CN102409068A - A kind of preparation method of (3aS,6aR)-biotin chiral lactone - Google Patents

Abstract

The invention discloses a method for preparing a (3a S , 6a R )-biotin chiral lactone, which is characterized in that esterase is produced by fermentation with Microbacteriumchocolatum SIT101CGMCCNO.4436, and then meso-dimethyl ester is used as a substrate for the enzyme conversion. The product in the enzyme conversion reaction liquid is acidified, solvent extracted, concentrated, and dried to obtain (4 S , 5 R )-semimethyl ester, which is then subjected to reduction reaction with a reducing agent, sodium borohydride or potassium borohydride, in anhydrous ethanol for 18-30 hours. The obtained reduction product is lactonized, precipitated, and dried to finally obtain (3a S , 6a R )-biotin chiral lactone. The preparation method of the invention has the characteristics of mild reaction conditions, high catalytic efficiency, high final product yield, and low production cost, and at the same time has low energy consumption, material consumption, and three wastes output, and is suitable for large-scale production.

Description

A kind of preparation method of (3aS,6aR)-biotin chiral lactone

technical field

The present invention relates to a preparation method of (3a S ,6a R )-biotin chiral lactone, in particular to a method for producing enzyme by fermentation using Microbacterium chocolatum SIT101 CGMCC NO.4436, and performing enzyme-catalyzed internal Asymmetric hydrolysis of racemic diester to prepare (4 S ,5 R )-hemimethyl ester, and hemimethyl ester is reduced by reducing agent and lactonized to obtain (3a S , 6a R )-biotin chiral lactone.

Background technique

It is known that (3a S , 6a R )-biotin chiral lactone shown in formula (I) is a key chiral intermediate for the synthesis of d -biotin ( d -Biotin, also known as vitamin H and coenzyme R), which Structural formula (I) is as follows:

d -Biotin, also known as vitamin H, is one of the water-soluble B vitamins. It is a coenzyme of carboxylase and an important coenzyme in the intermediate metabolism of sugar, protein and fat. Except some bacteria, yeast and plants can synthesize biotin by themselves, almost all animals and some microorganisms need to obtain biotin from exogenous sources. Biotin has a wide range of uses, 20-30% is used for medical raw materials, 50% is used for feed, and it is also used as cosmetic raw materials and nutritional additives.

The reported synthesis methods of (3a S ,6a R )-biotin chiral lactone mainly include resolution method, asymmetric synthesis and biosynthesis.

Chemical resolution, such as using optically active menthol or chiral amine ephedrine to resolve precursor substances, and then converting to obtain D-biotin intermediate lactone. The disadvantages of the chemical resolution method are that the resolution agent is expensive, the separation is not complete, and there are environmental pollution and toxicity problems. Physical separation, such as the use of induced crystallization to resolve the precursor hemimethyl ester, and then reduction to obtain the D-biotin intermediate lactone, requires high condition control, poor optical purity, and low yield. the

At present, the industry mainly adopts the method of reducing asymmetric cyclic imide to prepare (3a S ,6a R )-biotin chiral lactone (Tetrahedron. Lett., 1999, 40, 8873-8876.). During the synthesis process, a chiral amine with the same molar equivalent as the substrate is introduced to form a chiral cyclic imide with a diacid substrate, and then undergoes chemical reduction and lactonization ring closure to obtain a biotin chiral lactone. Because the method introduces expensive chiral amines, the cost is high, and the treatment of the three wastes is relatively complicated.

