Oligonucleotide synthesis method using highly dispersible liquid-phase support

• US 9,284,344 B2
• Filed: 05/30/2012
• Issued: 03/15/2016
• Est. Priority Date: 05/30/2012
• Status: Active Grant

• Current Assignee: Mitsubishi Gas Chemical Company Incorporated (Mitsubishi Electric Corporation)
• Original Assignee: Hokkaido System Science Co., Ltd.
• Inventors: Kim, Shokaku, Matsumoto, Masanori
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US14/404,562
• Publication Number:
  US 20150112053A1
• Time in Patent Office: 1,385 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 1/02 Phosphorylation
• CPC Class Codes: C07H 1/04 Introducing polyphosphoric acid radicals
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• 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: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A nucleic acid synthesis method enabling a reaction in a fluid (flow) with a highly dispersible liquid-phase support to improve coupling efficiency is provided.

The method for synthesizing an oligonucleotide comprising: sequentially condensing and oxidizing a nucleoside phosphoramidite compound in the presence of an acid/azole complex compound using a starting raw material, i.e., hydrophobic group-bonded nucleoside represented by Formula (1):

where R¹: an alkylene group having 1 to 12 carbon atoms, R²: an alkylene group having 1 to 22 carbon atoms, R³ and R⁴ each independently represent an alkyl group having 1 to 22 carbon atoms or the like, R⁵: a single bond or an alkylene group having 1 to 22 carbon atoms, R⁶: each independently an alkyl group having 6 to 30 carbon atoms, n represents an integer of 2 to 6, X represents a hydrogen atom, hydroxyl group, or the like, Y: a protecting group deprotectable under an acidic condition, and Z: an adenyl group, a guanyl group, or the like having a polar group optionally protected by a protecting group,

• • wherein a condensation reaction is performed by preliminarily dissolving the hydrophobic group-bonded nucleoside or hydrophobic group-bonded oligonucleotide and the nucleoside phosphoramidite compound in a non-polar solvent, and contacting the resulting solution with the acid/azole complex compound or a solution containing the complex compound.

Claims

1. A method for synthesizing an oligonucleotide comprising:

sequentially condensing and oxidizing a nucleoside phosphoramidite compound in the presence of an acid/azole complex compound using a starting raw material comprising a hydrophobic group-bonded nucleoside represented by Formula (1);

2. The method for synthesizing an oligonucleotide according to claim 1, wherein a dissolution solution of the hydrophobic group-bonded nucleoside or hydrophobic group-bonded oligonucleotide and the nucleoside phosphoramidite compound in a non-polar solvent and a dissolution solution of the acid/azole complex compound in a non-polar solvent are separately prepared, and are mixed.

3. The method for synthesizing an oligonucleotide according to claim 1, wherein a dissolution solution of the hydrophobic group-bonded nucleoside or hydrophobic group-bonded oligonucleotide and the nucleoside phosphoramidite compound in a non-polar solvent and a dissolution solution of the acid/azole complex compound in a non-polar solvent are separately fed into a flow path to continuously conduct the condensation reaction in the flow path.

4. A method for synthesizing an oligonucleotide, wherein the starting raw material hydrophobic group-bonded nucleoside is a compound represented by Formula (3):

5. The method for synthesizing an oligonucleotide according to claim 4, wherein in Formula (3), R^6′

is an octadecyl group.