.62-2.51 (m, 1H),
2.45-2.35 (td, J=8.7, 6.5 Hz, 1H), 2.32-2.24 (m, 2H), 1.93-1.82 (m,
6H), 1.20-1.15 (d, J=2.1 Hz, 9H); .sup.13C NMR (126 MHz,
CD.sub.3OD) .delta. 179.65, 166.28, 152.30, 152.28, 152.22, 137.90,
137.81, 137.79, 130.07, 130.04, 129.26, 129.24, 111.93, 111.88,
111.87, 87.26, 87.22, 86.96, 86.90, 86.76, 86.54, 86.12, 86.07,
85.98, 85.92, 85.88, 85.82, 80.54, 80.49, 80.46, 80.41, 71.84,
71.67, 68.71, 68.66, 68.45, 68.40, 62.58, 62.50, 60.72, 40.51,
40.44, 39.70, 39.52, 39.48, 28.67, 28.64, 28.61, 27.53, 12.64,
12.48; .sup.31P NMR (162 MHz, CDCl.sub.3) .delta. 29.23, 28.97; MS
(ESI+ve): calc (M+H): 717.22. found: 717.23. R.sub.f=0.5 (10%
MeOH/DCM).
[0642] Compound 103:
[0643] Compound 100 (400 mg, 0.352 mmol) was converted to compound
103 by a procedure analogous to that described for compound 101
(379 mg, 83%).
[0644] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 9.48 (s, 1H),
9.41-9.29 (m, 1H), 7.60-7.48 (dd, J=9.0, 1.0 Hz, 1H), 7.46-7.40
(dt, J=6.9, 1.2 Hz, 2H), 7.39-7.17 (m, 17H), 6.89-6.79 (m, 8H),
6.44-6.31 (m, 2H), 5.27-5.20 (t, J=6.5 Hz, 1H), 4.30-4.24 (t, J=6.1
Hz, 1H), 4.19-4.15 (m, 2H), 4.13-4.07 (t, J=7.1 Hz, 1H), 3.99-3.90
(m, 2H), 3.79-3.74 (m, 12H), 3.70-3.58 (m, 1H), 3.51-3.43 (td,
J=8.8, 7.2, 2.3 Hz, 1H), 3.40-3.32 (m, 1H), 3.02-2.85 (m, 2H),
2.61-2.49 (dt, J=18.5, 7.0 Hz, 1H), 2.47-2.33 (m, 1H), 1.98-1.90
(dt, J=10.2, 5.0 Hz, 1H), 1.85-1.81 (m, 3H), 1.74-1.62 (td, J=14.2,
7.1 Hz, 1H), 1.42-1.36 (m, 3H), 1.19-1.13 (d, J=4.9 Hz, 9H);
.sup.31P NMR (162 MHz, CDCl.sub.3) .delta. 29.36, 29.18; .sup.13C
NMR (126 MHz, CDCl.sub.3) .delta. 177.97, 177.89, 163.94, 163.91,
163.90, 163.86, 158.91, 158.87, 150.63, 150.54, 150.53, 150.50,
144.88, 144.85, 144.10, 144.04, 136.09, 135.99, 135.52, 135.50,
135.24, 135.16, 135.12, 135.04, 135.00, 130.31, 130.29, 130.20,
130.16, 130.13, 128.34, 128.20, 128.18, 128.14, 127.39, 127.31,
124.89, 113.55, 113.52, 113.43, 111.84, 111.38, 87.58, 87.42,
87.36, 85.30, 84.98, 84.95, 84.40, 84.33, 84.27, 83.98, 83.91,
83.84, 79.31, 79.27, 78.88, 78.84, 74.16, 74.08, 67.56, 67.50,
67.46, 67.41, 63.33, 63.24, 62.79, 62.75, 55.34, 39.21, 39.16,
39.04, 39.00, 38.85, 38.82, 29.95, 29.92, 29.66, 29.63, 27.17,
12.53, 11.80, 11.72; MS (ESI+ve): calc (M+H): 1312.69. found:
1312.49. R.sub.f=0.4 (5% MeOH/DCM).
[0645] Compound 203:
[0646] Compound 103 (200 mg, 0.154 mmol) was converted to compound
203 by a procedure analogous to that described for compound 201
(103 mg, 98%).