In recent years, there are also literatures reporting the method of biocatalytic preparation. For example, Japanese scholars (Agric. Biol Chem., 1982, 46(7), 1907) used pig liver esterase to asymmetrically hydrolyze prochiral dipropyl ester to obtain optically active monoester, and then reduced it with lithium borohydride to obtain D-biotin Intermediate lactone, the yield is 85%, and the ee value is 75%. Chen Fen'er et al. (Adv.Synth.Catal.2005,347,549) used immobilized porcine liver esterase to catalyze the hydrolysis of prochiral dimethyl esters, and the product chiral hemimethyl esters (ee value 91%) were recrystallized and chemically reduced The chiral lactone is obtained after lactonization, and the yield is about 60%. The method of using esterase to catalyze the asymmetric hydrolysis of meso diester can theoretically obtain optically pure monoester products with 100% yield, which overcomes the theoretical limitation of 50% of traditional kinetic resolution and avoids the traditional resolution. Disadvantages of chemical racemization are required to divide the remaining substrate. Compared with the method of chemical reduction of asymmetric cyclic imide, there is no need to introduce chiral amine, so it will effectively reduce the cost of production materials and the content of organic matter in production wastewater, combined with the mild reaction conditions and high selectivity unique to enzyme catalysis , low energy consumption and other advantages, is a promising new method to replace the existing chemical process. The main problem existing at present is that the selectivity of pig liver esterase is not high enough, and the obtained hemimethyl ester needs to be recrystallized before it can be chemically reduced, which leads to a low yield of chiral lactone, only about 60%, and pig liver Liver esterase is expensive, and its animal origin limits the large-scale acquisition and use of catalysts, thereby limiting the large-scale production of (3a S ,6a R )-biotin chiral lactone.

Contents of the invention

The purpose of the present invention is to provide a preparation method of (3a S ,6a R )-biotin chiral lactone in order to solve the above technical problems, that is to use Microbacterium chocolate SIT101 CGMCC NO.4436 as a biocatalyst with high selectivity Catalyzed asymmetric hydrolysis of meso dimethyl ester (III) to produce (4 S ,5 R )-hemimethyl ester (II), the obtained (4 S ,5 R )-hemimethyl ester (II) was chemically reduced, internally Esterification into (3a S ,6a R )-biotin chiral lactone (I).

Wherein the structural formula of (4 S ,5 R )-hemimethyl ester (II) is as follows:

The structural formula of meso dimethyl ester (III) is as follows:

。

.

The technical scheme adopted in the present invention

A method for preparing (3aS, 6aR)-biotin chiral lactone, namely using Microbacterium chocolatum SIT101 CGMCC NO.4436 with high esterase yield Strain Preservation Management Committee General Microbiology Center) to produce esterase by fermentation, and then use meso-dimethyl ester as the substrate for the esterase conversion, and the product in the esterase conversion reaction solution is acidified, solvent extracted, concentrated, and dried to obtain ( 4S,5R)-hemimethyl ester (II), (4S,5R)-hemimethyl ester (II) and reducing agent sodium borohydride or potassium borohydride in absolute ethanol for 18-30h, the resulting reduction product After lactonization, precipitation and drying, the (3aS,6aR)-biotin chiral lactone (I) with high optical purity was finally obtained.

Description of drawings

The reaction schematic diagram of the above (3a S ,6a R )-biotin chiral lactone preparation process is shown in Fig. 1 .

The (3a S ,6a R )-biotin chiral lactone obtained by the above preparation method has an ee value of 98.0% and a melting point of 121.0-121.7°C. (C=1, chloroform).

Technical effect of the present invention

A preparation method of (3a S , 6a R )-biotin chiral lactone of the present invention, that is, the intermediate product (4 S , 5 R )-hemimethyl ester is produced by microbial enzyme hydrolysis, and then reacted with a reducing agent and then internally The method of esterification reaction to generate (3a S ,6a R )-biotin chiral lactone, due to the use of highly active and selective microbial esterase as a catalyst, the reaction conditions are mild, the catalytic efficiency is high, and the optical purity of hemimethyl ester is high , can be directly reduced by a reducing agent without crystallization, and (3a S , 6a R )-biotin chiral lactone with high optical purity can be generated after lactonization with a high yield. Therefore, it has lower energy consumption, material consumption and output of three wastes than the existing chemical method.

