# US 8,569,476 B2 · Method for preparing oligonucleotide

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 8,569,476 B2
- Document ID: US8569476B2
- Status: Granted
- Kind: B2
- Application number: 13/003,195
- Filed: 2009-09-22
- Published: 2011-09-29
- Granted: 2013-10-29
- Earliest claimed: 2008-09-23
- Assignees: Suzhou Ribo Life Science Co., Ltd.
- Applicants: Zhen Xi; Jinyu Huang; Junbin Zhang
- Inventors: Zhen Xi; Jinyu Huang; Junbin Zhang
- Web page: https://patentgrep.com/patent/US8569476B2

## CPC

- 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
- C07H 19/067: with ribosyl as the saccharide radical
- C07H 19/10: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- C07H 19/167: with ribosyl as the saccharide radical
- C07H 19/20: with the saccharide radical esterified by phosphoric or polyphosphoric acids
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

A method for preparing oligonucleotide comprising reacting the compound of Formula (1) with the compound of Formula (2) in a liquid reaction medium under the condition of condensation reaction to obtain the compound of formula (3) is provided. 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) is applied as condensing agent. Oligonucleotides synthesized in the liquid reaction medium could be obtained on a large scale.

## Claims

### Claim 1

A method for preparing an oligonucleotide, comprising reacting the compound represented by Formula (1) with the compound represented by Formula (2) in a liquid reaction medium under the condition of condensation reaction to obtain the compound represented by Formula (3), wherein 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) is applied as condensing agent,
and wherein: R1 represents 4,4′-dimethoxytriphenylmethyl, or a covalently attached 5′-substituent derived from RNA or from DNA;
R2 and R3 independently represent a sterically hindered trisubstituted silyl protective group;
R4 represents a halogen atom;
A+ represents a tri-alkyl ammonium ion;
B1 and B2 independently represent 9-guaninyl substituted with N-acyl, 9-adeninyl substituted with N-acyl, 1 cytosinyl substituted with N-acyl, 1-thyminyl or 1-uracilyl;
and with the proviso that in Formula (1) or (3), —CH2—N3 is at an ortho-position.

### Claim 2 (depends on 1)

The method as in claim 1, wherein the condition of the condensation reaction comprises: the reaction medium is pyridine; relative to 1 mol of the compound represented by Formula (1), the amount of the compound represented by Formula (2) is 0.8-3 mol, the amount of the condensing agent is 2-5 mol, and the amount of the reaction medium is 5-50 L; the reaction temperature is 10-50° C. and the reaction time is 0.5-10 h.

### Claim 3 (depends on 1)

The method as in claim 1, wherein R2 and R3 independently represent tert-butyl dimethyl silyl, phenyl dimethyl silyl, tert-butyl diphenyl silyl or triisopropyl silyl; R4 represents Cl or Br; each of the alkyl groups in the tri-alkyl ammonium ion has 1-6 carbon atoms; the acyl is benzoyl, isobutyryl or acetyl.

### Claim 4 (depends on 1)

The method as in claim 1, wherein in Formula (2) or (3), R4 is at the ortho-position.

### Claim 5 (depends on 1)

The method as in claim 1 further comprising wherein the compound represented by Formula (1) is prepared by reacting the compound represented by Formula (4) with the compound represented by Formula (5) under the condition of condensation reaction, and then followed by hydrolytic removal of the 5′-protective group, N-methyl imidazole is applied as an adjuvant;
Where where: R2 represents a sterically hindered trisubstituted silyl protective group;
R5 represents 4,4′-dimethoxytriphenylmethyl;
R6 represents a halogen atom;
B1 represents 9-guaninyl substituted with N-acyl, 9-adeninyl substituted with N-acyl, 1-cytosinyl substituted with N-acyl, 1-thyminyl or 1-uracilyl;
and with the proviso that in Formula (5), —CH2—N3 is at an ortho-position.

