CN112195202B

CN112195202B - Biocatalytic preparation method of S-indoline-2-carboxylic acid

Info

Publication number: CN112195202B
Application number: CN202010961795.8A
Authority: CN
Inventors: 薛吉军; 李毅; 余玉奎; 王志刚
Original assignee: Gansu Haotian Pharma Tech Co ltd
Current assignee: Gansu Haotian Pharma Tech Co ltd
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2022-07-05
Anticipated expiration: 2040-09-14
Also published as: CN112195202A

Abstract

The invention discloses a biocatalytic preparation method of S-indoline-2-carboxylic acid. The method comprises the following specific steps: carrying out alkylation reaction on the compound I and dialkyl malonate under alkaline conditions to obtain a compound II; aminolysis is carried out on the compound II to obtain a diamide compound III, and the compound is subjected to asymmetric amide hydrolysis reaction under the catalysis of penicillin acylase to obtain a stereospecific amide carboxylic acid compound IV; and (3) carrying out Hofmann degradation and intramolecular cyclization reaction on the compound IV to obtain a key intermediate S-indoline-2-carboxylic acid compound VI. The method is green, safe and economic, the optical selectivity of the product is strong, the chiral purity is more than 99 percent, and the concentration of the enzyme catalysis reaction substrate is high, so that the method is suitable for industrial production.

Description

Biocatalytic preparation method of S-indoline-2-carboxylic acid

Technical Field

The invention relates to the technical field of biological enzyme catalysis and organic synthesis, in particular to a preparation method of perindopril intermediate S-indoline-2-carboxylic acid.

Background

Perindopril (known under the trade name Acertil) is a long-acting ACE inhibitor and has a potent arterial vasodilating effect, not only improving the systemic circulation and cardiac function of patients with congestive heart failure, but also promoting redistribution of blood flow to the forearm and renal vascular bed. The preparation has the advantages of other ACE inhibitors, longer action time, smaller side effect, better tolerance and good market prospect.

S-indoline-2-carboxylic acid is an important intermediate for the synthesis of perindopril. Currently, the synthesis of S-indoline-2-carboxylic acid mainly comprises two routes of chemical synthesis and enzymatic synthesis. Chemical synthesis adopts chemical resolution and chemical asymmetric synthesis to prepare a chiral intermediate, and the two methods have various problems, such as the need of using a large amount of resolving agents for chemical resolution, long reaction steps, high energy consumption and large waste discharge; a catalytic hydrogenation system based on a transition metal chiral ligand has the problems of expensive catalyst, complex ligand synthesis and the like. This has caused the traditional chemical synthesis routes to be greatly impacted by environmental and cost issues.

Studies on the enzymatic synthesis of S-indoline-2-carboxylic acid have been reported since the past decade. At first, chiral resolution was carried out by using lipase instead of transition metal catalyst, but the problem was that the resolution efficiency was low and the other enantiomer could not be used although the contamination was greatly reduced. The subsequent research utilizes aryl cinnamic acid to carry out double bond ammonia addition reaction under the catalysis of cells with Phenylalanine Ammonia Lyase (PAL) activity to prepare chiral amino acid, and then S-indoline-2-carboxylic acid is synthesized through catalytic cyclization. Professor Yangtze Shunjiao having phenylalanine ammonia-lyase activity to obtain L-o-chlorophenylalanine with chiral purity of 96.4% (chem. 1997, 55, 196); disemann pharmaceutical company (WO2006069799) discloses that L-o-halophenylalanine is directly synthesized by catalyzing bacterial sludge with phenylalanine ammonia lyase activity expressed in Escherichia coli, and S-indoline-2-carboxylic acid is synthesized by catalyzing and cyclizing with cuprous chloride, and the chiral purity reaches 99%. However, in the research of chiral amino acid preparation by using phenylalanine ammonia lyase, the substrate concentration is low (less than 30g/L), the productivity is limited, and the industrial application of the chiral amino acid is limited.

The amidohydrolase has the advantages of high efficiency, specificity, good stereoselectivity and mild reaction conditions, and is more green, environment-friendly and economical than a chemical method and a biological enzyme catalysis method. However, since the enzyme has substrate specificity, the amidohydrolase which can stereoselectively hydrolyze the specific substrate compound III mentioned in the application has not been reported in the literature.

