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Phosphoramidite reagent and applications thereof in oligonucleotide synthesis

An oligonucleotide and phosphorous technology, which is used in phosphorylation reagents and its application in oligonucleotide synthesis, and can solve the problems of reduced solubility of intermediates and difficult molecular chain growth reactions.

Active Publication Date: 2020-04-21
GUANGZHOU TROJAN PHARMATEC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problem that the solubility of intermediates in the existing process of synthesizing oligonucleotides leads to the difficulty of molecular chain growth reaction, provide a phosphitylation reagent and its preparation method, and use the phosphitylation reagent to synthesize Oligonucleotides

Method used

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  • Phosphoramidite reagent and applications thereof in oligonucleotide synthesis
  • Phosphoramidite reagent and applications thereof in oligonucleotide synthesis
  • Phosphoramidite reagent and applications thereof in oligonucleotide synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] This embodiment provides a phosphorylation reagent: bis(diisopropylamino)(2-cyano-2-(isodecyl)ethoxy)phosphine, whose structural formula is:

[0059] The route of synthesizing this phosphitylation reagent is as follows:

[0060]

[0061] Concrete synthesis method comprises the following steps:

[0062] 1-a) Mix isomeric decanol (compound 1.1, 100mL) and 37% HBr (300mL) in a three-necked flask, add concentrated sulfuric acid (30mL) slowly under mechanical stirring, and heat the mixed reaction solution to 115-120 °C for the reaction. The reaction was stopped after GC analysis of <0.5% isomeric decanol. Then the temperature was lowered to below 50°C, and petroleum ether (100 mL) was added for extraction. The petroleum ether layer was first neutralized with water, and the petroleum ether was removed under reduced pressure to obtain 120.0 g of isomeric dedecyl bromide (compound 1.2).

[0063] 1-b) Dissolve isomeric dedecyl bromide (compound 1.2, 22.1 g, 0.1 mol) and ...

Embodiment 2

[0070] This embodiment provides a phosphitylation reagent: bis(diisopropylamino)(1-propyl-2-cyano-2-(isodecyl)ethoxy)phosphine, whose structural formula is:

[0071] The route of synthesizing this phosphitylation reagent is as follows:

[0072]

[0073] Concrete synthesis method comprises the following steps:

[0074] 2-a) Dissolve ethyl 2-(isodecyl)-2-cyanoacetate (compound 1.4, 50.7g, 0.2mol) in DMF (100mL), add potassium acetate (19.6g, 0.2mol) And water (7.2g, 0.2mol), heated to reflux for 5h. Cool to room temperature, add ethyl acetate (150 mL) and water (200 mL) for extraction, wash the ethyl acetate layer with water (100 mL) and saturated brine (50 mL), and concentrate to obtain 2-isodecyl-acetonitrile (compound 2.1) The crude product was purified by column chromatography to obtain 31.0 g.

[0075] 2-b) Under nitrogen protection, diisopropylamine (10.2 g, 0.1 mol) was dissolved in anhydrous tetrahydrofuran (200 mL), and cooled to -78° C. with a dry ice-acetone b...

Embodiment 3

[0081] This embodiment provides a phosphorylation reagent: bis(diisopropylamino)(1-phenyl-2-cyano-2-(isodecyl)ethoxy)phosphine, whose structural formula is:

[0082] The route of synthesizing this phosphitylation reagent is as follows:

[0083]

[0084] The specific synthesis method is similar to that of Example 2, except that benzaldehyde is used instead of butyraldehyde in step 2-b).

[0085] Carry out to product P-003 1 H-NMR, MS test, the results are as follows:

[0086] 1 H-NMR (400MHz, CDCl 3 ): δ0.8-1.65 (m, 45H), 2.50-2.65 (m, 1H), 3.50-3.65 (m, 4H), 4.80-4.90 (m, 1H), 7.30-7.45 (m, 5H).

[0087] MS-ESI[M+1] + : 518.40.

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Abstract

The invention provides a phosphoramidite reagent and applications thereof. According to the invention, the phosphoramidite reagent is a 2-nitrile ethoxy phosphoramidite compound with a large non-polarsubstituent group on a 2-nitrile ethoxy group, and can react with a nucleoside monomer or a nucleotide fragment 3'-OH or 5'-OH to generate phosphoramidite diester, the phosphoramidite diester continuously reacts with another group of nucleoside monomers or nucleotide fragments 3'-OH or 5'-OH under the action of a proper activating agent to generate phosphoramidite trimester, oxidizing or vulcanizing is performed to obtain phosphoric acid triester or thiophosphoric acid trimester, and deprotection is performed to obtain oligonucleotides, wherein in the whole process, all phosphite intermediates or phosphate intermediates are easily dissolved in a solvent with medium and low polarity and have low solubility in a high-polar solvent while other reaction raw materials or by-products are easilydissolved in the high-polar solvent, such that the purification can be performed through non-polar / polar solvent extraction so as not to hinder the subsequent chain propagation reaction; the method is suitable for one-by-one nucleotide linking and reactions linked by a fragment method.

Description

technical field [0001] The invention belongs to the technical field of nucleotide synthesis, and in particular relates to a phosphorylation reagent and its application in oligonucleotide synthesis. Background technique [0002] The application of phosphitylation reagents in the synthesis of oligonucleotides began in the mid-1970s, and oligonucleotides were obtained by sequentially reacting two active groups on the phosphitylation reagents with protected nucleosides. The method of using phosphorylation reagents to synthesize oligonucleotides is called the phosphoramidite method, also known as the "phosphite triester method" in the early days, and is the most commonly used method in the synthesis of oligonucleotides. Reagents are key raw materials for the phosphoramidite method. [0003] Dichlorophosphite monoester was an early phosphitylation reagent. Phosphorous chlorophosphite monoester is an active intermediate in the reaction process, which has high activity. After the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F9/24C07H19/10C07H19/207C07H1/00
CPCC07F9/2475C07F9/2408C07F9/2429C07F9/2412C07H19/10C07H19/207C07H1/00Y02P20/55
Inventor 黄世福郑庆泉梁伟周李浓滔
Owner GUANGZHOU TROJAN PHARMATEC LTD
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