IPC분류정보
국가/구분 |
United States(US) Patent
등록
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국제특허분류(IPC7판) |
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출원번호 |
US-0811233
(1997-03-03)
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발명자
/ 주소 |
- Cheruvallath Zacharia S.
- Ravikumar Vasulinga T.
- Cole Douglas L.
- Capaldi Daniel C.
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출원인 / 주소 |
- ISIS Pharmaceuticals, Inc.
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대리인 / 주소 |
Oblon, Spivak, McClelland, Maier & Neustadt, P.C.
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인용정보 |
피인용 횟수 :
7 인용 특허 :
41 |
초록
▼
The present invention is directed to a method of synthesizing sulfurized oligonucleotide analogs by reacting an oligonucleotide analog containing a phosphorous(III) linkage with a dithiocarbonic acid diester polysulfide having the formula [ STR 1 ] to produce a sulfurized oligonucleotide analog. The
The present invention is directed to a method of synthesizing sulfurized oligonucleotide analogs by reacting an oligonucleotide analog containing a phosphorous(III) linkage with a dithiocarbonic acid diester polysulfide having the formula [ STR 1 ] to produce a sulfurized oligonucleotide analog. The diester polysulfide reagent is useful in solution and solid phase oligonucleotide analog synthesis.
대표청구항
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[ What is claimed as new and desired to be secured by Letters Patent of the United States is:] [1.] A method, comprising:reacting an oligonucleotide analog comprising at least one phosphorus(III) linkage with a thiodicarbonic acid diester polysulfide having the formula: [ STR 14 ] to produce a sulfu
[ What is claimed as new and desired to be secured by Letters Patent of the United States is:] [1.] A method, comprising:reacting an oligonucleotide analog comprising at least one phosphorus(III) linkage with a thiodicarbonic acid diester polysulfide having the formula: [ STR 14 ] to produce a sulfurized oligonucleotide analog, wherein each R is independently selected from the group consisting of C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms; andn is 2, 3 or 4. [10.] A method, comprising:(a) providing a nucleoside analog having a blocked hydroxyl group;(b) deblocking said blocked hydroxyl group to produce a free hydroxyl group;(c) reacting said free hydroxyl group with a protected nucleoside analog phosphoramidite having a blocked hydroxyl group to produce an oligonucleotide analog comprising a phosphorous(III) linkage and a blocked hydroxyl group;(d) reacting said phosphorous(III) linkage with a reagent selected from the group consisting of an oxidizing agent and a dithiocarbonic acid diester polysulfide having the formula: [ STR 17 ] to produce an oxidized or sulfurized phosphorous(V) linkage, where at least one linkage is a sulfurized phosphorous(V) linkage; and(e) repeating steps (b) through (d) at least once to produce a sulfurized oligonucleotide analog, reineach R is independently selected from the group consisting of C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, C.sub.6 -.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms; andn is 2, 3, or 4. The method of claim 10, whereineach R is independently selected from the group consisting of C.sub.2 -C.sub.8 alkyl, substituted C.sub.2 -C.sub.8 alkyl, C.sub.6 -C.sub.14 aryl and C.sub.6 -C.sub.14 substituted aryl; andn is 2. The method of claim 10, wherein said nucleoside analog is attached to a solid support. The method of claim 10, wherein said oligonucleotide analog comprising a phosphorous(III) linkage and a blocked hydroxyl group has the formula: [ STR 18 ] wherein each Pg is independently a group labile to a base and/or a nucleophile or an allyl group;R.sup.6 is a labile blocking group;each B is independently an unprotected or a protected heterocyclic base;each X is selected from the group consisting of hydrogen, hydroxyl, F, Cl, Br, C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms, substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms, O--C.sub.1 -C.sub.8 alkyl, substituted O--C.sub.1 -C.sub.8 alkyl, O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted O--C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, O--C.sub.6 -C.sub.14 aryl, substituted O--C.sub.6 -C.sub.14 