US5623068A
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Synthesis of DNA using substituted phenylacetyl-protected nucleotides
Assignee: Beckman Coulter Inc
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Filing: 1995-03-01
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Granted: 1997-04-22
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Expires: 2015-03-01
Abstract
Abstract Deoxyribonucleotide and ribonucleotide derivatives of the general formula I ##STR1## wherein R 1 represents --CR'R"--Ar, in which Ar is substituted aryl (as hereinafter defined) and R' and R" are independently selected from the group consisting of hydrogen and lower alkyl; one of --R 2 and R 3 is a hydroxyl-protecting group and the other is a group suitable for synthesis of polynucleotides or for attachment of the nucleotide to a solid support; R 4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base. When synthesis is carried out using these derivatives, the deprotection procedure is reduced to an essentially instantaneous process. The derivatives have acceptable shelf life and are very stable to conventional DNA synthesis conditions. Particularly preferred are those compounds wherein Ar is mono- and dihalo-substituted phenyl.
Inventors
- 👤 M. Parameswara Reddy
- 👤 Firdous Farooqui
- 👤 Naeem B. Hanna
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 22:23
CPC Classifications
C; C07; C07H; C07H19/00; C07H19/02; C07H19/04; C07H19/06; C07H19/16; C07H21/00; Y; Y02; Y02P; Y02P20/00; Y02P20/50; Y02P20/55
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
120 words
A compound of general formula I ##STR11## wherein R 1 represents --CR'R"--Ar, in which Ar is selected from the group consisting of mono- and dihalo- substituted phenyl, and methoxy phenyl, and R' and R" are independently selected from the group consisting of hydrogen and lower alkyl; one of R 2 and R 3 is a hydroxyl-protecting group and the other is selected from the group consisting of phosphoramidites, phosphonates, and groups suitable for attachment of the nucleoside to a solid support; R 4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base and wherein --CO--R 1 is attached to an exocyclic N of B.
Claim 2
Dependent Claim
120 words
A compound of general formula I ##STR11## wherein R 1 represents --CR'R"--Ar, in which Ar is selected from the group consisting of mono- and dihalo- substituted phenyl, and methoxy phenyl, and R' and R" are independently selected from the group consisting of hydrogen and lower alkyl; one of R 2 and R 3 is a hydroxyl-protecting group and the other is selected from the group consisting of phosphoramidites, phosphonates, and groups suitable for attachment of the nucleoside to a solid support; R 4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base and wherein --CO--R 1 is attached to an exocyclic N of B.
Claim 3
Dependent Claim
27 words
A compound according to claim 1, wherein B is selected from the group consisting of ##STR12## and wherein --CO--R 1 is attached to the exocyclic base N.
Claim 4
Dependent Claim
40 words
A compound according to claim 1, wherein the hydroxyl-protecting group is selected from the group consisting of 4,4'-dimethoxytrityl, 4,4',4"-tris-(benzyloxy)trityl, 4,4',4"-tris-(4,5-dichlorophthalimido)trityl, 4,4',4"-tris(levulinyloxy)trityl, 3-(imidazolylmethyl)-4,4'-dimethoxytrityl, pixyl (9-phenylxanthen-9-yl), 9-(p-methoxyphenyl)xanthen-9-yl, 4-decyloxytrityl, 4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xanthene-9-yl, 1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl, p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,4-dinitrophenylethoxycarbonyl, 4-(methylthiomethoxy)butyryl, 2-(methylthiomethoxymethyl)-benzoyl, 2-(isopropylthiomethoxymethyl)benzoyl, 2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl and levulinyl groups.
Claim 5
Dependent Claim
27 words
A compound according to claim 1, wherein the radical suitable for attachment to a solid support is selected from the group consisting of succinate and oxalyl groups.
Claim 6
Dependent Claim
34 words
A compound according to claim 1, wherein one of R 2 and R 3 is selected from the group consisting of 4,4'-dimethoxytrityl, pixyl and levulinyl and the other is a phosphoramidite or phosphonate group.
Claim 7
Dependent Claim
14 words
A compound according to claim 5, wherein the phosphoramidite group is a (N,N-diisopropyl)-β-cyanoethyl-phosphoramidite group.
Claim 8
Dependent Claim
20 words
A compound according to claim 1, wherein Ar is selected from the group consisting of mono- and dihalo- substituted phenyl.
Claim 9
Dependent Claim
13 words
A compound according to claim 1, wherein R' and R" are both hydrogen.
