3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds

• US 5,872,244 A
• Filed: 06/07/1995
• Issued: 02/16/1999
• Est. Priority Date: 09/02/1994
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Andrew C. Hiatt, Floyd D. Rose
• Inventors: Hiatt, Andrew C., Rose, Floyd
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/486,535
• Time in Patent Office: 1,350 Days
• Field of Search: 435/6, 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34, 536/26.26, 536/26.6, 536/26.7
• US Class Current: 536/26.26
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12P 19/30 Nucleotides
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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.

Claims

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.

2. The mononucleoside 5'"'"'-triphosphate of claim 1, wherein R₁ is selected from the group consisting of bis(2-chloroethoxy)methyl, 4-methoxytetrahydropyranyl, tetrahydrofuranyl, 1-ethoxyethyl, tri(p-methoxyphenyl)methyl, di(p-methoxy)phenylmethyl and t-butyldimethylsilyl.

3. The mononucleoside 5'"'"'-triphosphate of claim 1, wherein R₁ is selected from the group consisting of methyl, CH₃ (CH₂)_N, wherein N is an integer from 1-10, methoxymethyl, 2-methoxyethoxymethyl, trimethylsilyl and triethylsilyl.

4. The mononucleoside 5'"'"'-triphosphate of claim 1, wherein R₁ is a 1-ethoxyethyl group and said mononucleoside 5'"'"'-triphosphate is a deoxynucleoside.

5. The mononucleoside 5'"'"'-triphosphate of claim 1, wherein R₁ is a 1-ethoxyethyl group.

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.