EP-4741411-A2 - OLIGONUCLEOTIDE PRODUCTION METHOD, AND NUCLEOSIDE, NUCLEOTIDE, OR OLIGONUCLEOTIDE

EP4741411A2EP 4741411 A2EP4741411 A2EP 4741411A2EP-4741411-A2

Abstract

The present invention provides a production method oligonucleotide, including (1) a step of condensing a nucleoside, nucleotide or oligonucleotide (a) or the like wherein a 5'-hydroxy group is not protected and a nucleoside, nucleotide or oligonucleotide (b) wherein a 5'-hydroxy group is protected in a non-polar solvent to give a reaction solution containing a phosphite triester product (c); (3) a step of 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) a step of deprotecting the oligonucleotide (d) to give a reaction solution containing an oligonucleotide (e) wherein the 5'-hydroxy group is not protected; and (6) a step of adding a polar solvent to the reaction solution containing the oligonucleotide (e) and purifying the oligonucleotide (e) by solid-liquid separation or extraction.

Inventors

HIRAI, Kunihiro
KATAYAMA, SATOSHI
HIROSE, NAOKO
ICHIMARU, Taisuke
YAMASHITA, KEN
TAKAHASHI, DAISUKE

Assignees

AJINOMOTO CO., INC.

Dates

Publication Date: 20260513
Application Date: 20161216

Claims (3)

A nucleoside or oligonucleotide represented by the formula (a-IV) : wherein m is an integer of not less than 0; Base in the number of m+1 are each independently an optionally protected nucleic acid base; X in the number of m+1 are each independently a hydrogen atom, a halogen atom, an optionally protected hydroxy group, or a divalent organic group bonded to a 2-position carbon atom and a 4-position carbon atom; R 10 in the number of m are each independently an oxygen atom or a sulfur atom; R p1 in the number of m are each independently a protecting group of phosphoric acid group; L is a single bond, or a group represented by the formula (a1) or (a1'): wherein * indicates the bonding position to Y; ** is the bonding position to an oxygen atom; R 1 and R 2 are each independently a C 1-22 hydrocarbon group; L 1 is an optionally substituted divalent C 1-22 hydrocarbon group; L 2 is a single bond or a group represented by ***C(=O)N(R 3 )-R 4 -N(R 5 )**** wherein *** is the bonding position to L 1 , **** is the bonding position to C=O, R 4 is a C 1-22 alkylene group, R 3 and R 5 are each independently a hydrogen atom or a C 1-22 alkyl group, or R 3 and R 5 are optionally joined to form a ring; Y is a single bond, 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 the formula (a2"): wherein * indicates a bonding position; R 6 is a hydrogen atom, or when R b is a group represented by the following formula (a3), it optionally shows, together with R 8 , a single bond or -O- to form, together with ring B and ring C, a fused ring; k is an integer of 1 - 4; Q in the number of k are each independently -O-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NH- or -NHC(=O)-; R 7 in the number of k are each independently a hydrocarbon group wherein a linear aliphatic hydrocarbon group having a carbon number of not less than 10 is bonded via a single bond or a linker, or an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of not less than 14 and not more than 300; ring B optionally has, in addition to QR 7 in the number of k, a substituent selected from the group consisting of a halogen atom, a C 1-6 alkyl group optionally substituted by a halogen atom, and a C 1-6 alkoxy group optionally substituted by a halogen atom; R a is a hydrogen atom; and R b is a hydrogen atom, or a group represented by the formula (a3): wherein * indicates a bonding position; j is an integer of 0 to 4; Q in the number of j are each independently as defined above; R 9 in the number of j are each independently a hydrocarbon group wherein a linear aliphatic hydrocarbon group having a carbon number of not less than 10 is bonded via a single bond or a linker, or an organic group having at least one aliphatic hydrocarbon group having one or more branched chains and having a total carbon number of not less than 14 and not more than 300; R 8 is a hydrogen atom, or optionally shows, together with R 6 , a single bond or -O- to form, together with ring B and ring C, a fused ring; and ring C optionally has, in addition to QR 9 in the number of j, a substituent selected from the group consisting of a halogen atom, a C 1-6 alkyl group optionally substituted by a halogen atom, and a C 1-6 alkoxy group optionally substituted by a halogen atom, or R a and R b are joined to form an oxo group.
The nucleoside or oligonucleotide according to claim 1, wherein R p1 is a group represented by -CH 2 CH 2 WG wherein WG is an electron-withdrawing group.
The nucleoside according to claim 1 or 2, wherein m is 0.