Disclosure of Invention

The invention aims to: aiming at the problems of expensive catalyst, complex ligand synthesis, low resolution efficiency and low substrate concentration in the existing preparation method, the invention provides a preparation method of perindopril intermediate S-indoline-2-carboxylic acid, which has high efficiency, specificity, good stereoselectivity and mild reaction conditions.

The synthetic route of the method is as follows:

the technical scheme adopted by the invention is as follows:

(1) and (3) reacting the dialkyl malonate with a compound I to obtain a compound II.

Wherein, the X group of the compound I is chlorine, bromine, iodine, methylsulfonyloxy or p-toluenesulfonyloxy. The R group of the compound II is methyl, ethyl, propyl, isopropyl or alkyl with less than five carbon atoms. The alkaline condition is sodium methoxide, sodium ethoxide, sodium hydrogen, sodium tert-butoxide, sodium hydroxide, potassium tert-butoxide, potassium hydroxide or potassium carbonate. The reaction solvent is methanol, ethanol, isopropanol, cyclohexane, toluene, tetrahydrofuran or dioxane.

(2) And (2) carrying out ammonolysis reaction on the compound II obtained in the step (1) to obtain a compound III.

Wherein the ammonolysis reaction condition is ammonia water or ammonia gas organic solvent solution; the organic solvent is methanol, ethanol, isopropanol, dioxane or tetrahydrofuran.

(3) And (3) carrying out enzymatic reaction on the compound III obtained in the step (2) in an aqueous solution reaction system under the catalysis of penicillin acylase to obtain a high-selectivity compound IV.

Wherein the concentration of the compound III is 50-1000 g/L; in the reaction system, the penicillin acylase is free enzyme, immobilized enzyme or enzyme in the form of thalli; the penicillin acylase gene is constructed on an expression vector, and the weight ratio of the bacterial dosage to the substrate is 0.01: 1; the reaction temperature range is 10-50 ℃, the reaction time is 2-24h, and the reaction pH is 7.5-9.5.

(4) And (4) degrading the compound IV obtained in the step (3) by Hofmann to obtain a compound V.

Wherein the reaction solvent is water, methanol, ethanol, toluene, dioxane, dichloromethane or any combination of the above solvents; the reaction temperature is 0-70 ℃, and the alkali used in the reaction is sodium hydroxide, potassium hydroxide or lithium hydroxide.

(5) And (3) carrying out intramolecular cyclization reaction on the compound V obtained in the step (4) under the catalysis of copper salt in the presence of an acid-binding agent to obtain S-indoline-2-carboxylic acid (VI).

Wherein the copper salt is cuprous chloride, cuprous bromide, cuprous iodide, cuprous oxide or cupric acetate; the acid-binding agent is sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium bicarbonate, potassium carbonate or potassium hydroxide; the reaction solvent is water, toluene, dioxane, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide or any combination of the above solvents; the reaction temperature range is 50-150 ℃.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) the novel biological enzyme catalysis reaction system derived from penicillin acylase (Escherichia coli, Ec) is utilized to carry out high-efficiency production through the biological catalysis selective deamination hydroxyl group conversion reaction.

(2) The method is suitable for industrial production, utilizes the high selectivity and specificity of the biological enzyme, converts the fixed acylamino into the carboxyl by single catalysis, generates the compound IV with high chiral purity, and then carries out subsequent reaction to produce the S-indoline-2-carboxylic acid.

(3) The method and the reaction system have high stereoselectivity, high catalytic activity and higher tolerance to the substrate, so that large-scale production can be carried out under extremely high substrate concentration; meanwhile, the production efficiency can be improved and the production cost can be reduced in production.

(4) Compared with a chemical synthesis method, the method obviously reduces or eliminates the use of various polluting chemicals, does not need to use expensive chiral resolving agents or noble metal catalysts, has low synthesis cost and obviously reduces the risk of environmental pollution.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The biological catalysis of S-indoline-2-carboxylic acid has the following chemical structural formula:

the synthetic route is as follows:

example 1

Construction of penicillin acylase engineering bacteria:

the PGA target gene and amidase gene are entrusted to a commercial company for complete gene synthesis, cloned into a pET28K (+) vector, transformed into escherichia coli DH5a competent cells, subjected to plate culture, a single colony of a positive transformant is selected, a plasmid determination series is extracted, a recombinant plasmid is extracted and introduced into a BL21(DE3) strain, and the strain is cultured in an LB culture medium to obtain the genetically engineered bacterium capable of inducing and expressing recombinant penicillium acylase.

