阅读说明：本技术 制备(1r，3s)-3-氨基环戊醇的方法、整合酶抑制剂、应用 (Method for preparing (1R, 3S) -3-aminocyclopentanol, integrase inhibitor and application ) 是由 石淑敏 章兆琪 陈承 黄勇开 陈海滨 于 2020-07-14 设计创作，主要内容包括：本发明具体公开了一种制备(1R,3S)-3-氨基环戊醇的方法、整合酶抑制剂、应用,所述制备(1R,3S)-3-氨基环戊醇的方法包括以下步骤：以R-3-羟基环戊酮为反应物,加入氨基供体、辅酶和转氨酶进行混合,并置于水相缓冲液中进行酶促反应,得到(1R,3S)-3-氨基环戊醇。同时本发明提供了催化R-3-羟基环戊酮生成(1R,3S)-3-氨基环戊醇的转氨酶的氨基酸序列,本公开内容还提供了编码转氨酶的核苷酸序列,制备方法及反应工艺,本发明公开的酶催化制备(1R,3S)-3-氨基环戊醇的反应条件温和,绿色环保,解决了常规化学法制备(1R,3S)-3-氨基环戊醇过程中存在的化学反应步骤多,操作繁琐,生产成本较高,且易造成环境污染严重的问题。(The invention specifically discloses a method for preparing (1R, 3S) -3-aminocyclopentanol, an integrase inhibitor and application, wherein the method for preparing the (1R, 3S) -3-aminocyclopentanol comprises the following steps: taking R-3-hydroxycyclopentanone as a reactant, adding an amino donor, coenzyme and transaminase for mixing, and placing the mixture in an aqueous phase buffer solution for enzymatic reaction to obtain (1R, 3S) -3-aminocyclopentanol. The invention also provides an amino acid sequence of the transaminase for catalyzing R-3-hydroxycyclopentanone to generate (1R, 3S) -3-aminocyclopentanol, and the disclosure also provides a nucleotide sequence for encoding the transaminase, a preparation method and a reaction process.)

1. A method for preparing (1R, 3S) -3-aminocyclopentanol, comprising the steps of: taking R-3-hydroxycyclopentanone as a reactant, adding an amino donor, coenzyme and transaminase for mixing, and placing the mixture in an aqueous phase buffer solution for enzymatic reaction to obtain (1R, 3S) -3-aminocyclopentanol.

2. The process for preparing (1R, 3S) -3-aminocyclopentanol according to claim 1, wherein said transaminase is an enzyme encoded by a transaminase gene;

wherein the nucleotide sequence of the transaminase gene is shown as SEQ ID No.1, and the amino acid sequence of the transaminase gene is shown as SEQ ID No. 2.

3. The production method according to claim 1, wherein the reaction temperature of the enzymatic reaction is 30 to 40 ℃.

4. The production method according to claim 3, wherein the temperature of the enzymatic reaction is 30 ℃.

5. The method according to claim 1, wherein the aqueous buffer is a phosphate buffer or a triethanolamine buffer.

6. The method of claim 5, wherein the aqueous buffer has a pH ranging from 6 to 9.

7. The method according to claim 6, wherein the aqueous buffer has a pH of 7.

8. The process according to claim 1, wherein the coenzyme is pyridoxal-5-phosphate, the amino donor is isopropylamine or an amino acid, and the transaminase is reacted in a reaction system in the form of at least one of wet cells of Escherichia coli, cell disruption supernatant or enzyme powder.

9. An integrase inhibitor prepared by the method for preparing (1R, 3S) -3-aminocyclopentanol according to any one of claims 1 to 7.

10. The integrase inhibitor according to claim 9, characterized in that (1R, 3S) -3-aminocyclopentanol is used for the preparation of a medicament for the treatment of HIV infection.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for preparing (1R, 3S) -3-aminocyclopentanol, an integrase inhibitor and application.

Background

The (1R, 3S) -3-aminocyclopentanol is an important medical intermediate and has very wide market prospect. The main active ingredient of a new anti-AIDS (Acquired Immune Deficiency Syndrome) drug Biktarrvy developed by Gillede science corporation is an integrase inhibitor Bictegravir. Bictegravir is obtained by constructing a bridged ring by using (1R, 3S) -3-aminocyclopentanol hydrochloride as a raw material, and is the only chiral source for forming a chiral molecule, so that the preparation of (1R, 3S) -3-aminocyclopentanol is one of the key steps for synthesizing Bictegravir.

At present, the preparation method of (1R, 3S) -3-aminocyclopentanol is generally prepared by a resolution method, and is partially prepared by using an oxidant (such as a carbamide peroxide-trifluoroacetic anhydride system) or a catalyst (such as a copper catalyst) and the like and performing multi-step reaction, for example, taking an amide formed by chiral carboxylic acid and hydroxylamine as a chiral source, obtaining a chiral diels-alder reaction product in a copper-catalyzed oxidation reaction system, and then obtaining a target product after reduction reaction, alkaline deprotection reaction and acidification reaction.