aryl, O--C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted O--C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, O--C.sub.7 -C.sub.18 aralkyl, substituted O--C.sub.7 -C.sub.18 aralkyl, O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms, substituted O--C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms, O--C.sub.1 -C.sub.8 -alkyl-O--C.sub.1 -C.sub.8 -alkyl, O--C.sub.1 -C.sub.8 alkenyl, O--C.sub.1 -C.sub.8 alkoxyamino, O-tri-C.sub.1 -C.sub.8 -alkyl silyl, substituted O-tri-C.sub.1 -C.sub.8 -alkyl silyl, NH--C.sub.1 -C.sub.8 alkyl, N--(C.sub.1 -C.sub.8).sub.2, NH--C.sub.1 -C.sub.8 alkenyl, N--(C.sub.1 -C.sub.8).sub.2 alkenyl, S--C.sub.1 -C.sub.8 alkyl, S--C.sub.1 -C.sub.8 alkenyl, NH.sub.2, N.sub.3, NH--C.sub.1 -C.sub.8 -alkyl-NH.sub.2, polyalkylamino and an RNA cleaving group;each Y is independently O or S;each Z is independently O or S;L is a group labile to a nucleophile and/or a base;S.sup.p is a solid support; andm is 0 or a positive integer. The method of claim 13, whereineach step (d) is reacting said phosphorous(III) linkage with said dithiocarbonic acid diester polysulfide;each R is independently selected from the group consisting of C.sub.2 -C.sub.8 alkyl, substituted C.sub.2 -C.sub.8 alkyl, C.sub.6 -C.sub.14 aryl and substituted C.sub.6 -C.sub.14 aryl;n is 2; andm is an integer between 0 and 198. The method of claim 14, whereineach Pg is independently selected from the group consisting of .beta.-cyanoethyl, 4-cyano-2-butenyl, 2-diphenylmethylsilyl and a 2-N-amidoethyl group having the formula R.sup.1 CONR.sup.2 CHR.sup.3 CHR.sup.4 --;each R.sup.1 is independently selected from the group consisting of C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms;each R.sup.2, R.sup.3 and R.sup.4 is independently selected from the group consisting of hydrogen, C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms, orR.sup.3 and R.sup.4 together with the carbon atoms they are bonded to form a C.sub.3 -C.sub.8 cycloalkyl group, a substituted C.sub.3 -C.sub.8 cycloalkyl group, a C.sub.2 -C.sub.8 heterocycloalkyl group containing up to three heteroatoms or a substituted C.sub.2 -C.sub.8 heterocycloalkyl group containing up to three heteroatoms;R.sup.6 is selected from the group consisting of 4,4'-dimethoxytrityl, monomethoxytrityl, diphenylmethyl, phenylxanthen-9-yl and 9-(p-methoxyphenyl)xanthen-9-yl; andeach B is independently selected from the group consisting of adenine, guanine, cytosine, thymine, uracil, 2-aminopurine, inosine and 5-methylcytosine, where the exocyclic amino group of each base is protected with an acyl group. The method of claim 12, further comprising:(f) removing said sulfurized oligonucleotide analog from said solid support. The method of claim 13, further comprising:(f) removing said sulfurized oligonucleotide analog from said solid support, wherein said sulfurized oligonucleotide analog removed from said solid support has the formula: [ STR 19 ] The method of claim 14, further comprising: (f) removing said sulfurized oligonucleotide analog from said solid support, wherein said sulfurized oligonucleotide analog removed from said solid support has the formula: [ STR 20 ] The method of claim 1, wherein the reacting step is conducted at a temperature of 0 to 40.degree. C. [23.] A composition, comprising: (a) an oligonucleotide analog comprising at least one phosphorus(III) linkage; and(b) an effective amount of a thiodicarbonic acid diester polysulfide having the formula: [ STR 21 ] for sulfurizing said at least one phosphorus(III) linkage of the oligonucleotide analog, whereineach R is independently selected from the group consisting of C.sub.1 -C.sub.8 alkyl, substituted C.sub.1 -C.sub.8 alkyl, C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, substituted C.sub.2 -C.sub.8 heterocycloalkyl containing up to three heteroatoms, C.sub.6 -C.sub.14 aryl, substituted C.sub.6 -C.sub.14 aryl, C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, substituted C.sub.3 -C.sub.11 hetaryl containing up to three heteroatoms, C.sub.7 -C.sub.18 aralkyl, substituted C.sub.7 -C.sub.18 aralkyl, C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms and substituted C.sub.4 -C.sub.15 heterocycloaralkyl containing up to three heteroatoms; andn is 2, 3 or 4.
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