Claim 10
Dependent Claim
143 words
In a method of synthesizing oligonucleotides by a condensation method, the improvement which comprises: using as a starting material at least one nucleotide derivative of general formula I ##STR13## where in R 1 represents CR'R"--Ar, in which Ar is selected from the group consisting of mono- and dihalo- substituted phenyl, and methoxy phenyl and R' and R" are independently selected from the group consisting of hydrogen and lower alkyl; one of R 2 and R 3 is a hydroxyl-protecting group and the other is selected from the group consisting of phosphoramidites, phosphonates, and groups suitable for attachment of the nucleotide to a solid support; R 4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base and wherein --CO--R 1 is attached to an exocyclic N of B.
Claim 11
Dependent Claim
143 words
In a method of synthesizing oligonucleotides by a condensation method, the improvement which comprises: using as a starting material at least one nucleotide derivative of general formula I ##STR13## where in R 1 represents CR'R"--Ar, in which Ar is selected from the group consisting of mono- and dihalo- substituted phenyl, and methoxy phenyl and R' and R" are independently selected from the group consisting of hydrogen and lower alkyl; one of R 2 and R 3 is a hydroxyl-protecting group and the other is selected from the group consisting of phosphoramidites, phosphonates, and groups suitable for attachment of the nucleotide to a solid support; R 4 is selected from the group consisting of hydrogen, --OH and protected hydroxyl; and B represents a divalent radical corresponding to a purine or pyrimidine base and wherein --CO--R 1 is attached to an exocyclic N of B.
Claim 12
Dependent Claim
47 words
A method according to claim 9, further comprising deprotecting the oligonucleotide using a reagent selected from the group consisting of agents which are at least 5 times more nucleophilic than ammonia, agents which are at least 1.5 times less polar than water, aqueous ammonia and mixtures thereof.
Claim 13
Dependent Claim
27 words
The method according to claim 9, wherein B is selected from the group consisting of ##STR14## and wherein --CO--R 1 is attached to the exocyclic base N.
Claim 14
Dependent Claim
39 words
A method according to claim 9, wherein the hydroxyl-protecting group is selected from the group consisting of 4,4'-dimethoxytrityl, 4,4',4"-tris-(benzyloxy)trityl, 4,4',4"-tris-(4,5-dichlorophthalimido)trityl, 4,4',4"-tris(levulinyloxy)trityl, 3-(imidazolylmethyl)-4,4'-dimethoxytrityl, pixyl (9-phenylxanthen-9-yl), 9-(p-methoxyphenyl)xanthen-9-yl, 4-decyloxytrityl, 4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xanthene-9-yl, 1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl, p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,4-dinitrophenylethoxycarbonyl, 4-(methylthiomethoxy)butyryl, 2-(methylthiomethoxymethyl)-benzoyl, 2-(isopropylthiomethoxymethyl)benzoyl, 2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl and levulinyl.
Claim 15
Dependent Claim
27 words
A method according to claim 9, wherein the radical suitable for attachment to a solid support is selected from the group consisting of succinate and oxalyl groups.
Claim 16
Dependent Claim
34 words
A method according to claim 9, wherein one of R 2 and R 3 is selected from the group consisting of 4,4'-dimethoxytrityl, pixyl and levulinyl and the other is a phosphoramidite or phosphonate group.
Claim 17
Dependent Claim
14 words
A method according to claim 14, wherein the phosphoramidite group is a (N,N-diisopropyl)-β-cyanoethyl-phosphoramidite group.
Claim 18
Dependent Claim
20 words
A method according to claim 9, wherein Ar is selected from the group consisting of mono- and dihalo- substituted phenyl.
Claim 19
Dependent Claim
13 words
A method according to claim 9, wherein R' and R" are both hydrogen.
Claim 20
Dependent Claim
81 words
A compound of general formula I ##STR15## wherein R 1 represents --CH 2 --Ar, in which Ar is selected from the group consisting of mono- and dihalo- substituted phenyl, and methoxyphenyl; one of R 2 and R 3 is a hydroxyl-protecting group and the other is selected from the group consisting of phosphoramidites, phosphonates, and groups suitable for attachment of the nucleoside to a solid support; and R 4 is selected from the group consisting of hydrogen, --OH, and protected hydroxyl.
Claim 21
Dependent Claim
81 words
A compound of general formula I ##STR15## wherein R 1 represents --CH 2 --Ar, in which Ar is selected from the group consisting of mono- and dihalo- substituted phenyl, and methoxyphenyl; one of R 2 and R 3 is a hydroxyl-protecting group and the other is selected from the group consisting of phosphoramidites, phosphonates, and groups suitable for attachment of the nucleoside to a solid support; and R 4 is selected from the group consisting of hydrogen, --OH, and protected hydroxyl.
Claim 22
Dependent Claim
27 words
The compound of claim 18 wherein groups suitable for attachment of the nucleoside to a solid support are selected from the group consisting of succinate and oxalyl.
Claim 23
Dependent Claim
19 words
The compound of claim 18 wherein protected hydroxyl is selected from the group consisting of 4,4'-dimethoxytrityl, pixyl and levulinyl.
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