[0647] .sup.1H NMR (399 MHz, CD.sub.3OD) .delta. 7.80-7.76 (dd,
J=8.2, 1.2 Hz, 1H), 7.55-7.51 (dd, 7.1, 1.2 Hz, 1H), 6.32-6.24 (m,
2H), 5.26-5.19 (m, 1H), 4.46-4.20 (m, 6H), 4.10-4.05 (m, 1H),
3.82-3.78 (dd, J=6.5, 3.2 Hz, 2H), 3.22-3.14 (ddd, J=16.6, 7.0, 5.8
Hz, 2H), 2.61-2.51 (tdd, J=13.0, 5.9, 2.1 Hz, 1H), 2.46-2.37 (ddd,
J=14.3, 8.3, 6.0 Hz, 1H), 2.31-2.26 (t, J=5.8 Hz, 2H), 1.91-1.86
(dt, J=11.0, 1.2 Hz, 6H), 1.21-1.17 (m, 9H); .sup.31P NMR (162 MHz,
CD.sub.3OD) .delta. 30.15; .sup.13C NMR (100 MHz, CD.sub.3OD)
.delta. 179.45, 179.42, 166.29, 152.31, 152.29, 152.23, 137.82,
137.80, 137.78, 111.91, 111.88, 87.21, 87.17, 86.94, 86.87, 86.63,
86.52, 86.11, 86.06, 85.92, 85.84, 85.77, 80.67, 80.60, 80.49,
80.43, 71.79, 71.64, 68.80, 68.74, 68.58, 68.52, 64.11, 64.07,
64.02, 62.54, 62.44, 40.48, 40.43, 39.81, 39.71, 39.68, 39.52,
39.47, 30.74, 30.72, 30.68, 27.52, 12.65, 12.50; MS (ESI+ve): calc
(M+H): 691.21. found: 691.09. R.sub.f=0.5 (10% MeOH/DCM).
[0648] Compound 104:
[0649] Compound 100 (400 mg, 0.352 mmol) was converted to compound
104 by a procedure analogous to that described for compound 101
(451 mg, 94%).
[0650] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 9.17-9.01 (m, 2H),
7.51-7.46 (dd, J=7.8, 1.5 Hz, 1H), 7.45-7.38 (m, 2H), 7.37-7.09 (m,
19H), 7.01-6.90 (m, 2H), 6.87-6.78 (m, 8H), 6.39-6.27 (m, 2H),
5.15-5.01 (m, 1H), 4.20-4.13 (m, 1H), 3.96-3.90 (m, 1H), 3.90-3.83
(m, 2H), 3.80-3.68 (m, 14H), 3.52-3.20 (m, 3H), 2.45-2.16 (m, 2H),
2.01-1.88 (ddd, J=23.3, 13.6, 5.6 Hz, 1H), 1.85-1.79 (dd, J=9.3,
1.2 Hz, 3H), 1.69-1.53 (m, 1H), 1.40-1.31 (m, 12H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 176.46, 176.37, 163.84, 163.78,
158.90, 158.87, 150.52, 150.50, 150.43, 149.38, 149.28, 144.95,
144.88, 144.16, 144.10, 136.13, 136.11, 136.09, 136.03, 135.57,
135.49, 135.37, 135.26, 135.21, 135.08, 135.04, 130.83, 130.74,
130.29, 130.21, 130.16, 129.51, 129.49, 129.40, 129.36, 129.35,
129.31, 128.38, 128.35, 128.27, 128.23, 128.19, 128.14, 127.39,
127.33, 126.05, 125.94, 122.94, 122.86, 113.53, 113.42, 111.77,
111.73, 111.39, 111.28, 87.55, 87.52, 87.37, 87.32, 85.33, 84.95,
84.90, 84.29, 84.20, 84.00, 83.92, 83.87, 83.79, 79.05, 79.00,
74.29, 74.24, 67.31, 67.24, 67.17, 67.11, 63.37, 55.37, 55.35,
39.37, 39.32, 39.15, 39.10, 38.64, 30.51, 30.41, 30.36, 27.28,
27.24, 12.59, 12.51, 11.75, 11.67; .sup.31P NMR (162 MHz,
CDCl.sub.3) .delta. 29.12, 28.49; MS (ESI+ve): calc (M+NH.sub.4):
1374.51. found: 1374.74. R.sub.f=0.4 (5% MeOH/DCM).