### Claim 6 (depends on 5)

The method as in claim 5, wherein the condition of the condensation reaction further comprises: one or more of dichloromethane and pyridine is applied as reaction medium; relative to 1 mol of the compound represented by Formula (4), the amount of the compound represented by Formula (5) is 1-5 mol, the amount of the adjuvant is 1.8-3.5 mol, and the amount of the reaction medium is 20-200 L; the reaction temperature is −10° C.˜10° C. and the reaction time is 5-100 h.

### Claim 7 (depends on 5)

The method as in claim 5, wherein the process for preparing the compound represented by Formula (5) further comprises the following steps:
(1) in the presence of benzoyl peroxide, the compound represented by Formula (9) reacts with N-halogenated succinimide to obtain the compound represented by Formula (10);
(2) the compound represented by Formula (10) reacts with alkali metal azide to obtain the compound represented by Formula (11);
(3) the compound represented by Formula (11) is hydrolyzed and acylated to obtain the compound represented by Formula (5),
Where where: R9 represents a halogen atom; R10 represents C1-C4 alkyl.

### Claim 8 (depends on 7)

The method as in claim 7, wherein in step (1), the reaction medium is one or more of carbon tetrachloride, chloroform, benzene, toluene and heptane; relative to 1 mol of the compound represented by Formula (9), the amount of N-halogenated succinimide is 1-3 mol, the amount of benzoyl peroxide is 0.01-0.1 mol, the amount of the reaction medium is 5-20 L; the reaction temperature is 80˜120° C. and the reaction time is 0.5-6 h;
in step (2), the reaction medium is one or more of ethanol, acetone, N,N-dimethyl formamide and dimethyl sulfoxide; relative to 1 mol of the compound represented by Formula (10), the amount of alkali metal azide is 1-3 mol, the amount of the reaction medium is 3-10 L; the reaction temperature is 0° C.˜80° C. and the reaction time is 2-30 h;
in step (3), the hydrolysis comprises the reaction between the compound represented by Formula (11) and the alcohol-water mixed solution of alkali metal hydroxide wherein relative to 1 mol of the compound represented by Formula (10), the amount of alkali metal hydroxide is 5-100 mol, the reaction temperature is 0° C.˜50° C., the reaction time is 0.1-2 h and the concentration of alkali metal hydroxide in the alcohol-water mixed solution is 5-10 wt %.

### Claim 9 (depends on 1)

The method as in claim 1, further comprising wherein the compound represented by Formula (2) is obtained through the reaction of the compound represented by Formula (6), the compound represented by Formula (7) and trialkyl amine in a reaction medium in the presence of a catalyst, the catalyst being one or more of 1,2,4-triazole, triethylamine and pyridine, the reaction medium being one or more of dichloromethane, dioxane and tetrahydrofuran,
where: R1 represents 4,4′ dimethoxytriphenylmethyl, or a covalently attached 5′-substituent derived from RNA or from DNA;
R3 represents a sterically hindered trisubstituted silyl protective group;
R4, R7 and R8 independently represent a halogen atom;
B2 represents 9-guaninyl substituted with N-acyl, 9-adeninyl substituted with N-acyl, 1-cytosinyl substituted with N-acyl, 1-thyminyl or 1-uracilyl.

### Claim 10 (depends on 9)

The method as in claim 9 further comprising wherein the temperature of the reaction is −10° C.˜10° C. and the reaction time is 0.5-10 h; relative to 1 mol of the compound represented by Formula (6), the amount of the compound represented by Formula (7) is 1-5 mol, the amount of trialkyl amine is 1-50 mol and the amount of catalyst is 2-10 mol.

### Claim 11 (depends on 6)

The method as in claim 6, wherein the process for preparing the compound represented by Formula (5) further comprises the following steps:
(1) in the presence of benzoyl peroxide, the compound represented by Formula (9) reacts with N-halogenated succinimide to obtain the compound represented by Formula (10);
(2) the compound represented by Formula (10) reacts with alkali metal azide to obtain the compound represented by Formula (11);
(3) the compound represented by Formula (11) is hydrolyzed and acylated to obtain the compound represented by Formula (5),
where: R9 represents a halogen atom; R10 represents C1-C4 alkyl.