Moreover, the preparation method of a (3a S , 6a R )-biotin chiral lactone of the present invention does not need to add any chiral resolving agent, which further saves the cost. Compared with the method of preparing (3a S , 6a R )-biotin chiral lactone by using pig liver esterase, the method for preparing (3a S , 6a R )-biotin chiral lactone of the present invention has the advantages of low production cost and can be selected Higher in nature and can be mass-produced at a lower cost, so it has higher application value than the pig liver esterase method.

Figure 1. Schematic diagram of the preparation process of (3a S ,6a R )-biotin chiral lactone.

Detailed ways

The present invention is described in further detail below by way of examples, but the present invention is not limited. the

The reagents used in the present invention are all analytically pure or chemically pure. the

The preparation method of the meso-dimethyl ester used in the present invention :

The meso-diacid (28.32g, 0.08mol), anhydrous methanol (16.2mL, 0.4mol), concentrated sulfuric acid (0.4mL, 7.5mmol) and benzene (65mL) were heated to reflux for 6 h under stirring conditions, and The solvent was removed by pressure, and water (40mL) and ethyl acetate (40mL) were added to the residue. After the organic phase was separated, the aqueous phase was extracted three times with ethyl acetate (10mL). The ethyl acetate phases were combined and washed with 2M Na ₂ CO ₃ (3 times in total, 5ml each time) and water (4 times in total, 5ml each time) for washing. After drying with anhydrous sodium sulfate, the solvent was removed under reduced pressure to obtain meso-dimethyl ester (III), with a yield of 92% and a purity of 99%.

The detection method of SR hemimethyl ester : chiral liquid chromatography (the chromatographic column is Chiralcel OJ-H chromatographic column, the mobile phase is n-hexane:isopropanol:trifluoroacetic acid=92:8:0.1, the ultraviolet detection wavelength is 220nm, the flow rate 0.5ml/min);

(3a S ,6a R )-lactone detection method: Chiral liquid chromatography (the chromatographic column is a Chiralcel OD-H chromatographic column, the mobile phase is n-hexane:isopropanol=80:20, the ultraviolet detection wavelength is 210nm, and the flow rate is 1.0ml/min);

The yield of (4 S ,5 R )-hemimethyl ester was calculated using meso-dimethyl ester as substrate;

The yield of (3a S , 6a R )-biotin chiral lactone was calculated using (4 S ,5 R )-hemimethyl ester as substrate.

Example 1

A preparation method of (3a S , 6a R )-biotin chiral lactone, comprising the following preparation steps:

(1) Preparation of (4 S ,5 R )-hemimethyl ester (II)

① Incline culture of strains

Take the Microbacterium chocolatum SIT101 CGMCC NO.4436 strain stored in the refrigerator at 4°C, take it out, inoculate it on the slant medium, and cultivate it at 30 ^o C for 48 hours to obtain the slant seeds of Microbacterium chocolatum SIT101 , and store it in a refrigerator at 4°C;

The slant medium: glucose 15.0 g/L, yeast extract 5.0 g/L, peptone 10.0 g/L, MgSO ₄ 1g/L, agar 15-20 g/L, pH7.0;

②, Enzyme production fermentation culture

Take Microbacterium chocolatum SIT101 CGMCC NO.4436 slant seeds preserved at 4 ^o C, pick a loop and inoculate it into a 250ml shaker flask containing 50ml of fermentation medium, and culture it at 30°C with shaking at 160rpm for 48h. Centrifuge at 10,000 g for about 15 minutes to obtain resting cells of Microbacterium chocolatum SIT101 containing esterase;

The fermentation medium: glucose 15.0g/L, yeast extract 5.0g/L, peptone 10.0g/L, MgSO ₄ 1g/L, pH7.0;

③. Preparation of (4 S ,5 R )-hemimethyl ester (II)

