阅读说明：本技术 一种2-脱氧-2,2-二氟-3,5-二苯甲酰基-d-呋喃核糖的生物合成方法 (Biosynthesis method of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose ) 是由 付自强 陈恬 程加铭 卢文才 顾学新 张锦涛 于 2021-09-28 设计创作，主要内容包括：本发明涉及一种2-脱氧-2,2-二氟-3,5-二苯甲酰基-D-呋喃核糖的生物合成方法,属于药物中间体技术领域。为了解决合成路线长和污染大的问题,提供一种2-脱氧-2,2-二氟-3,5-二苯甲酰基-D-呋喃核糖的生物合成方法,该方法包括在醇溶剂中,使式Ⅱ化合物2-脱氧-2,2-二氟戊呋喃糖-1-酮-3,5-二安息香酸盐在催化量的CIR酶的催化作用下进行酶催化反应生成产物式Ⅰ化合物2-脱氧-2,2-二氟-3,5-二苯甲酰基-D-呋喃核糖。本发明具有高产物收率和高纯度的优点,产物收率达到98％以上,纯度达到96％以上。环境污染少的优点。(The invention relates to a biosynthesis method of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, belonging to the technical field of drug intermediates. In order to solve the problems of long synthesis route and large pollution, a biosynthesis method of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose is provided, which comprises the step of carrying out enzyme catalysis reaction on 2-deoxy-2, 2-difluoropentofuranose-1-one-3, 5-dibenzoate of a compound shown in a formula II in an alcohol solvent under the catalysis of catalytic action of catalytic amount of CIR enzyme to generate a product, namely the compound shown in the formula I, namely 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose. The method has the advantages of high yield and high purity, the yield of the product reaches more than 98%, and the purity reaches more than 96%. Less environmental pollution.)

1. A method for the biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, comprising the steps of:

carrying out an enzyme catalysis reaction on a compound 2-deoxy-2, 2-difluoropentofuranose-1-keto-3, 5-di-benzoate shown in a formula II in an alcohol solvent under the catalysis of catalytic amount of CIR enzyme to generate a product, namely a compound 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose shown in a formula I;

the protein sequence of the CIR enzyme is shown in SEQ ID NO. 1.

2. The method of claim 1, wherein the CIR enzyme is added in an amount of 10% to 25% by weight of the compound of formula II, 2-deoxy-2, 2-difluoropentofuranose-1-ulose-3, 5-dibenzoate.

3. The method of claim 2, 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose biosynthesis process of claim 2, wherein the CIR enzyme is added in an amount of 18% to 20% by weight of the compound of formula ii, 2-deoxy-2, 2-difluoropentofuranose-1-ulose-3, 5-dibenzoate.

4. The method for the biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose according to claim 1, wherein the temperature of the enzymatic reaction is 20 ℃ to 30 ℃.

5. The method for the biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose according to claim 1 or 2 or 3 or 4, wherein the alcoholic solvent is selected from one or more of ethanol, propanol and isopropanol.

6. The method for the biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose according to claim 1, 2, 3 or 4, wherein the enzymatic reaction is further followed by filtration, and the collected filtrate is subjected to distillation to remove the solvent.

7. The method for biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose according to claim 6, wherein the temperature of the distillation treatment is controlled to 50 ℃ or lower.

Technical Field

The invention relates to a biosynthesis method of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, belonging to the technical field of drug intermediates.

Background

Gemcitabine hydrochloride is a cell cycle specific antimetabolite, mainly acts on tumor cells in a DNA synthesis phase, namely S phase cells, can prevent the progression from the G1 phase to the S phase under certain conditions, and has obvious cytotoxic activity on various cultured human and mouse tumors.

The synthesis of gemcitabine hydrochloride reported in the prior literature is mainly performed by taking 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose as an intermediate of gemcitabine, and further synthesizing the final gemcitabine hydrochloride product. For example, the catalyst is prepared by using 2-deoxy-2, 2-difluoropentofuranose-1-one-3, 5-di-benzoate as a raw material and performing catalytic hydrogenation reduction on the raw material by palladium carbon. The catalyst palladium carbon adopted in the synthesis method is expensive, the process risk in the hydrogenation reaction process is high, the yield and purity of the obtained product are low, and the synthesis method is not suitable for industrial production.

And the 2-deoxy-2, 2-difluoropentofuranose-1-one 3, 5-di-benzoate is used as a raw material, and lithium aluminum hydride or red aluminum is adopted for carrying out reduction reaction to obtain a corresponding product.