[0651] Compound 204:
[0652] Compound 104 (200 mg, 0.147 mmol) was converted to compound
204 by a procedure analogous to that described for compound 201 (98
mg, 88%).
[0653] .sup.1H NMR (399 MHz, CD.sub.3OD) .delta. 7.77-7.73 (m, 1H),
7.51-7.43 (m, 2H), 7.38-7.31 (m, 1H), 7.25-7.19 (ddd, J=9.2, 5.4,
1.6 Hz, 1H), 7.08-7.02 (ddd, J=8.0, 3.8, 1.3 Hz, 1H), 6.28-6.17 (m,
2H), 5.10-5.01 (m, 1H), 4.30-4.16 (m, 3H), 4.11-4.03 (m, 3H),
4.03-3.97 (d, J=5.3 Hz, 2H), 3.74-3.63 (m, 2H), 2.48-2.11 (m, 5H),
1.90-1.82 (m, 6H), 1.43-1.36 (d, J=3.4 Hz, 9H); .sup.13C NMR (100
MHz, CD.sub.3OD) .delta. 178.05, 166.26, 152.25, 152.19, 150.78,
137.80, 137.76, 132.13, 132.09, 130.61, 130.56, 127.24, 124.10,
111.92, 111.84, 111.79, 87.14, 87.09, 86.80, 86.71, 86.50, 85.98,
85.95, 85.92, 85.87, 85.83, 85.75, 80.55, 80.48, 80.32, 80.27,
71.97, 71.73, 68.67, 68.61, 68.35, 68.29, 62.51, 62.42, 40.41,
40.36, 40.32, 39.66, 39.64, 39.35, 39.29, 31.08, 31.04, 27.61,
12.68, 12.65, 12.49; .sup.31P NMR (162 MHz, CD.sub.3OD) .delta.
29.54, 29.29; MS (ESI+ve): calc (M+H): 753.22. found: 753.12.
R.sub.f=0.5 (10% MeOH/DCM).
[0654] Compound 105:
[0655] Compound 100 (200 mg, 0.176 mmol) was converted to compound
105 by using compound 14 in a procedure analogous to that described
for compound 101 (158 mg, 70%).
[0656] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.46-7.39 (m, 2H)
7.38-7.16 (m, 18H), 6.90-6.77 (m, 8H), 6.43-6.27 (m, 1H), 5.39-5.18
(m, 2H), 4.31-4.23 (dd, J=12.0, 6.2 Hz, 1H), 4.20-4.12 (m, 1H),
3.98-3.86 (m, 1H), 3.82-3.70 (m, 12H), 3.69-3.52 (m, 1H), 3.50-3.43
(td, J=9.9, 8.9, 2.7 Hz, 1H), 3.41-3.29 (ddd, J=17.2, 10.8, 2.5 Hz,
1H), 2.59-2.49 (m, 1H), 2.44-2.30 (m, 1H), 2.03-1.93 (m, 1H),
1.86-1.79 (d, J=2.9 Hz, 3H), 1.75-1.67 (m, 4H), 1.43-1.36 (d, 3H),
1.16-1.08 (d, J=9.3 Hz, 9H); .sup.31P NMR (162 MHz, CDCl.sub.3)
.delta. 28.14, 27.81 (two diastereomers). MS (ESI+ve): calc (M+H):
1281.4. found: 1281.1 (M+H).sup.+ and 1298.6
(M+NH.sub.4).sup.+.
[0657] Compound 205:
[0658] Compound 105 (137 mg, 0.107 mmol) was converted to compound
205 by a procedure analogous to that described for compound 201 (66
mg, 91%).