Therefore, the above technical solution has the following disadvantages in practical operation: most of the existing methods for preparing (1R, 3S) -3-aminocyclopentanol have complex chemical process operation, higher production cost and great environmental pollution.

Disclosure of Invention

The embodiment of the invention aims to provide a method for preparing (1R, 3S) -3-aminocyclopentanol, so as to solve the problems that most of the existing methods for preparing (1R, 3S) -3-aminocyclopentanol in the background art are complex in operation, high in production cost and high in environmental pollution.

In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

a process for preparing (1R, 3S) -3-aminocyclopentanol comprising the steps of:

taking R-3-hydroxycyclopentanone as a reactant, adding an amino donor, coenzyme and transaminase for mixing, and placing the mixture in an aqueous phase buffer solution for enzymatic reaction to obtain the (1R, 3S) -3-aminocyclopentanol.

As a further scheme of the invention: the transaminase is an enzyme encoded by a transaminase gene, wherein the nucleotide sequence of the transaminase gene is shown in SEQ ID No.1, and the amino acid sequence of the transaminase is shown in SEQ ID No. 2.

Specifically, the nucleotide sequence shown in SEQ ID No.1 is:

further, the amino acid sequence shown in SEQ ID No.2 is:

as still further aspect of the present invention, the method for preparing (1R, 3S) -3-aminocyclopentanol comprises: the specific synthetic route of the method for preparing (1R, 3S) -3-aminocyclopentanol from R-3-hydroxycyclopentanone is as follows:

preferably, the transaminase is a product from Ningbo enzyme Sai bioengineering, Inc., which is a protein recombinantly expressed in E.coli.

Further, the reaction temperature of the enzymatic reaction is 30 to 40 ℃, preferably 30 ℃.

Further, the coenzyme is pyridoxal 5-phosphate.

Further, the amino donor is isopropylamine or amino acid, etc., preferably, the amino donor is isopropylamine.

Further, the aqueous phase buffer solution is phosphate or triethanolamine buffer solution.

Further, the pH range of the aqueous phase buffer solution is 6-9, and the concentration is 0.08-0.12 mol/L.

Preferably, the concentration of the aqueous phase buffer solution is 0.1mol/L, and the pH of the aqueous phase buffer solution is 7.

Further, the transaminase used in the reaction system is escherichia coli wet cells, cell disruption supernatant or enzyme powder expressing the transaminase.

Further, the specific implementation process of the method for preparing the (1R, 3S) -3-aminocyclopentanol is as follows: adding the substrate R-3-hydroxycyclopentanone, transaminase, amino donor and coenzyme into a reactor, mixing in an aqueous buffer solution with pH of 6-9 and temperature of 30-40 ℃ to form a reaction system, carrying out reaction, inactivating with 50% acetonitrile, and detecting the reaction result by HPLC (high performance Liquid Chromatography).

Further, the total reaction volume of the reaction system is 4-6mL, and the input final concentration of each raw material is as follows: 3-5g/L of R-3-hydroxycyclopentanone substrate, 15-25g/L of transaminase, 0.05-1.15mmol/L of coenzyme, 0.4-0.6mol/L of amino donor and 0.08-0.12mol/L of aqueous buffer.

The invention provides the (1R, 3S) -3-aminocyclopentanol prepared by the method for preparing the (1R, 3S) -3-aminocyclopentanol.

Further, the (1R, 3S) -3-aminocyclopentanol can be used as a pharmaceutical intermediate.

The invention provides an integrase inhibitor prepared from the (1R, 3S) -3-aminocyclopentanol.

The invention provides application of the integrase inhibitor in preparing a medicine for treating HIV (human immunodeficiency Virus, namely AIDS Virus) infection.

Compared with the prior art, the invention has the beneficial effects that:

the method for preparing (1R, 3S) -3-aminocyclopentanol provided by the embodiment of the invention takes R-3-hydroxycyclopentanone as a reactant, and utilizes an amino donor, coenzyme, transaminase and the like to carry out enzymatic reaction to obtain (1R, 3S) -3-aminocyclopentanol, so that the synthesis steps are greatly simplified, the conversion rate can reach more than 95% at most, and the chiral value ee is more than or equal to 99%; in addition, because the raw materials such as transaminase and the like are adopted, the reaction condition is mild, the environment is protected, and the problems that in the prior art, the chemical reaction steps are more, most operations are complicated, the production cost is higher, and the environmental pollution is serious because a large amount of organic reagents are used in the process of preparing the (1R, 3S) -3-aminocyclopentanol by adopting a chemical method are solved.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.