It can be seen that the synthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose is mainly carried out by chemical synthesis, so that the method has high risk and great environmental pollution. Therefore, it is of great significance to find a new synthetic route.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a biosynthesis method of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, and solves the technical problem of how to provide a new synthesis route which can reduce pollution and has high product yield.

The invention aims to realize the following technical scheme, and the biosynthesis method of the 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose is characterized by comprising the following steps:

carrying out an enzyme catalysis reaction on a compound 2-deoxy-2, 2-difluoropentofuranose-1-keto-3, 5-di-benzoate shown in a formula II in an alcohol solvent under the catalysis of catalytic amount of CIR enzyme to generate a product, namely a compound 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose shown in a formula I;

the protein sequence of the CIR enzyme is shown in SEQ ID NO. 1.

The compound is synthesized by adopting an enzyme method, and a specific CIR enzyme with a protein sequence shown in SEQ ID NO.1 is adopted, so that the raw material 2-deoxy-2, 2-difluoropentofuranose-1-ketone-3, 5-di-benzoate can be effectively subjected to enzyme catalysis to efficiently reduce and convert ketone carbonyl in a molecular structure into hydroxyl, and the hydroxyl can be converted into a corresponding product 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, and the compound has the advantages of high conversion efficiency, high yield and high purity. And a hydrogenation reduction mode with high potential safety hazard is not needed. Meanwhile, the method is carried out in an alcohol solvent system, so that the reaction can be carried out mildly, the post-treatment is easy to operate, a large amount of waste liquid and the like cannot be generated after the reaction is finished, the pollution to the environment is reduced, the production operation is greatly simplified, and the industrial production is easy to realize.

In the above-mentioned method for biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, preferably, the CIR enzyme is added in an amount of 10 to 25% by weight based on the weight of 2-deoxy-2, 2-difluoropentofuranose-1-ulose-3, 5-di-benzoate as the compound of formula II. The method can more effectively realize carbonyl conversion in the catalytic reduction raw material, better avoid the generation of other byproducts, and has better production yield and purity quality requirements, so that the yield of the product reaches more than 98 percent, and the purity reaches more than 99 percent. More preferably, the CIR enzyme is added in an amount of 18% to 20% by weight based on the weight of 2-deoxy-2, 2-difluoropentofuranose-1-ulose-3, 5-di-benzoate as a compound of formula II.

In the above-mentioned method for biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, the temperature of the enzyme-catalyzed reaction is preferably 20 to 30 ℃. By adopting the CIR enzyme of the protein sequence to carry out enzyme catalysis, the reaction can be carried out mildly, the reaction can be promoted to be carried out efficiently under the condition of lower reaction temperature, the reaction is mild, the generation of impurities can be reduced, and the purity and quality requirements of products can be further ensured.

In the biosynthesis method of the 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, the alcohol solvent has the advantages of low toxicity and high safety. Preferably, the alcohol solvent is selected from one or more of ethanol, propanol and isopropanol. Can ensure the mild reaction, and the solvent is easy to recover, which is beneficial to reducing the production cost.

In the method for biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, the step of filtering after completion of the enzyme-catalyzed reaction, and distilling the collected filtrate to remove the solvent is further included. The enzyme catalytic reduction and alcohol solvent system are adopted for reaction, so that the yield and purity quality of the product can be efficiently ensured, and the corresponding product can be obtained only by filtering to remove residual particles in the enzyme and the system and directly removing the solvent. The method does not need to be refined, better realizes the effect of ensuring the yield and the product purity, and greatly reduces the production operation.

In the above-mentioned method for biosynthesis of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, the temperature of the distillation treatment is controlled to 50 ℃ or lower. The influence of overhigh temperature on the product in the distillation process can be better ensured, and the purity and quality effects of the product can be better ensured.

The biosynthesis method of 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose of the present invention can be represented by the following chemical reaction equation:

in summary, compared with the prior art, the invention has the following advantages:

by adopting a specific CIR enzyme with a protein sequence shown in SEQ ID NO.1 and an alcohol solvent system, the method has the advantages of high raw material conversion rate, high product yield and high purity. The method has the advantages of mild reaction, easy operation of post-treatment, no generation of a large amount of waste liquid after the reaction is finished, no need of adopting a hydrogenation reduction mode with palladium carbon and high potential safety hazard, reduced environmental pollution, great reduction of production operation, easy industrial production, and final product yield of more than 98 percent and purity of more than 96 percent.

Detailed Description

The technical solution of the present invention is further specifically described below by way of specific examples, but the present invention is not limited to these examples.