Take the esterase-containing Microbacterium chocolatum ( Microbacterium chocolatum ) SIT101 resting cells obtained in step (2) with a wet weight of 7 g, suspend them in 95 ml of sodium phosphate buffer solution with a concentration of 0.1 M, pH 8.0, and add internal Spin 7 mmol of dimethyl ester, then add 5 ml of dimethyl sulfoxide, shake the reaction mixture on a constant temperature shaker at 30 °C and 160 r/min for 48 h, add 2 mol/L hydrochloric acid to adjust the pH value of the water phase to 2.0, and use ethyl acetate Ester extraction acidified liquid 3 times, the consumption of each ethyl acetate is 30ml, combine organic phase and dry with anhydrous sodium sulfate, then evaporate organic phase under reduced pressure to obtain (4 S , 5 R )-hemimethyl ester, its yield 88%, the purity is 98%, and the ee value is greater than 99%;

(2) Preparation of (3a S ,6a R )-biotin chiral lactone

Dissolve 0.78g (2mmol) of ( 4S , 5R )-hemimethylester and 0.22g (2mmol) of anhydrous calcium chloride in 10mL of absolute ethanol, and then control the temperature at 0°C, add 0.23g (6mmol) sodium borohydride, remove the ice bath after the addition is complete, stir the reaction at room temperature for 30h, raise the temperature to 55~60°C, add 5% hydrochloric acid solution 6mL, react for 30min, TLC monitors the reaction process, and waits until the bottom After the product was completely reacted, 10 mL of distilled water was added, and then cooled in an ice-water bath, a colorless precipitate appeared, and after filtration and drying, 0.52 g of (3a S , 6a R )-biotin chiral lactone was finally obtained, with a yield of 89%. , ee value is 97.8%.

Example 2

A preparation method of (3a S , 6a R )-biotin chiral lactone, comprising the following preparation steps:

(1), the preparation of (4 S ,5 R )-hemimethyl ester (II), the same as in Example 1;

(2) Preparation of (3a S ,6a R )-biotin chiral lactone

(C=1,氯仿)。 Take (4 S ,5 R )-hemimethyl ester 7.8g (20mmol) and anhydrous calcium chloride 2.22g (20mmol) prepared by the method of step (1) and dissolve them in 100mL absolute ethanol, then control the temperature at 0°C, add 3.2g (60mmol) potassium borohydride, remove the ice bath after the addition, stir and react at room temperature for 18h, heat up to 55~60°C, add 60mL of 5% hydrochloric acid solution, react for 30min, monitor the reaction progress by TLC, wait until the bottom After the product was completely reacted, 100 mL of distilled water was added, and then cooled in an ice-water bath, a colorless precipitate appeared, and after filtration and drying, 5.3 grams of (3a S , 6a R )-biotin chiral lactone was finally obtained, with a yield of 91%. , ee value is 98.0%, melting point is 121.0-121.7℃,

(C=1, chloroform).

The above content is only a basic description of the concept of the present invention, and any equivalent transformation made according to the technical solution of the present invention shall fall within the scope of protection of the present invention. the

Claims (7)

1. microbial enzyme manufactured (3a S, 6a RThe method of)-lactone is characterized in that comprising the steps:

(1), the production of esterase

Adopt chocolate microbacterium ( Microbacterium chocolatum) SIT101 CGMCC NO.4436 fermentative prodn esterase;

(2), intermediate product (4 S, 5 RThe preparation of)-half methyl esters

With the meso dimethyl ester is the substrate that the esterase of step (1) gained transforms, the product in the esterase conversion reaction liquid through acidifying, SX, concentrate, after the drying intermediate product (4 S, 5 R)-half methyl esters;

(3), (3a S, 6a RThe preparation of)-vitamin H asymmetric lactone

The intermediate product (4 of step (2) gained S, 5 R)-half methyl esters carries out reduction reaction with borane reducing agent sodium hydride or POTASSIUM BOROHYDRIDE 97MIN again in absolute ethyl alcohol, the gained reduzate through lactonize, precipitate and drying after finally obtain (the 3a of high-optical-purity S, 6a R)-vitamin H asymmetric lactone.

