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CN-121991151-A - Synthesis process of 2-TBS adenosine phosphoramidite monomer

CN121991151ACN 121991151 ACN121991151 ACN 121991151ACN-121991151-A

Abstract

The application relates to the field of pharmaceutical chemistry, in particular to a synthesis process of a 2-TBS adenosine phosphoramidite monomer. The method comprises the following steps of S1, carrying out silicon-based protection on adenosine 2', 3' and 5 'hydroxyl groups to obtain a compound A, S2, reacting the compound A with benzoyl chloride to obtain a compound B, S3, selectively deprotecting the compound B and a hydrogen fluoride-pyridine complex to obtain a compound C, S4, reacting the compound C with 4,4' -dimethoxy trityl chloride to obtain a compound D, and S5, carrying out phosphitylation reaction on the compound D with a phosphorus reagent to obtain a compound E. The problems of low total yield of the 2-TBS adenosine phosphoramidite monomer, poor stability of an intermediate product and low purity are effectively solved, the high selectivity of a synthesized target product is realized through a high-selectivity reaction system, the yield and the purity are improved, and the separation and purification steps are simplified through stabilizing the intermediate product group.

Inventors

  • LI QIANG
  • LU JIANGPING

Assignees

  • 康羽生命科学技术(苏州)有限公司

Dates

Publication Date
20260508
Application Date
20260119

Claims (10)

  1. A process for synthesizing 2-TBS adenosine phosphoramidite monomer is characterized by comprising the following steps of S1, carrying out silicon-based protection on adenosine 2', 3' and 5 'hydroxyl groups to obtain a compound A, S2, reacting the compound A with benzoyl chloride to obtain a compound B, S3, selectively deprotecting the compound B and hydrogen fluoride-pyridine complex to obtain a compound C, S4, reacting the compound C with 4,4' -dimethoxytrityl chloride to obtain a compound D, S5, carrying out a phosphoramidition reaction on the compound D and a phosphorus reagent to obtain a compound E, and synthesizing the following steps: 。
  2. 2. The process for synthesizing the 2-TBS adenosine phosphoramidite monomer according to claim 1, wherein the S1 silicon-based protection comprises the steps of adding adenosine and imidazole into DMF, dropwise adding 1,3 dichloro-1, 3-tetraisopropyl disiloxane at a temperature of 0-5 ℃, stirring for 30-45min, detecting the adenosine content by HPLC to be less than 0.5%, adding imidazole and TBSCl, continuously stirring for 1-2h, dropwise adding into 8-10 times volume water for crystallization, stirring for 8-12h at a speed of 200-300rpm at a temperature of 5-8 ℃, filtering and drying to obtain the 2-TBS adenosine phosphoramidite monomer.
  3. 3. The process for synthesizing 2-TBS adenosine phosphoramidite monomer according to claim 1, wherein in S4, pyridine pretreatment liquid is added with compound C and 4,4' -dimethoxy trityl chloride, stirred and reacted for 8-12 hours at 15-25 ℃, and then extracted, washed, concentrated and subjected to column chromatography to obtain compound D.
  4. 4. The process for synthesizing 2-TBS adenosine phosphoramidite monomer according to claim 3, wherein the preparation method of the pyridine pretreatment liquid is characterized in that pyridine dried by anhydrous magnesium sulfate is added with methylene dichloride dehydrated by a molecular sieve, and after stirring, 18-crown-6 is added, and stirring is carried out for 20-30min at 15-25 ℃.
  5. 5. The process for synthesizing 2-TBS adenosine phosphoramidite monomer according to claim 4, wherein the molar ratio of 18-crown-6 to compound C is 1 (4-6).
  6. 6. The process for synthesizing 2-TBS adenosine phosphoramidite monomer according to claim 1, wherein the S5 phosphorus reagent is N, N-diisopropylamino-2-cyanoethoxy-phosphine chloride.
  7. 7. The process for synthesizing the 2-TBS adenosine phosphoramidite monomer according to claim 1, wherein in the step S5, acetonitrile is added into the compound D, a phosphorus reagent, tetrazole and pyridinium triflate are added under the protection of nitrogen, the reaction is carried out for 2-3 hours at 15-25 ℃, and the compound E is obtained through concentration, washing, stirring crystallization and centrifugation.
  8. 8. The process for synthesizing 2-TBS adenosine phosphoramidite monomer according to claim 7, wherein the stirring crystallization comprises adding ethyl acetate and methyl tert-butyl ether, stirring, dropwise adding n-heptane, and stirring at 5-8 ℃ for crystallization for 12-15h.
  9. 9. The process for synthesizing the 2-TBS adenosine phosphoramidite monomer according to claim 1, wherein in the S2, pyridine is taken, dichloromethane is added, compound A is added after stirring, benzoyl chloride is slowly added dropwise at the temperature of 5-8 ℃, the content of the compound A is detected by sampling HPLC and is less than 0.5%, water quenching is carried out, ammonia water is added for regulating the pH value to be 10-11, extraction is carried out, and concentration is carried out, so that the compound B is obtained.
  10. 10. The process for synthesizing the 2-TBS adenosine phosphoramidite monomer according to claim 1, wherein in the step S3, the compound B is taken, tetrahydrofuran is added and stirred, N-methylimidazole is added, the temperature is minus 5 to minus 8 ℃, the fluoropyridine complex is slowly added dropwise, stirring is carried out for 18 to 26 hours, extraction, washing, drying and pulping are carried out overnight, and the compound C is obtained by filtering.