Preparation of penicillin acylase:

inoculating the genetic engineering bacteria preserved in the glycerin pipe in the last step into an LB liquid culture medium (37 ℃, 180rpm, 12h) containing kanamycin sulfate, inoculating the obtained strain liquid into a fermentation embryo culture medium containing 50 mu g/ml kanamycin sulfate according to the proportion of 1.5%, culturing until OD600 is more than or equal to 30, and adding IPTG to induce fermentation for 12 h. The cells were collected by high speed centrifugation and prepared for microbial transformation.

The fermentation formula is as follows:

raw and auxiliary materials	Feeding proportion (g/L)
		Peptone	10
Yeast powder	5
		MgSO4·7H2O	3.63
KH2PO4	13.8
		KOH	2.5
Glucose	15
		Glycerol	6
Defoaming agent	0.5

Example 2

In the synthetic route shown in formula 1, X is a chlorine group, R is methyl, and alkali is sodium methoxide.

Preparation of compound ii:

methanol (2100mL) and dimethyl malonate (600g,4.54mol) were added to a reaction flask, and 25% sodium methoxide solution (1040mL,4.52mol) was added dropwise and stirred at room temperature for 30 min. Then controlling the temperature below 30 ℃, and dropwise adding o-chlorobenzyl chloride (488g,3.03 mol); the reaction was stirred for 1.5h, quenched with saturated ammonium chloride, extracted with ethyl acetate (3X 2000mL), the combined organic phases washed with saturated brine, dried and concentrated under reduced pressure to give compound II as an oil (778g, 83% yield).

The chlorine in this embodiment may also be selected from bromine, iodine, methanesulfonyloxy, or p-toluenesulfonyloxy.

The dimethyl malonate of the embodiment, wherein the R group is methyl, ethyl, propyl, isopropyl or alkyl with less than five carbon atoms can also be selected.

The sodium methoxide in this embodiment may also be sodium ethoxide, sodium hydride, sodium tert-butoxide, sodium hydroxide, potassium tert-butoxide, potassium hydroxide or potassium carbonate.

The solvent in this embodiment is methanol, and may also be ethanol, isopropanol, cyclohexane, toluene, tetrahydrofuran or dioxane.

Preparation of compound iii:

methanol (1480mL) and compound II (436g,1.70mol) were added to a reaction flask, and after dissolving with stirring, 34% aqueous ammonia (2400mL) was added. Stirring and reacting for 20h, and filtering; the filter cake was added to tert-butyl methyl ether (1500mL), refluxed for 30min, cooled, filtered and the filter cake was dried to give compound III (347g, 90% yield).

The ammonia water of the embodiment can also adopt ammonia organic solvent solution; the organic solvent can also be selected from methanol, ethanol, isopropanol, dioxane and tetrahydrofuran.

Preparation of Compound IV:

compound iii (100g,0.39mol) was added to a reaction flask, dissolved in 1000mL of phosphate buffer (0.02M, pH 7.8), 20g of the cell obtained by fermentation in example 1 was added, the temperature was controlled at 28 to 30 ℃, the reaction was stirred, and 0.1M sodium hydroxide was added to control pH 8.0. Stopping the reaction when the substrate residue is monitored by HPLC to be less than or equal to 5.0 percent; the reaction mixture was centrifuged at high speed, and the supernatant was taken, the pH was adjusted to 3.5 to 3.8 with 6M hydrochloric acid, and after precipitating a solid, the solid was filtered, washed with a small amount of ice water, and dried to obtain compound iv (77.2g, yield 87%).

Preparation of Compound V:

adding water and sodium hydroxide into a reaction bottle to prepare a 20% sodium hydroxide solution, stirring, cooling to 15-20 ℃, adding a compound IV (56.8g,0.25mol), continuously cooling to 0-5 ℃, starting to slowly dropwise add a 10% sodium hypochlorite solution (236g, 0.32mol), and controlling the temperature to 0-5 ℃. After the dropwise addition, the temperature is restored to 25-30 ℃, and the mixture is stirred. The temperature is raised to 50-55 ℃ and then the temperature is kept for 1.5 h. When the HPLC monitors that the intermediate is less than or equal to 1.0 percent, the temperature is reduced to 15 to 20 ℃, hydrochloric acid is used for adjusting the PH value to be 6.8 to 7.0, and the compound V (40.9g, the yield is 82 percent) is obtained after ultrafiltration, nanofiltration, concentration and crystallization.

