EP0219342A2
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Method and reagents for in vitro oligonucleotide synthesis
Assignee: Genentech Inc
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Filing: 1986-10-14
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Granted: —
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Expires: 2006-10-14
Abstract
Abstract A method is provided for the high fidelity. rapid and economical in vitro synthesis of oligonucleotides. Nucleoside H-phosphonates are condensed in seriatim using a dehydrating agent to produce a poly (nucleoside H-phosphonate). The product is oxidized to yield the desired oligonucleotide. A novel reagent is provided for multiple nucleoside additions in single cycles.
Inventors
- 👤 Brian Carl Froehler
- 👤 Mark Douglas Matteucci
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 15:54
CPC Classifications
C; C07; C07H; C07H21/00
Claims Analysis
Total Claims: 30
Claim 1
Independent Claim
94 words
A method for the synthesis of a polynucleotide comprising (a) providing a nucleoside bound to a carrier (b) condensing a blocked nucleoside H-phosphonate with the 5' hydroxyl of the carrier-bound nucleoside in the presence of a dehydrating agent, (c) removing the 5' blocking group of the nucleoside H-phosphonate of step b) from the carrier, (d) sequentially repeating steps b) and c) using nucleoside H-phosphonates until a polynucleotide H-phosphonate having greater than two nucleotides has been obtained, (e) oxidizing the polynucleotide H-phosphonate to form the corresponding polynucleotide, and (f) separating the polynucleotide from the carrier.
Claim 2
Independent Claim
94 words
A method for the synthesis of a polynucleotide comprising (a) providing a nucleoside bound to a carrier (b) condensing a blocked nucleoside H-phosphonate with the 5' hydroxyl of the carrier-bound nucleoside in the presence of a dehydrating agent, (c) removing the 5' blocking group of the nucleoside H-phosphonate of step b) from the carrier, (d) sequentially repeating steps b) and c) using nucleoside H-phosphonates until a polynucleotide H-phosphonate having greater than two nucleotides has been obtained, (e) oxidizing the polynucleotide H-phosphonate to form the corresponding polynucleotide, and (f) separating the polynucleotide from the carrier.
Claim 3
Dependent Claim
15 words
The method of claim 1 wherein the dehydrating agent is an acylating or phosphorylating agent.
Claim 4
Dependent Claim
25 words
The method of claim 2 wherein the dehydrating agent is other than an aryl sulfonyl chloride or aryl sulfonyl tetrazolide, diphenyl chlorophosphate, or a carbodiimide.
Claim 5
Dependent Claim
13 words
A method of claim 2 wherein the dehydrating agent is an acylating agent.
Claim 6
Dependent Claim
58 words
The method of claim 4 wherein the acylating agent has the structure wherein A is aryl, heteroaryl or in which R is the same or different normal or branched alkyl, heteroalkyl, aryl, heteroaryl or halogen and X is halogen or a pseudohalogen selected from the group of azide, heterocyclic secondary amines or groups capable of forming active esters.
Claim 7
Dependent Claim
9 words
The method of claim 5 wherein X is chloride.
Claim 8
Dependent Claim
12 words
The method of claim 5 wherein the acylating agent is pivaloyl chloride.
Claim 9
Independent Claim
34 words
The method according to any one of the preceding claims wherein the dehydrating agent results in premature chain termination in less than about 20 mole % of the total oligomer population after four cycles.
Claim 10
Independent Claim
15 words
The method of any preceding claim wherein the dehydrating agent is an organic acid halide.
Claim 11
Independent Claim
13 words
The method according to any preceding claim wherein the nucleoside is a deoxyribonucleoside.
Claim 12
Independent Claim
19 words
The method according to any preceding claim wherein steps b) and c) are repeated through at least 2 cycles.
Claim 13
Dependent Claim
20 words
The method according to claim 11 wherein steps b) and c) are repeated through about from 4 to 125 cycles.
Claim 14
Independent Claim
15 words
The method according to any preceding claim wherein the dehydrating agent is an acid chloride.
Claim 15
Independent Claim
19 words
The method according to any preceding claim wherein the dehydrating agent is capable of capping aborted oligonucleotide phosphonate chains.