[0659] .sup.1H NMR (399 MHz, CD.sub.3OD) .delta. 7.83-7.76 (m, 1H),
7.56-7.50 (m, 1H), 6.34-6.22 (m, 2H), 5.51-5.43 (m, H), 5.28-5.20
(qt, 0.1=7.8, 1.8 Hz, 1H), 4.47-4.31 (m, 3H), 4.29-4.21 (m, 1H),
4.10-4.05 (m, 1H), 3.87-3.73 (dd, J=7.6, 3.1 Hz, 2H), 2.62-2.50
(tdd, J=16.9, 5.7, 1.9 Hz, 1H), 2.45-2.36 (m, 1H), 2.32-2.25 (ddd,
J=6.9, 5.4, 1.5 Hz, 3H), 1.92-1.84 (m, 6H), 1.22-1.18 (d, J=5.3 Hz,
9H); .sup.31P NMR (162 MHz, CD.sub.3OD) .delta. 28.71, 28.42 (two
diastereomers). MS (ESI+ve): calc (M+H): 677.2. found: 677.2
(M+H).sup.+, 694.2 (M+NH.sub.4).sup.+.
[0660] Compound 106:
[0661] Compound 100 (405 mg, 0.357 mmol) was converted to compound
106 by using compound 19 and following a procedure analogous to
that described for compound 101 (0.35 g, 71%). .sup.1H NMR (399
MHz, CDCl.sub.3) .delta. 9.97-9.42 (m, 2H), 7.58-7.47 (m, 1H),
7.46-7.39 (m, 2H), 7.39-7.13 (m, 17H), 6.87-6.78 (m, 8H), 6.44-6.29
(dtd, J=20.4, 9.2, 4.7 Hz, 2H), 5.27-5.16 (dt, J=14.7, 7.3 Hz, 1H),
4.30-4.22 (m, 1H), 4.22-4.12 (m, 1H), 4.02-3.90 (q, J=3.8, 3.4 Hz,
2H), 3.80-3.73 (m, 12H), 3.72-3.65 (m, 5H), 3.51-3.43 (m, 1H),
3.40-3.31 (m, 1H), 3.14-2.93 (m, 2H), 2.85-2.72 (m, 4H), 2.67-2.59
(m, 2H), 2.57-2.34 (m, 6H), 1.97-1.87 (td, J=13.7, 13.1, 5.7 Hz,
1H), 1.84 (s, 3H), 1.73-1.61 (td, J=14.1, 6.8 Hz, 1H), 1.42-1.37
(d, J=6.7 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
163.97, 163.94, 163.91, 158.88, 158.84, 150.64, 150.60, 150.52,
144.86, 144.83, 144.09, 144.04, 136.06, 136.04, 135.95, 135.93,
135.54, 135.19, 135.09, 135.03, 134.99, 130.28, 130.17, 130.13,
128.29, 128.17, 128.14, 127.38, 127.31, 113.51, 113.42, 111.82,
111.79, 111.44, 111.38, 87.53, 87.38, 87.33, 85.29, 85.26, 84.89,
84.85, 84.41, 84.36, 84.29, 84.25, 83.88, 83.85, 83.80, 83.76,
79.28, 79.23, 78.72, 78.67, 74.04, 67.53, 67.46, 67.37, 67.29,
66.77, 63.33, 63.21, 57.84, 55.34, 53.41, 53.34, 39.23, 39.09,
39.01, 38.92, 38.55, 38.51, 38.46, 38.42, 35.64, 35.59, 30.35,
30.30, 30.26, 12.60, 11.79, 11.74; .sup.31P NMR (162 MHz,
CDCl.sub.3) .delta. 29.30, 29.14; MS (ESI+ve): calc (M+H): 1372.44.
found: 1372.79. R.sub.f=0.4 (5% MeOH/DCM).
[0662] Compound 206:
[0663] Compound 106 (200 mg, 0.146 mmol) was converted to compound
206 by a procedure analogous to that described for compound 201
(110 mg, 98%).