Preparation of Compound VI:

purified water (750g) was added to the reaction flask, and potassium carbonate (83g, 0.6mol) was added and dissolved with stirring. Compound V (100g,0.5mol) is then added, and a catalytic amount of cuprous chloride (0.3g, 0.003mol) is added. Heating and refluxing, cooling to room temperature when the substrate residue is less than or equal to 1.0% by HPLC (high performance liquid chromatography), adjusting the pH value to 4.5-5.0 by hydrochloric acid, stirring for 30min, filtering the solution, washing a filter cake by ice water, and drying to obtain a compound VI (58.7g, yield 72%).

Cuprous chloride in this embodiment can also be cuprous bromide, cuprous iodide, cuprous oxide, or cupric acetate.

The potassium carbonate in this embodiment may also be sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium bicarbonate, or potassium hydroxide.

The water in this embodiment may also be toluene, dioxane, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, or any combination of the above solvents.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, substitutions and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A biocatalytic preparation method of S-indoline-2-carboxylic acid is characterized by comprising the following steps:

(1) reacting dialkyl malonate in a reaction solvent under an alkaline condition to generate dialkyl malonate alkali metal salt, and reacting the dialkyl malonate alkali metal salt with a compound I to obtain a compound II;

(2) carrying out ammonolysis reaction on the compound II obtained in the step (1) in a reaction solvent to obtain a compound III;

(3) carrying out enzymatic reaction on the compound III obtained in the step (2) in an aqueous solution reaction system under the catalysis of penicillin acylase to obtain a high-selectivity compound IV, wherein the penicillin is derived from penicillin acylase PGA of escherichia coli; the reaction temperature is 10-50 ℃; the reaction time is 2-24 h; the reaction pH is 7.5-9.5;

(4) dissolving the compound IV obtained in the step (3) in a reaction solvent, and performing Hofmann degradation under an alkaline condition to obtain a compound V; the reaction temperature is 0-70 ℃;

(5) carrying out intramolecular cyclization reaction on the compound V obtained in the step (4) in a reaction solvent under the catalysis of copper salt in the presence of an acid-binding agent to obtain S-indoline-2 carboxylic acid (VI); the X group in the compound I is chlorine; the R group of the compound II is methyl, ethyl, propyl, isopropyl or alkyl with less than five carbon atoms; the reaction temperature is 50-150 ℃;

2. the method according to claim 1, wherein the basic conditions in step (1) are performed using sodium methoxide, sodium ethoxide, sodium hydrogen, sodium tert-butoxide, sodium hydroxide, potassium tert-butoxide, potassium hydroxide or potassium carbonate; the reaction solvent is methanol, ethanol, isopropanol, cyclohexane, toluene, tetrahydrofuran or dioxane.

3. The preparation method according to claim 1, wherein the reagent used in the ammonolysis reaction in step (2) is ammonia water or ammonia gas organic solvent solution; the reaction solvent is methanol, ethanol, isopropanol, dioxane or tetrahydrofuran.

4. The process according to claim 1, wherein the penicillin acylase in the step (3) is an enzyme in a free form, an immobilized enzyme or an enzyme in a bacterial form.

5. The process according to claim 1, wherein the concentration of the compound III in the step (3) is 50 to 1000 g/L; the weight ratio of the dosage of the penicillin acylase thallus to the substrate is 0.01: 1.

6. The method according to claim 1, wherein the reaction solvent in step (4) is water, methanol, ethanol, toluene, dioxane, dichloromethane or any combination thereof; the alkali used in the alkaline condition is sodium hydroxide, potassium hydroxide or lithium hydroxide.

7. The process according to claim 1, wherein the copper salt in the step (5) is cuprous chloride, cuprous bromide, cuprous iodide, cuprous oxide or cupric acetate; the acid-binding agent is sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium bicarbonate, potassium carbonate or potassium hydroxide; the reaction solvent is water, toluene, dioxane, N-dimethylformamide, N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide or any combination of the above solvents.