Example 1

Adding 50g of isopropanol solvent and 25g of 2-deoxy-2, 2-difluoropentofuranose-1-one 3, 5-diantimonate into a clean reaction bottle, adding 5g of CIR enzyme, wherein the protein sequence of the CIR enzyme is shown in SEQ ID NO.1, then carrying out heat preservation reaction for 5 hours under the condition of controlling the reaction temperature to be 20-25 ℃, carrying out filter pressing after the reaction is finished, collecting filtrate, carrying out reduced pressure distillation on the obtained filtrate to remove the solvent in the reaction liquid, heating the obtained filtrate by using a water bath in the reduced pressure distillation process, controlling the temperature to be below 50 ℃, and obtaining 24.8g of corresponding gemcitabine intermediate 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, wherein the yield is 98.8%, and the content of a liquid phase (HPLC) is 96.5%.

Example 2

Adding 50g of isopropanol solvent and 25g of 2-deoxy-2, 2-difluoropentofuranose-1-one 3, 5-diantimonate into a clean reaction bottle, adding 2.5g of CIR enzyme, wherein the protein sequence of the CIR enzyme is shown in SEQ ID NO.1, then, controlling the reaction temperature to be 25-30 ℃ for carrying out heat preservation reaction for 6 hours, carrying out filter pressing after the reaction is finished, collecting filtrate, carrying out reduced pressure distillation on the obtained filtrate to remove the solvent in the reaction liquid, heating the obtained filtrate by using a water bath in the reduced pressure distillation process, and controlling the temperature to be below 50 ℃ to obtain 24.7g of corresponding gemcitabine intermediate 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, wherein the yield is 98.5%, and the content of a liquid phase (HPLC) is 96.1%.

Example 3

Adding 80g of isopropanol solvent and 25g of 2-deoxy-2, 2-difluoropentofuranose-1-one 3, 5-diantimonate into a clean reaction bottle, adding 4.5g of CIR enzyme, wherein the protein sequence of the CIR enzyme is shown in SEQ ID NO.1, then, controlling the reaction temperature to be 20-25 ℃ for carrying out heat preservation reaction for 5 hours, carrying out filter pressing after the reaction is finished, collecting filtrate, carrying out reduced pressure distillation on the obtained filtrate to remove the solvent in the reaction liquid, heating the obtained filtrate by using a water bath in the reduced pressure distillation process, controlling the temperature to be below 50 ℃, and obtaining 24.85g of corresponding gemcitabine intermediate 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, wherein the yield is 99%, and the content of a liquid phase (HPLC) is 98.5%.

Example 4

Adding 50g of absolute ethanol solvent and 25g of 2-deoxy-2, 2-difluoropentofuranose-1-one 3, 5-di-benzoate into a clean reaction bottle, adding 4g of CIR enzyme, wherein the protein sequence of the CIR enzyme is shown in SEQ ID NO.1, then carrying out heat preservation reaction for 6h under the condition that the temperature is controlled to be 25-28 ℃, carrying out filter pressing after the reaction is finished, collecting filtrate, carrying out reduced pressure distillation on the obtained filtrate to remove the solvent in the reaction liquid, heating the obtained filtrate by using a water bath in the reduced pressure distillation process, controlling the temperature to be below 50 ℃, and obtaining 24.6g of corresponding gemcitabine intermediate 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, wherein the yield is 98%, and the content of a liquid phase (HPLC) is 98.4%.

Example 5

Adding 80g of absolute ethyl alcohol solvent and 25g of 2-deoxy-2, 2-difluoropentofuranose-1-one 3, 5-di-benzoate into a clean reaction bottle, adding 6.25g of CIR enzyme, wherein the protein sequence of the CIR enzyme is shown in SEQ ID NO.1, then carrying out heat preservation reaction for 4h under the condition of controlling the reaction temperature to be 20-25 ℃, carrying out filter pressing after the reaction is finished, collecting filtrate, carrying out reduced pressure distillation on the obtained filtrate to remove the solvent in the reaction liquid, heating the obtained filtrate by using a water bath in the reduced pressure distillation process, controlling the temperature to be below 50 ℃, and obtaining 24.7g of corresponding gemcitabine intermediate 2-deoxy-2, 2-difluoro-3, 5-dibenzoyl-D-ribofuranose, wherein the yield is 98.3%, and the content of a liquid phase (HPLC) is 98.2%.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Sequence listing

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Ile Gly Arg Met Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr

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Leu Cys Ser Asp Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met

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Asp Gly Gly Phe Ser Gln Val

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