[0664] .sup.1H NMR (399 MHz, CD.sub.3OD) .delta. 7.83-7.75 (dd,
J=7.6, 1.4 Hz, 1H), 7.56-7.48 (d, J=1.6 Hz, 1H), 6.35-6.23 (m, 2H),
5.27-5.20 (m, 1H), 4.48-4.31 (m, 3H), 4.28-4.21 (dd, J=9.7, 2.1 Hz,
1H), 4.11-4.04 (t, J=4.0 Hz, 1H), 3.97-3.84 (br, 4H), 3.83-3.77
(dd, J=6.0, 3.2 Hz, 2H), 3.43-3.36 (m, 2H), 3.29-3.18 (m, 6H),
3.11-3.00 (m, 4H), 2.62-2.51 (tdd, J=11.7, 5.7, 1.7 Hz, 1H),
2.47-2.38 (ddd, J=14.3, 8.4, 6.0 Hz, 1H), 2.38-2.25 (q, J=5.3, 4.8
Hz, 2H), 1.91 (s, 3H), 1.88 (s, 3H); .sup.31P NMR (162 MHz,
CD.sub.3OD) .delta. 30.19, 30.12; .sup.13C NMR (100 MHz,
CD.sub.3OD) .delta. 166.28, 166.24, 166.23, 152.32, 152.27, 152.24,
138.05, 138.00, 137.77, 137.75, 112.08, 112.03, 111.97, 111.94,
87.28, 87.24, 87.01, 86.96, 86.62, 86.51, 86.10, 86.06, 85.76,
85.68, 71.73, 71.51, 68.91, 68.58, 68.51, 65.44, 62.60, 62.50,
57.50, 53.50, 40.25, 40.16, 39.64, 39.57, 39.20, 39.16, 39.06,
32.56, 32.55, 31.04, 31.00, 12.73, 12.69, 12.52; MS (ESI+ve): calc
(M+H): 768.18. found: 768.14. R.sub.f=0.3 (10% MeOH/DCM).
[0665] Compound 107:
[0666] Using compound 22 in place of compound 5, compound 100 is
converted to compound 107 by a procedure analogous to that
described for compound 101.
[0667] Compound 207:
[0668] Compound 107 is converted to compound 207 by a procedure
analogous to that described for compound 201.
[0669] Compound 108:
[0670] Using compound 25 in place of compound 5, compound 100 is
converted to compound 108 by a procedure analogous to that
described for compound 101.
[0671] Compound 208:
[0672] Compound 108 is converted to compound 208 by a procedure
analogous to that described for compound 201.
[0673] Compound 109:
[0674] Using compound 27 in place of compound 5, compound 100 is
converted to compound 109 by a procedure analogous to that
described for compound 101.
[0675] Compound 209:
[0676] Compound 109 is converted to compound 209 by a procedure
analogous to that described for compound 201.
[0677] Compound 110:
[0678] Using compound 29 in place of compound 5, compound 100 is
converted to compound 110 by a procedure analogous to that
described for compound 101.
[0679] Compound 210:
[0680] Compound 110 is converted to compound 210 by a procedure
analogous to that described for compound 201.
[0681] Compound 111:
[0682] Using compound 31 in place of compound 5, compound 100 is
converted to compound 111 by a procedure analogous to that
described for compound 101.
[0683] Compound 211:
[0684] Compound 111 is converted to compound 211 by a procedure
analogous to that described for compound 201.
[0685] Compound 112:
[0686] Using compound 33 in place of compound 5, compound 100 is
converted to compound 112 by a procedure analogous to that
described for compound 101.
[0687] Compound 212:
[0688] Compound 112 is converted to compound 212 by a procedure
analogous to that described for compound 201.
[0689] Compound 113:
[0690] Using compound 38 in place of compound 5, compound 100 is
converted to compound 113 by a procedure analogous to that
described for compound 101.
[0691] Compound 213:
[0692] Compound 113 is converted to compound 213 by a procedure
analogous to that described for compound 201.
[0693] Compound 114:
[0694] Using compound 41 in place of compound 5, compound 100 is
converted to compound 114 by a procedure analogous to that
described for compound 101.
[0695] Compound 214:
[0696] Compound 114 is converted to compound 214 by a procedure
analogous to that described for compound 201.
[0697] Compound 115:
[0698] Using compound 43 in place of compound 5, compound 100 is
converted to compound 115 by a procedure analogous to that
described for compound 101.
[0699] Compound 215:
[0700] Compound 115 is converted to compound 215 by a procedure
analogous to that described for compound 201.
Example 3
Alternative Synthesis of Phosphorothiotriesters Using
##STR00059## ##STR00060## ##STR00061##
[0702] Compound 150:
[0703] Compound 100 (300 mg, 0.264 mmol) was converted to compound
150 by a procedure analogous to that described for compound 101
(170 mg, 50%).
