US2003153743A1PendingUtilityA1
Processes for the synthesis of oligomeric compounds
Priority: Apr 24, 1998Filed: Jan 3, 2003Published: Aug 14, 2003
Est. expiryApr 24, 2018(expired)· nominal 20-yr term from priority
C07H 21/00C07B 2200/11Y02P20/55
53
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Claims
Abstract
Methods for the preparation of oligonucleotides having bioreversible phosphate blocking groups are disclosed. The oligonucleotides are prepared utilizing amidite type chemistry wherein the bioreversible phosphorus protecting group is formed as an integral part of the amidite reagent.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method for preparing an oligomeric compound comprising a moiety having the Formula I:
wherein:
Z is aryl having 6 to about 14 carbon atoms or alkyl having from one to about six carbon atoms;
Y 1 is O or S;
Y 2 is O or S;
Y 3 is C(═O) or S;
q is 2 to about 4;
R 1 is H, OH, F, or a group of formula R 7 —(R 8 ) n ;
R 7 is C 3 -C 20 alkyl, C 4 -C 20 alkenyl, C 2 -C 20 ; alkynyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyloxy, or C 2 -C 20 alkynyloxy;
R 8 is hydrogen, amino, protected amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, or a group of formula (—O—X 1 ) p , where p is 2 to about 10 and X 3 is alkyl having from one to about 10 carbons;
B is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
comprising the steps of:
providing a compound having the Formula II:
wherein:
R 3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
M is an optionally protected internucleotide linkage;
each B, independently is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
n is 0 to about 50;
R 5 is —N(R) 6 , 2 or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R 6 is straight or branched chain alkyl having from 1 to 10 carbons; and
reacting the compound of Formula II with a compound having Formula III:
wherein:
R 3a is hydrogen;
m is 0 to about 50;
R 2 is a hydroxyl protecting group, or a linker connected to a solid support, provided that R 2 and R 3 are not both simultaneously a linker connected to a solid support;
thereby forming the oligomeric compound.
2 . The method of claim 1 further comprising the step of oxidizing or sulfurizing the oligomeric compound to form a compound having Formula III, wherein R 3a is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support; and where m is increased by n+1.
3 . The method of claim 2 further comprising a capping step.
4 . The method of claim 3 wherein the capping step is performed prior to oxidation.
5 . The method of claim 3 further comprising the step of cleaving the oligomeric compound to produce a compound having the Formula IV:
wherein R 2 is H.
6 . The method of claim 5 wherein the cleaving step occurs enzymatically.
7 . The method of claim 5 wherein the cleaving step occurs in vivo.
8 . The method of claim 1 wherein q is 2; and Y 3 is C(═O).
9 . The method of claim 1 wherein Z is methyl, phenyl or t-butyl.
10 . The method of claim 9 wherein Z is t-butyl.
11 . The method of claim 8 wherein n is 0.
12 . The method of claim 8 wherein R 2 is a linker to a solid support.
13 . The method of claim 8 wherein Y 1 and Y 2 are each 0.
14 . The method of claim 8 wherein Y 1 and Y 2 are each S.
15 . The method of claim 8 wherein Y 1 is O and Y 2 is S.
16 . The method of claim 8 wherein each R 6 is isopropyl.
17 . The method of claim 8 wherein n is 0; R 3 is H, R 5 is diisopropylamino; Y 1 is O; Y 2 is S; and Z is methyl or t-butyl.
18 . The method of claim 17 wherein Z is t-butyl.
19 . The method of claim 1 wherein B is a radiolabeled nucleobase.
20 . The method of claim 19 wherein the radiolabeled nucleobase has the formula:
wherein * denotes a 14 C atom.
21 . The method of claim 17 wherein B is a radiolabeled nucleobase of formula:
wherein * denotes a 14 C atom.
22 . The method of claim 1 wherein the compound of Formula II is formed by reaction of a compound having Formula V:
with a compound having the Formula VI:
in the presence of an acid.
23 . The method of claim 1 wherein the compound of Formula II is obtained by reaction of a compound having Formula V:
with a chlorophosphine compound of formula ClP[i-Pr 2 N] 2 , followed by reaction with a compound of Formula XX:
in the presence of an acid.
24 . The method of claim 1 wherein M is an optionally protected phosphodiester, phosphorothioate, phosphorodithioate, or alkyl phosphonate internucleotide linkage.
25 . A method for preparing a phosphoramidite of Formula:
wherein:
R 3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
Z is aryl having 6 to about 14 carbon atoms or alkyl having from one to about six carbon atoms;
Y 1 is O or S;
Y 2 is O or S;
Y 3 is C(═O) or S;
q is 2 to about 4;
M is an optionally protected internucleotide linkage;
each B, independently is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
n is 0 to about 50;
R 1 is H, OH, F, or a group of formula R 7 —(R 8 ) n ;
R 7 is C 3 -C 20 alkyl, C 4 -C 2 , alkenyl, C 2 -C 20 alkynyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyloxy, or C 2 -C 20 alkynyloxy;
R 8 is hydrogen, amino, protected amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, or a group of formula (—O—X 3 ) p , where p is 1 to about 10 and X 3 is alkyl having from one to about 10 carbons;
R 5 is —N(R 6 ) 2 , or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R 6 is straight or branched chain alkyl having from 1 to 10 carbons;
comprising the steps of:
providing a compound Formula:
and reacting the compound with a diaminohalophosphine of Formula:
wherein X is halogen;
thereby providing a phosphordiamidite of Formula:
and
contacting the nucleoside phosphordiamidite with a reagent of Formula XX:
to produce the phosphoramidite.