Description

Synthesis process of 2-TBS adenosine phosphoramidite monomer Technical Field The application relates to the field of oligonucleotides, in particular to a synthesis process of a 2-TBS adenosine phosphoramidite monomer. Background Artificially synthesized oligonucleotides have been widely used in research on targeted gene therapy in the past decades. Oligonucleotides mainly include antisense oligonucleotides, ASODNs, small interfering RNAs, transcription factor decoys, ribozymes, deoxyribozymes, antigenes, cpG oligonucleotides, nucleic acid aptamers, and the like. Among them, ASODN and siRNA are the most commonly used gene regulation tools, are widely used, and have been developed as gene therapy drugs. The oligonucleotide is prepared from nucleoside phosphoramidite monomer by classical solid phase synthesis, and is widely applied to the fields of novel therapeutic strategy development, gene editing, nucleic acid medicines and the like. In the cost of raw materials and auxiliary materials for synthesizing the oligonucleotide, the nucleoside phosphoramidite monomer accounts for a main part, and the adenosine phosphoramidite monomer is one of core raw materials for synthesizing the oligonucleotide and is widely applied to the fields of gene diagnosis, drug research and development, nucleic acid vaccine preparation and the like. The existing preparation method of the 2-TBS adenosine phosphoramidite monomer has obvious defects, mainly because the synthesis steps are complicated, and the conversion between multi-step protection and deprotection is needed, the total yield is low, and the requirement of industrial mass production is difficult to meet. And the stability of the intermediate product is poor, so that the purity of the final product is reduced, and the difficulty of subsequent purification is increased. In addition, the phosphitylation reaction conditions are harsh, the reaction selectivity is poor, byproducts such as phosphate esters and the like are easy to generate, and the yield and purity of the target monomer are further reduced. These problems severely limit the broader application and development of 2-TBS adenosine phosphoramidite monomers in the relevant field. Disclosure of Invention In order to effectively improve the problems of low total yield of the 2-TBS adenosine phosphoramidite monomer, poor stability of intermediate products and low purity, the application provides a synthesis process of the 2-TBS adenosine phosphoramidite monomer. In a first aspect, the application provides a synthesis process of a 2-TBS adenosine phosphoramidite monomer, the reaction formula of which is shown as formula I, comprising the following steps of S1, carrying out silicon-based protection on adenosine 2', 3' and 5 'hydroxyl groups to obtain a compound A, S2, reacting the compound A with benzoyl chloride to obtain a compound B, S3, selectively deprotecting the compound B and a hydrogen fluoride-pyridine complex to obtain a compound C, S4, reacting the compound C with 4,4' -dimethoxytrityl chloride to obtain a compound D, S5, carrying out phosphorous acylation on the compound D and a phosphorus reagent to obtain a compound E, and the synthesis route is as follows: 。 The application takes adenosine as an initial raw material, realizes the synthesis of an adenosine phosphoramidite monomer protected by 2-tertiary butyl dimethyl silicon based through five steps of reaction, and sequentially carries out the silicon-based protection of adenosine hydroxyl, the reaction with benzoyl chloride, the selective deprotection, the reaction with 4,4' -dimethoxy trityl chloride and the phosphoramidition reaction, and the steps are tightly connected and matched to optimize the product structure, thereby reducing the occurrence of side reaction, effectively improving the purity of the product, reducing unnecessary side reaction and impurity generation, finally obtaining the 2-TBS adenosine phosphoramidite monomer, and guaranteeing the high yield and high purity of the product. Specifically, the 2', 3' and 5' hydroxyl groups of the adenosine are subjected to silicon-based protection, a substrate with stable structure and strong reaction selectivity is provided for subsequent reaction, the increase of byproducts caused by the indifferent reaction of the hydroxyl groups is avoided, the separation of intermediates is reduced, and the requirement of industrial production is met. Then in the S2 step, benzoyl protection is carried out on amino in the adenosine molecule, benzoyl chloride is used as a protecting reagent to carry out nucleophilic addition reaction with amino in the adenosine molecule to form a stable benzoyl protecting structure, the selectivity of the molecular structure is improved, meanwhile, the intermediate structure protected by benzoyl is more stable, side reactions of amino in subsequent reactions are avoided, conditions are created for subsequent selective deprotection reactions, in the S3 step, the specific silic