[0704] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 9.34-9.30 (s, 1H),
9.28-9.17 (d, J=30.6 Hz, 1H), 7.57-7.47 (m, 1H), 7.47-7.40 (m, 2H),
7.38-7.18 (m, 17H), 7.18-7.07 (d, =1.4 Hz, 1H), 6.88-6.77 (dd,
J=9.0, 1.5 Hz, 8H), 6.44-6.34 (ddd, J=15.6, 8.9, 5.4 Hz, 1H),
6.32-6.21 (ddd, J=18.9, 8.5, 5.9 Hz, 1H), 5.27-5.19 (q, J=5.9 Hz,
1H), 4.46-4.33 (m, 2H), 4.31-4.16 (m, 2H), 4.03-3.91 (m, 2H),
3.81-3.67 (m, 12H), 3.54-3.46 (m, 1H), 3.42-3.34 (m, 1H), 3.34-3.25
(d, J=20.2 Hz, 3H), 2.64-2.53 (td, J=13.4, 5.4 Hz, 1H), 2.47-2.34
(dq, J=19.9, 6.5, 5.9 Hz, 1H), 1.99-1.91 (m, 1H), 1.85-1.80 (t,
J=1.5 Hz, 3H), 1.78-1.65 (tt, J=14.1, 7.5 Hz, 111), 1.44-1.37 (dd,
J=7.3, 1.2 Hz, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
171.27, 163.83, 163.80, 158.95, 158.93, 158.90, 150.64, 150.53,
150.46, 150.38, 144.91, 144.88, 144.09, 144.02, 136.00, 135.98,
135.94, 135.81, 135.11, 135.04, 134.98, 134.97, 130.34, 130.27,
130.20, 128.30, 128.23, 128.20, 127.46, 127.36, 113.59, 113.56,
113.48, 111.95, 111.38, 87.60, 87.47, 87.43, 86.03, 85.83, 84.44,
84.34, 83.81, 79.82, 79.58, 73.99, 73.91, 67.85, 67.78, 63.31,
63.20, 55.39, 51.77, 51.70, 39.16, 38.99, 38.90, 37.21, 37.16,
37.12, 37.05, 12.63, 12.57, 11.85, 11.80; .sup.31P NMR (162 MHz,
CDCl.sub.3) .delta. 26.15, 25.60; MS (ESI+ve): calc (M+H): 1308.37.
found: 1308.70. R.sub.f=0.5 (5% MeOH/DCM).
[0705] Compound 151:
[0706] A DCM (5 mL) solution of compound 150 (150 mg, 0.116 mmol)
was treated with 2-morpholinoethanethiol (17 mg, 0.116 mmol) at
r.t. with monitoring by TLC. After 0.5 h, the mixture was washed
with NaHCO.sub.3, extracting 5.times. into DCM. The organic
extracts were dried (MgSO.sub.4), filtered and reduced. Column
chromatography gave compound 151 as a colorless solid foam (81 mg,
51%).