Description

[Technical Field] The present invention relates to a production method of oligonucleotide, and nucleoside, nucleotide or oligonucleotide effectively usable in the aforementioned production method. [Background Art] As a production method of oligonucleotide, a solid phase method using a phosphoramidite method is widely used at present (non-patent document 1). The solid phase method is advantageous from the aspect of speed, since process has been optimized and automation has progressed. However, it is associated with defects in that scaling-up is limited due to facility restriction. In addition, a production method of oligonucleotide by a liquid phase method has also been studied. However, since the operation is complicated and the yield is low, and therefore, a large-scale, rapid synthesis of long oligonucleotide is difficult. In recent years, in an attempt to solve the respective defects of the solid phase method and liquid phase method, a production method using a hydrophobic group-linked nucleoside (patent document 1), a production method using a nucleoside or oligonucleotide having the 3'-hydroxy group protected by a particular organic group (patent document 2) and the like have been disclosed. The aforementioned patent document 2 describes a production method of oligonucleotide, including the following steps (i) - (iv): (i) removing a 5'-hydroxy-protecting group (e.g., dimethoxytrityl group) of a nucleoside or oligonucleotide (deprotection),(ii) condensing a nucleoside or oligonucleotide obtained by deprotection and a nucleoside or oligonucleotide wherein a 3'-hydroxy group is phosphoramidited and a 5'-hydroxy group is protected,(iii) oxidizing or sulfurizing a phosphite triester product obtained by the condensation, and(iv) solid-liquid separating an oligonucleotide having the protected 5'-hydroxy group, which is obtained by the oxidation or sulfurization. The oligonucleotide chain can be elongated by repeating the aforementioned steps (i) - (iv). [Document List] [Patent documents] patent document 1: JP-A-2010-275254patent document 2: WO 2012/157723 [non-patent document] non-patent document 1: S. L. Beaucage, D. E. Bergstorm, G. D. Glick, R. A. Jones, Current Protocols in Nucleic Acid Chemistry; John Wiley & Sons (2000) [SUMMARY OF THE INVENTION] [Problems to be Solved by the Invention] An object of the present invention is to provide a production method of oligonucleotide, capable of efficiently performing condensation. [Means of Solving the Problems] The present inventors have conducted intensive studies and found that condensation can be efficiently performed by changing the order of the steps described in patent document 2 and performing (I) condensing a nucleoside, nucleotide or oligonucleotide (a) wherein a 5'-hydroxy group is not protected, and at least one group of a 3'-hydroxy group and the like is protected by a protecting group, and a nucleoside, nucleotide or oligonucleotide (b) wherein a 3'-hydroxy group or 3'-amino group is phosphoramidited and a 5'-hydroxy group is protected,(II) oxidizing or sulfurizing a phosphite triester product (c) obtained by the condensation,(III) removing the 5'-hydroxy-protecting group from an oligonucleotide (d) obtained by the oxidation or sulfurization (deprotection), and(IV) purifying by solid-liquid separating or extracting an oligonucleotide (e) wherein the 5'-hydroxy group is not protected, which is obtained by the deprotection. To repeat the steps (i) - (iv) described in Patent document 2, condensation (step (ii)) is performed after deprotection (step (i)). To repeat the aforementioned steps (I) - (IV), condensation (step (I)) is performed after solid-liquid separation or extraction (step (IV)). Therefore, in the production method containing the aforementioned steps (I) - (IV), impurity produced by deprotection is absent during condensation and condensation can be performed efficiently. The present invention based on the above finding is as described below. [1] A method for producing an oligonucleotide, comprising the following steps (1), (3), (4) and (6): (1) condensing, in a non-polar solvent, a nucleoside, nucleotide or oligonucleotide (b) wherein a 3'-hydroxy group or 3'-amino group is phosphoramidited, a 5'-hydroxy group is protected by a temporary protecting group removable under acidic conditions, and other groups are optionally protected by a protecting group selected from protecting groups unremovable under acidic conditions but removable under basic conditions and protecting groups used for nucleic acid synthesis, anda nucleoside, nucleotide or oligonucleotide (a) wherein a 5'-hydroxy group is not protected, at least one group selected from an amino group and an imino group of a nucleic acid base, a 2'-hydroxy group, a 3'-hydroxy group and a 3'-amino group of a ribose residue, and a 3'-hydroxy group and a 3'-amino group of a deoxyribose residue is protected by a protecting group unremovable under acidic conditions but removable under basic condi