3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds
First Claim
1. A mononucleoside 5'"'"'-triphosphate having a removable blocking moiety protecting the 3'"'"' position which is an ether and which has the following formula:
- ##STR17## wherein R2 is a triphosphate group and R1 is selected from the group consisting of methyl, CH3 (CH2)N, wherein N is an integer from 1-10, methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butyoxymethyl, 2-methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, 3'"'"'-bromotetrahydropyranyl, tetrahydrothiopyranyl, 4'"'"'-methoxytetrahydropyranyl, 4'"'"'-methoxytetrahydrothiopyranyl, 4'"'"'-methoxytetrahydrothiopyranyl S,S-dioxido, tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl, tri(p-methoxyphenyl)methyl, di(p-methoxy)phenylmethyl, 2,2,2-trichloroethyl, 2-(phenylselenyl)ethyl, butyl, allyl, cinnamyl, p-chlorophenyl, benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, p-cyanobenzyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, α
-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, p-(p'"'"'-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl, 9-(9-phenyl-10-oxo)anthryl, benzisothiazolyl S,S-dioxido, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyl dimethylsilyl, (triphenylmethyl)dimethylsilyl, methyldiiosopropylsilyl, methyl di-t-butylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triisopropylsilyl and triphenylsilyl;
B is a nucleotide base.
4 Assignments
0 Petitions
Reexamination
Accused Products
Abstract
A method for the stepwise creation of phosphodiester bonds between desired nucleosides resulting in the synthesis of polynucleotides having a predetermined nucleotide sequence by preparing an initiation substrate containing a free and unmodified 3'"'"'-hydroxyl group; attaching a mononucleotide selected according to the order of the predetermined nucleotide sequence to the 3'"'"'-hydroxyl of the initiating substrate in a solution containing a catalytic amount of an enzyme capable of catalyzing the 5'"'"' to 3'"'"' phosphodiester linkage of the 5'"'"'-phosphate of the mononucleotide to the 3'"'"'-hydroxyl of the initiating substrate, wherein the mononucleotide contains a protected 3'"'"'-hydroxyl group, whereby the protected mononucleotide is covalently linked to the initiating substrate and further additions are hindered by the 3'"'"'-hydroxyl protecting group. Methods in which a mononucleotide immobilized on a solid support is added to a free polynucleotide chain are also disclosed.
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Citations
6 Claims
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1. A mononucleoside 5'"'"'-triphosphate having a removable blocking moiety protecting the 3'"'"' position which is an ether and which has the following formula:
- ##STR17## wherein R2 is a triphosphate group and R1 is selected from the group consisting of methyl, CH3 (CH2)N, wherein N is an integer from 1-10, methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butyoxymethyl, 2-methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, 3'"'"'-bromotetrahydropyranyl, tetrahydrothiopyranyl, 4'"'"'-methoxytetrahydropyranyl, 4'"'"'-methoxytetrahydrothiopyranyl, 4'"'"'-methoxytetrahydrothiopyranyl S,S-dioxido, tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl, tri(p-methoxyphenyl)methyl, di(p-methoxy)phenylmethyl, 2,2,2-trichloroethyl, 2-(phenylselenyl)ethyl, butyl, allyl, cinnamyl, p-chlorophenyl, benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, p-cyanobenzyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, α
-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, p-(p'"'"'-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl, 9-(9-phenyl-10-oxo)anthryl, benzisothiazolyl S,S-dioxido, trimethylsilyl, triethylsilyl, isopropyldimethylsilyl, t-butyl dimethylsilyl, (triphenylmethyl)dimethylsilyl, methyldiiosopropylsilyl, methyl di-t-butylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triisopropylsilyl and triphenylsilyl;
B is a nucleotide base. - View Dependent Claims (2, 3, 4, 5)
- ##STR17## wherein R2 is a triphosphate group and R1 is selected from the group consisting of methyl, CH3 (CH2)N, wherein N is an integer from 1-10, methoxymethyl, methylthiomethyl, benzyloxymethyl, t-butyoxymethyl, 2-methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl, tetrahydropyranyl, 3'"'"'-bromotetrahydropyranyl, tetrahydrothiopyranyl, 4'"'"'-methoxytetrahydropyranyl, 4'"'"'-methoxytetrahydrothiopyranyl, 4'"'"'-methoxytetrahydrothiopyranyl S,S-dioxido, tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl, 1-(isopropoxy)ethyl, tri(p-methoxyphenyl)methyl, di(p-methoxy)phenylmethyl, 2,2,2-trichloroethyl, 2-(phenylselenyl)ethyl, butyl, allyl, cinnamyl, p-chlorophenyl, benzyl, p-methoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, p-cyanobenzyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, α
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6. A mononucleoside 5'"'"'-triphosphate having a removable blocking moiety protecting the 3'"'"' position which is an ether and which has the following formula:
- ##STR18## wherein R2 is a triphosphate group;
R1 is a silyl group; and
B is a nucleotide base.
- ##STR18## wherein R2 is a triphosphate group;
Specification