26 . The method of claim 25 wherein q is 2 and Y 3 is C(═O).
27 . The method of claim 26 wherein n is 0.
28 . The method of claim 26 wherein each R 6 is alkyl.
29 . The method of claim 26 wherein each R 6 is isopropyl.
30 . The method of claim 26 wherein Y 1 and Y 2 are each O.
31 . The method of claim 26 wherein Y 1 and Y 2 are each S.
32 . The method of claim 26 wherein Y 1 is O and Y 2 is S.
33 . The method of claim 26 wherein Z is methyl, phenyl or t-butyl.
34 . The method of claim 33 wherein Z is t-butyl.
35 . The method of claim 26 wherein Z is methyl, phenyl or t-butyl, n is 0, Y 1 is O and Y 2 is S.
36 . The method of claim 26 wherein M is a optionally protected phosphodiester, phosphorothioate, phosphorodithioate, or alkyl phosphonate internucleotide linkage.
37 . The method of claim 26 wherein X is chlorine.
38 . The method of claim 25 wherein B is a radiolabeled nucleobase.
39 . The method of claim 35 wherein B is a radiolabeled nucleobase.
40 . The method of claim 25 or claim 35 wherein the radiolabeled nucleobase has the Formula:
wherein * denotes a 14 C atom.
41 . A method for preparing a phosphoramidite of Formula:
wherein:
R 3 is hydrogen, a hydroxyl protecting group, or a linker connected to a solid support;
Z is aryl having 6 to about 14 carbon atoms or alkyl having from one to about six carbon atoms;
Y 1 is O or S;
Y 2 is O or S;
Y 3 is C(═O) or S;
M is an optionally protected internucleotide linkage;
each B, independently is a naturally occurring or non-naturally occurring nucleobase that is optionally protected and optionally radiolabeled;
n is 0 to about 50;
R 1 is H, OH, F, or a group of formula R 7 —(R 8 ) n ;
R 7 is C 3 -C 10 alkyl, C 4 -C 20 alkenyl, C 2 -C 29 alkynyl, C 1 -C 20 alkoxy, C 2 -C 20 alkenyloxy, or C 2 -C 20 alkynyloxy;
R 8 is hydrogen, amino, protected amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S-aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, N-phthalimido, imidazole, azido, hydrazino, hydroxylamino, isocyanato, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocycle, carbocycle, intercalator, reporter molecule, conjugate, polyamine, polyamide, polyalkylene glycol, polyether, a group that enhances the pharmacodynamic properties of oligonucleotides, a group that enhances the pharmacokinetic properties of oligonucleotides, or a group of formula (—O—X 3 ) p , where p is 1 to about 10 and X 3 is alkyl having from one to about 10 carbons;
R 5 is —N(R 6 ) 2 , or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R 6 is straight or branched chain alkyl having from 1 to 10 carbons;
comprising the steps of:
providing a compound Formula:
and reacting the compound with a compound of Formula:
wherein:
R 5 is —N(R) 6 , 2 or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms and up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
R 6 is straight or branched chain alkyl having from 1 to 10 carbons;
thereby producing the phosphordiamidite.
42 . The method of claim 41 wherein q is 2 and Y 3 is C(═O).
43 . The method of claim 42 wherein n is 0.
44 . The method of claim 42 wherein each R 6 is alkyl.
45 . The method of claim 42 wherein each R 6 is isopropyl.
46 . The method of claim 42 wherein Y 1 and Y 2 are each O.
47 . The method of claim 42 wherein Y 1 and Y 2 are each S.
48 . The method of claim 42 wherein Y 1 is O and Y 2 is S.
49 . The method of claim 42 wherein Z is methyl, phenyl or t-butyl.
50 . The method of claim 49 wherein Z is t-butyl.
51 . The method of claim 42 wherein Z is methyl, phenyl or t-butyl, n is 0, Y 1 is O and Y 2 is S.
52 . The method of claim 42 wherein M is a optionally protected phosphodiester, phosphorothioate, phosphorodithioate, or alkyl phosphonate internucleotide linkage.
53 . The method of claim 41 wherein B is a radiolabeled nucleobase.
54 . The method of claim 42 wherein B is a radiolabeled nucleobase.
55 . The method of claim 41 wherein the radiolabeled nucleobase has the Formula:
wherein * denotes a 14 C atom.
56 . The method of claim 41 wherein the compound of Formula:
is formed by the reaction of a compound of Formula:
with a compound of Formula:
in the presence of an acid.Cited by (0)
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