Claim 16
Independent Claim
21 words
The method according to any preceding claim wherein the step e) occurs only after the entire polynucleotide H-phosphonate has been synthesized.
Claim 17
Independent Claim
42 words
The method according to any preceding claim which is such that if 49 cycles of step d) are followed by steps e) and f), a 50 nucleotide polynucleotide is present in a greater amount than any other polynucleotide separated from the carrier.
Claim 18
Independent Claim
13 words
The method according to any preceding claim wherein the nucleoside H-phosphonates are predetermined.
Claim 19
Dependent Claim
43 words
The method of claim 17 which is such that if 4 cycles of step d) are followed by steps e) and f), greater than about 80 mole percent of the polynucleotides separated from the carrier consists of a polynucleotide containing 5 predetermined nucleotides.
Claim 20
Dependent Claim
18 words
A composition having the formula wherein Y is a counterion, n is =1, and M is a nucleoside.
Claim 21
Dependent Claim
18 words
A composition having the formula wherein Y is a counterion, n is =1, and M is a nucleoside.
Claim 22
Dependent Claim
11 words
The composition of claim 19 wherein M is a protected nucleoside.
Claim 23
Dependent Claim
13 words
The composition of claim 19 wherein the protecting group is a dialkyl formamidine.
Claim 24
Independent Claim
87 words
A method for the preparation of internucleotide phosphate analogues of polynucleotides, comprising a) providing a carrier-bound polynucleoside H-phosphonate containing greater than two nucleotides, b) reacting the polynucleoside H-phosphonate with an agent selected from i) s 8 , ii) ROH wherein R is alkyl, heteroalkyl, heteroaryl or aryl, or iii) H- N- X wherein X and Y are the same or different and are Y hydrogen, aryl, heteroaryl, heteroalkyl, alkyl, or are taken together to form a cycloalkene, cycloalkane or heterocycle, and c) recovering the internucleotide phosphate analogue.
Claim 25
Independent Claim
87 words
A method for the preparation of internucleotide phosphate analogues of polynucleotides, comprising a) providing a carrier-bound polynucleoside H-phosphonate containing greater than two nucleotides, b) reacting the polynucleoside H-phosphonate with an agent selected from i) s 8 , ii) ROH wherein R is alkyl, heteroalkyl, heteroaryl or aryl, or iii) H- N- X wherein X and Y are the same or different and are Y hydrogen, aryl, heteroaryl, heteroalkyl, alkyl, or are taken together to form a cycloalkene, cycloalkane or heterocycle, and c) recovering the internucleotide phosphate analogue.
Claim 26
Dependent Claim
15 words
The method of claim 22 wherein ROH or H- N - X are fluorescent. Y
Claim 27
Dependent Claim
83 words
A composition comprising a polynucleotide with at least one substituted internucleotide phosphate, said polynucleotide having the formula wherein 1) -Z is -OH, - SH, -O-R, or - N-X and a is an integer greater than about 10; Y 2) at least 1 Z is not -OH; 3) R is aryl, heteroaryl, alkyl or heteroalkyl; 4) X or Y are hydrogen, heteroalkyl, alkyl, aryl or heteroaryl, or are taken together to form a heterocycle, heterocyclic alkane or alkene; and 5) M is a nucleoside.
Claim 28
Dependent Claim
83 words
A composition comprising a polynucleotide with at least one substituted internucleotide phosphate, said polynucleotide having the formula wherein 1) -Z is -OH, - SH, -O-R, or - N-X and a is an integer greater than about 10; Y 2) at least 1 Z is not -OH; 3) R is aryl, heteroaryl, alkyl or heteroalkyl; 4) X or Y are hydrogen, heteroalkyl, alkyl, aryl or heteroaryl, or are taken together to form a heterocycle, heterocyclic alkane or alkene; and 5) M is a nucleoside.
Claim 29
Dependent Claim
18 words
The composition of claim 24 wherein X and Y are taken together to form an oxygen-containing heteroaryl substitutent.
Claim 30
Dependent Claim
18 words
The composition of claim 24 or 25 wherein - OR or - N - X are fluorescent. Y
References Cited (Backward Citations)
DE2644432A1
Cited
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