2. a kind of microbial enzyme manufactured (3a as claimed in claim 1 S, 6a RThe method of)-lactone is characterized in that the production of the esterase described in the step (1):

Be about to get 4 ℃ of preservations chocolate microbacterium ( Microbacterium chocolatum) SIT101 CGMCC NO.4436 inclined-plane seed is inoculated in the fermention medium, under 30 ℃, 48h is cultivated in the 160rpm jolting, with 10000 gCentrifugal 15min promptly obtain containing said esterase chocolate microbacterium ( Microbacterium chocolatum) the SIT101 resting cell.

3. a kind of microbial enzyme manufactured (3a as claimed in claim 2 S, 6a RThe method of)-lactone, its spy

Levying fermention medium used in the production process that is the esterase described in the step (1) is glucose 15.0g/L, yeast extract paste 5.0g/L, peptone 10.0g/L, MgSO ₄1g/L, pH7.0.

4. like claim 1,2 or 3 described a kind of microbial enzyme manufactured (3a S, 6a RThe method of)-lactone,

It is characterized in that (4 described in the step (2) S, 5 RThe preparation of)-half methyl esters:

Be about to contain esterase chocolate microbacterium ( Microbacterium chocolatum) the SIT101 resting cell, being suspended in concentration is 0.1M, in the sodium phosphate salt buffered soln of pH8.0, adds meso dimethyl ester and DMSO 99.8MIN., gets reaction mixture; Again with the reaction mixture of gained behind jolting reaction 48h on 30 ℃, the constant temperature shaking table of 160r/min, use the ethyl acetate extraction acidizing fluid, organic phase with anhydrous sodium sulfate drying afterwards evaporated under reduced pressure obtain (4 S, 5 R)-half methyl esters.

5. a kind of microbial enzyme manufactured (3a as claimed in claim 4 S, 6a RThe method of)-lactone is characterized in that (4 described in the step (2) S, 5 RContain in the reaction mixture described in the preparation process of)-half methyl esters esterase chocolate microbacterium ( Microbacterium chocolatum) weight in wet base of SIT101 resting cell is 70g/L, meso dimethyl ester concentration is 10 ~ 20g/L, the content of DMSO 99.8MIN. is 5% (V/V) in the damping fluid.

6. a kind of microbial enzyme manufactured (3a as claimed in claim 5 S, 6a RThe method of)-lactone is characterized in that the described (3a of step (3) S, 6a RThe preparation of)-vitamin H asymmetric lactone:

Be under the condition of ice bath, with (4 of Calcium Chloride Powder Anhydrous and gained S, 5 R)-half methyl esters is dissolved in the absolute ethyl alcohol, adds borane reducing agent sodium hydride or POTASSIUM BOROHYDRIDE 97MIN, then under the room temperature behind the stirring reaction 18-30h; Add hydrochloric acid soln and carry out lactonization reaction, add zero(ppm) water behind the lactonization reaction again, ice bath cooling deposition; Filter back gained filter cake through vacuum-drying, promptly get (3a S, 6a R)-vitamin H asymmetric lactone.

7. a kind of microbial enzyme manufactured (3a as claimed in claim 6 S, 6a RThe method of)-lactone is characterized in that the described (3a of step (3) S, 6a RIn the preparation process of)-vitamin H asymmetric lactone (4 S, 5 RThe proportioning of)-half methyl esters, Calcium Chloride Powder Anhydrous, reductive agent and absolute ethyl alcohol is calculated by the mole volume ratio, and promptly (4 S, 5 R)-half methyl esters: Calcium Chloride Powder Anhydrous: reductive agent: absolute ethyl alcohol is 1mol:1mol:3mol:4 ~ 5L.