[0707] .sup.1H NMR (399 MHz, CDCl.sub.3) .delta. 9.68-9.54 (m, 1H),
9.44 (s, 1H), 7.59-7.48 (m, 1H), 7.47-7.40 (m, 2H), 7.40-7.13 (m,
17H), 6.90-6.76 (ddd, J=9.3, 4.4, 2.7 Hz, 8H), 6.45-6.27 (m, 2H),
5.32-5.22 (dd, J=8.5, 5.7 Hz, 1H), 4.34-4.25 (m, 1H), 4.23-4.14 (m,
1H), 4.07-3.89 (m, 2H), 3.79-3.74 (m, 12H), 3.74-3.65 (m, 6H),
3.51-3.33 (m, 2H), 2.90-2.79 (dd, J=14.2, 7.6 Hz, 2H), 2.73-2.55
(m, 3H), 2.55-2.34 (m, 6H), 2.02-1.91 (m, 1H), 1.87-1.81 (dd,
J=4.9, 1.2 Hz, 3H), 1.77-1.66 (ddd, J=14.2, 8.7, 6.4 Hz, 1H),
1.41-1.35 (dd, J=6.6, 1.2 Hz, 3H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 163.97, 163.93, 163.88, 158.90, 158.86, 158.71,
150.64, 150.59, 150.53, 150.50, 144.92, 144.88, 144.13, 144.08,
136.11, 136.07, 136.03, 136.00, 135.73, 135.60, 135.22, 135.14,
135.08, 135.04, 135.02, 130.32, 130.30, 130.23, 130.18, 128.33,
128.19, 128.17, 127.39, 127.33, 113.56, 113.52, 113.45, 111.85,
111.82, 111.38, 111.29, 87.56, 87.41, 87.38, 85.71, 85.35, 84.91,
84.38, 84.27, 84.22, 84.05, 83.97, 83.85, 83.78, 79.36, 79.11,
79.05, 74.25, 74.07, 67.39, 66.88, 66.79, 63.27, 57.80, 55.36,
53.55, 53.51, 53.40, 43.06, 40.72, 40.54, 39.25, 39.16, 39.01,
35.91, 12.64, 12.60, 11.78, 11.74; .sup.31P NMR (162 MHz,
CDCl.sub.3) .delta. 27.76, 27.46; MS (ESI+ve): calc (M+H): 1358.43.
found: 1358.74. R.sub.f=0.4 (5% MeOH/DCM).
[0708] Compound 251:
[0709] Compound 151 (75 mg, 0.055 mmol) was converted to compound
251 by a procedure analogous to that described for compound 201 (10
mg, 24%). MS (ESI+ve): calc (M+H): 754.17. found: 754.19.
R.sub.f=0.3 (10% MeOH/DCM).
[0710] Compound 152:
[0711] Compound 100 is converted to compound 152 by a procedure
analogous to that described for compound 101.
##STR00062## ##STR00063##
[0712] Compound 153:
[0713] Using 1-Thio-.beta.-D-glucose tetraacetate in place of
compound 4, compound 152 is converted to compound 153 by a
procedure analogous to that described for compound 151.
[0714] Compound 253:
[0715] Compound 153 is converted to compound 253 by a procedure
analogous to that described for compound 201.
[0716] Compound 154:
[0717] Compound 100 is converted to compound 154 by a procedure
analogous to that described for compound 101.
[0718] Compound 155:
[0719] Compound 154 is converted to compound 155 by a procedure
analogous to that described for compound 151.
[0720] Compound 255:
[0721] Compound 155 is converted to compound 255 by a procedure
analogous to that described for compound 201.
Example 4
Thioalkylation of H-Phosphonates to Provide Phosphorothiotriesters
in Solid Phase
[0722] Compound 300:
[0723] Synthesis of (Rp)-CAGT-H-phosphonate-oxalyl linker-CPG was
carried out on an Applied Biosystems 394 DNA/RNA synthesizer
according to the reported methods (Journal of American Chemical
Society 2008, 130, 16031-16037; Angewandte Chemie International
Edition 2009, 48, 496-499).
[0724] Compound 301:
[0725] (Sp)-CAGT-phosphorothioate (R.dbd.H):
(Rp)-CAGT-H-phosphonate-oxalyl linker-CPG was treated by 0.2 M
Beaucage Reagent/CH.sub.3CN--BSA (9:1, v/v), stirred for 1 h at rt,
then washed successively with CS.sub.2 and acetonitrile and dried
under reduced pressure. The resultant CPG was treated with 2 mL of
28% aqueous NH.sub.3 and stirred for 18 h at rt. After removal of
NH.sub.3 under reduced pressure, the resulting product was analyzed
by LC/MS and HPLC.
[0726] Compound 302:
[0727] (Sp)-CAGT-S-methyl phosphorothiotriester (R=Me): BSTFA (50
.mu.L, 188 .mu.mol) and acetonitrile (500 .mu.L) were added to
(Rp)-CAGT-H-phosphonate-oxalyl linker-CPG (14.7 mg, 1 .mu.mol) then
the mixture was shaken for 20 min at rt. S-methyl methane
sulfonothioate (20 .mu.L, 212 .mu.mol) and NEt.sub.3 (50 .mu.L)
were added and shaking was continued for 1 h at rt. The CPG was
washed with CH.sub.3CN then dried in vacuo. 20% PrNH.sub.2 in dry
CH.sub.3CN (2 mL) was added to the CPG and the mixture was stirred
for 16 h at rt. Solvents were removed under reduced pressure and
CH.sub.3CN was added to the mixture. The CPG was removed by
filtration and the filtrate was concentrated under reduced
pressure. CH.sub.3CN/DMSO/0.5 M AA buffer (1:1:1, v/v/v) was added,
the mixture was stirred for 16 h at rt, then analyzed by LC/MS and
HPLC.
