Ajinomoto Co., Ltd.

Kunihiro Hirai

Daisuke Takahashi

Satoshi Katayama

Taisuke Ichimaru

Naoko Hirose

Takayoshi Torii

Ryotaro Nakaya

Ken Yamashita

A method for producing an oligonucleotide, including a step of removing a temporary protecting group of a 5'-hydroxyl group by adding an acid to a protected oligonucleotide having a phosphorothioate (PS), phosphate (PO) or H-phosphonate moiety, and deacylating a by-product having an acylated 5′-hydroxyl group by solvolysis can complete a condensation reaction to improve a condensation yield, and can markedly reduce impurities such as single base deletion form (N-1mer) and the like.

Production method for oligonucleotides

Oligonucleotides may be produced by a process, including (1) condensing a nucleoside, nucleotide or oligonucleotide (b), and a nucleoside, nucleotide or oligonucleotide (a), or a substituted nucleotide or oligonucleotide (&#x3b1;) in a non-polar solvent to give a reaction solution containing a phosphite triester product (c); (3) oxidizing or sulfurizing the phosphite triester product (c) to give a reaction solution containing an oligonucleotide (d) wherein the 5'-hydroxy group is protected; (4) deprotecting the oligonucleotide (d) to give a reaction solution containing an oligonucleotide (e) wherein the 5&#x2032;-hydroxy group is not protected; and (6) adding a polar solvent to the reaction solution containing the oligonucleotide (e) and purifying the oligonucleotide (e) by solid-liquid separation, wherein said nucleoside, nucleotide or oligonucleotide (a) or said substituted nucleotide or oligonucleotide (&#x3b1;) is a compound represented by formula (a-i): wherein Base, R, R, m, L, Y, and Z are defined herein.

Oligonucleotide production method, and nucleoside, nucleotide, or oligonucleotide

Precipitation promoters, which are an organic compound having one or more linear aliphatic hydrocarbon groups having not less than 10 carbon atoms, wherein the aliphatic hydrocarbon group has not less than 20 carbon atoms in total are useful for precipitating an organic compound protected by an organic group having one or more aliphatic hydrocarbon groups having not less than 10 carbon atoms from a solvent.

Precipitation promoter and precipitation method in which same is used

The present invention provides a protected nucleotide for elongation, which can be purified efficiently and in a high yield by a liquid-liquid extraction operation, and can achieve an oligonucleotide production method by a phosphoramidite method. It has been found that the above-mentioned problem can be solved by a particular oligonucleotide comprising a protected base and/or particular oligonucleotide protected by a branched chain-containing aromatic group at 3'-position.

Oligonucleotide with protected base

A method of producing an n+p-mer oligonucleotide efficiently in a high yield, which includes use of, as a starting material, an n-mer oligonucleotide wherein the 3'-terminal hydroxyl group is protected, and the 5&#x2032;-terminal hydroxyl group is protected by a temporary protecting group, and continuously performing, in a solution, (1) a deprotection step of the 5&#x2032;-terminal hydroxyl group, (2) a 5&#x2032;-terminal elongation step by the addition of a p-mer oligonucleotide wherein the 3&#x2032;-position is phosphoramidited, and (3) an oxidation step or a sulfurization step of a phosphite triester moiety. The 3&#x2032;-hydroxyl group of the n-mer oligonucleotide is protected by a solubilizing protecting group represented by formula: -L-Y&#x2014;Z, where L is a group represented by formula (a1): Y is an oxygen atom, or NR wherein R is a hydrogen atom, an alkyl group or an aralkyl group, and Z is a group represented by formula (a2): 

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Kawasaki, Japan

Kunihiro Hirai