[0728] Compound 303:
[0729] Compound 303 is prepared by sulfurization of compound 300 on
support followed by cleavage. ACN (450 .mu.L), BSTFA (50 .mu.L) and
compound 12 (20 mg) are added to compound 300 (1 .mu.mol) which is
shaken for 18 h. The CPG is collected by filtration resuspended in
20% PrNH.sub.2 in dry CH.sub.3CN (2 mL) and shaken for 16 h at rt.
Solvents were removed under reduced pressure and the residue is
purified by RPHPLC to provide pure compound 303.
Example 5
Thioalkylation of H-Phosphonates to Provide Phosphorothiotriesters
in Solution Phase
##STR00064## ##STR00065## ##STR00066##
[0731] Compound 305:
[0732] Compound 300 (0.5 .mu.mol) was taken up in ACN (125 .mu.L)
then BSTFA (62 .mu.L) was added and the mixture was shaken for 20
min. PrNH.sub.2 (125 .mu.L) was added and the vial was rotated for
18 h. After filtration and washing with 1 mL ACN, the solvent was
removed in vacuo and the residue was co-evaporated 3.times. with
toluene to provide crude compound 304. The residue was redissolved
in pyridine (375 .mu.L) and treated with BSTFA for (16 .mu.l, 60.0
.mu.mol) followed by compound 9 (7.2 mg, 30.0 .mu.mol) with
stirring under Ar. After 2 h at r.t. the solvent was removed and
the residue was treated with MeOH (0.125 mL) for 1 h, then AA (0.5
M, 0.125 mL) was added and the mixture was stirred at r.t. for 2 h.
The product was purified by RPHPLC to provide compound 305.
[0733] Compound 303:
[0734] Substituting compound 12 for compound 9, compound 303 was
prepared by a procedure analogous to that described for compound
305.
[0735] Compound 306:
[0736] Substituting compound 12 for compound 14, compound 306 was
prepared by a procedure analogous to that described for compound
305.
[0737] Compound 307:
[0738] Substituting compound 12 for compound 29, compound 307 is
prepared by a procedure analogous to that described for compound
305.
[0739] Compound 308:
[0740] Substituting compound 12 for compound 31, compound 308 is
prepared by a procedure analogous to that described for compound
305.
[0741] Compound 309:
[0742] Substituting compound 12 for compound 38, compound 309 is
prepared by a procedure analogous to that described for compound
305.
Example 6
Stereoselective Thioalkylation of H-Phosphanates
[0743] Objective: To demonstrate that the reaction of MTS reagents
to H-phosphonate to generate phosphorothio triester is
stereospecific. .sup.31P NMR was used to trace the changes during
the course of the reaction.
##STR00067##
[0744] Experimental procedure: In an NMR tube was added compound
100S 5'-O-(4,4'-dimethoxytrityl)thymidin-3'-yl
3'-O-(4,4'-dimethoxytrityl)thymidin-5'-yl H-phosphonate (20 mg, 18
mol) in 0.8 mL CD.sub.3CN and the .sup.31P NMR spectrum was
recorded. BSTFA (17 .mu.L, 176 .mu.mol) was added to same NMR tube
and after 5 min .sup.31P NMR spectrum was recorded again.
Triethylamine (49 .mu.L, 352 .mu.mol) and S-methyl
methanethiosulfonate (22 .mu.L, 88 .mu.mol) were added to same NMR
tube and .sup.31P NMR spectrum was recorded immediately.
[0745] The same procedure was repeated for Rp isomer (compound
100R). The .sup.31P NMR spectrum recorded for the starting
material, intermediate and the product show that the
stereochemistry at phosphorus atom is retained during the
reaction.
[0746] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *