CN114540321A

CN114540321A - Preparation method of R-2-sulfonyl-1-phenylethanol derivative

Info

Publication number: CN114540321A
Application number: CN202210102497.2A
Authority: CN
Inventors: 游松; 秦斌; 张文鹤; 郭继阳; 高晓; 钱东
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2022-01-27
Filing date: 2022-01-27
Publication date: 2022-05-27
Anticipated expiration: 2042-01-27
Also published as: CN114540321B

Abstract

The invention discloses a preparation method of an R-2-sulfonyl-1-phenyl ethanol derivative, belonging to the field of synthesis of pharmaceutical and chemical intermediates and the field of biotechnology. The method takes 2-sulfonyl-1-phenyl ethanone compounds as initial substrates, takes R-selective ketoreductase (R-KRED) from Lactobacillus fermentum as a chiral biocatalyst, and takes Glucose Dehydrogenase (GDH) from Bacillus subtilis as a circulating cofactor NDAPH to asymmetrically reduce substrate carbonyl, thus obtaining R-type product alcohol; the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 1. The method utilizes the biocatalyst, has mild reaction conditions, avoids the catalysis of transition metal and the use of complex chiral ligands, is green and environment-friendly, has large substrate loading capacity and high conversion rate and stereoselectivity, and is beneficial to large-scale industrial production.

Description

Preparation method of R-2-sulfonyl-1-phenylethanol derivative

Technical Field

The invention belongs to the field of synthesis of pharmaceutical and chemical intermediates and the field of biotechnology, and relates to a preparation method of an R-2-sulfonyl-1-phenylethanol derivative.

Background

Chiral 2-sulfonyl-1-phenylethanol compounds are useful components in organic chemistry and have been widely used in the synthesis of pharmaceutical molecules and biologically active compounds, such as gamma-butenolide, gamma-butyrolactone, 2, 5-disubstituted tetrahydrofuran delta-valerolactone. Meanwhile, the compound can be used as a drug intermediate for drug synthesis.

Thus, several methods of synthesizing these compounds have been developed over the last two decades, such as asymmetric conjugated boronation of α, β -unsaturated sulfones and asymmetric reduction of β -ketosulfones, including borane reduction, transition metal catalyzed asymmetric hydrogenation, and asymmetric transfer hydrogenation. However, the generality of these processes is often plagued by high pressure conditions, the use of complex chiral ligand catalysts, or heavy metal contamination.

Ketone Reductase (KRED), also known as carbonyl reductase or alcohol dehydrogenase, is an enzyme that stereoselectively reduces a prochiral ketone to a chiral secondary alcohol using reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) or reduced Nicotinamide Adenine Dinucleotide (NADH) as a hydrogen donor. With the rapid development of protein engineering, including site-directed saturation mutagenesis and random mutagenesis, tailored KREDs have become the catalysts of choice for ketone reduction by many pharmaceutical companies and are increasingly used in the synthesis of key chiral alcohol intermediates for use in pharmaceuticals, such as montelukast sodium and atorvastatin calcium. Ketoreductases as biocatalysts not only possess the advantages of high stereoselectivity and high catalytic efficiency, but also: the reaction can be carried out in aqueous solution, and the reaction condition is mild; the enzyme is easy to prepare and degradable, and is green and environment-friendly; and metal and complex ligand catalyst are not needed, so that subsequent product purification is facilitated.

Disclosure of Invention

Aiming at the defects of the existing chemical catalysis method, the invention provides a preparation method of an R-2-sulfonyl-1-phenylethanol derivative. 2-sulfonyl-1-phenyl ethyl ketone compounds are used as initial substrates, R-selective ketoreductase (R-KRED) from Lactobacillus fermentum is used as a chiral biocatalyst, and Glucose Dehydrogenase (GDH) circulating cofactor NDAP from Bacillus subtilis is used to asymmetrically reduce substrate carbonyl, so that R-type product alcohol is obtained. The method utilizes the biocatalyst, has mild reaction conditions, avoids the catalysis of transition metal and the use of complex chiral ligands, is green and environment-friendly, has large substrate loading capacity and high conversion rate and stereoselectivity, and is beneficial to large-scale industrial production.

A biocatalyst comprises ketoreductase, glucose dehydrogenase, and NADP⁺Or NAD⁺And glucose, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 1.

Further, in the above technical means, the ketoreductase: glucose dehydrogenase: NADP⁺Or NAD⁺: the glucose is 0.02-40 g/L: 0.02-40 g/L: 0-0.5 mmol: 6-200 g/L.

Namely, the dosage of the ketoreductase is 0.02-40g/L (freeze-dried cells), the dosage of the glucose dehydrogenase is 0.02-40g/L (freeze-dried cells), the dosage of NADP + is 0-0.5mmol, and the dosage of glucose is 6-200 g/L.

The application of the biocatalyst in the catalytic preparation of the R-2-sulfonyl-1-phenylethanol derivative is characterized in that the 2-sulfonyl-1-phenylethanone derivative is used as an initial substrate, and under the action of the biocatalyst, a substrate carbonyl group is asymmetrically reduced into a hydroxyl group to obtain the R-2-sulfonyl-1-phenylethanol derivative.

Further, in the technical scheme, the structural formula of the 2-sulfonyl-1-phenyl ethyl ketone derivative is shown as a formula A, and the structural formula of the R-2-sulfonyl-1-phenyl ethyl alcohol derivative is shown as a formula B;

in the formula, R₁Is mono-or poly-substituted C1-C4 alkyl, or C1-C4 alkoxy, or halogen; r₂Is C1-C4 alkyl or phenyl.

Further, in the technical scheme, the pH value of the catalytic reaction is 5.5-8. Preferably 7.0.

Further, in the technical scheme, the temperature of the catalytic reaction is 28-45 ℃. Preferably 30 deg.c.

Further, in the above technical scheme, the concentration of the substrate is 0.5-200 g/L.

Further, in the above technical scheme, the 2-sulfonyl-1-acetophenone derivative is selected from any one of the following:

a biocatalytic preparation method of an R-2-sulfonyl-1-phenylethanol derivative comprises the following steps:

the structural compound shown in the formula A is asymmetrically reduced into an R-2-sulfonyl-1-phenylethanol derivative in a buffer salt solution under the catalysis of R-type ketoreductase, and a cofactor NADPH in a reaction system is catalyzed by glucose dehydrogenase;

wherein the compound R has a structure shown in formula A₁Is mono-or poly-substituted C1-C4 alkyl, C1-C4 alkoxy, or halogen;

R₂is C1-C4 alkyl or phenyl.

The buffer salt solution is selected from phosphate, sodium citrate and phosphate, and is preferably phosphate.

The R-2-sulfonyl-1-phenyl ethanol derivative biocatalytic preparation method has the advantages of mild reaction conditions, environmental friendliness, high enantiomer selectivity of the product and the like.

Drawings

FIG. 1 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 4 of the present invention; a is racemic 2-methylsulfonyl-1- (3-ethoxy-4-methoxyphenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (3-ethoxy-4-methoxyphenyl) ethanol.

FIG. 2 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 5 of the present invention. A is racemic 2-methylsulfonyl-1-phenyl ethanol standard control; b is R-2-methylsulfonyl-1-phenylethanol.

FIG. 3 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 6 of the present invention. A is racemic 2-methylsulfonyl-1- (4-methylphenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (4-methylphenyl) ethanol.

FIG. 4 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 7 of the present invention. A is racemic 2-methylsulfonyl-1- (3-methoxyphenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (3-methoxyphenyl) ethanol.

FIG. 5 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 8 of the present invention. A is racemic 2-methylsulfonyl-1- (4-methoxyphenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (4-methoxyphenyl) ethanol.

FIG. 6 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 9 of the present invention. A is racemic 2-methylsulfonyl-1- (4-fluorophenyl) ethanol standard substance contrast; b is R-2-methylsulfonyl-1- (4-fluorophenyl) ethanol.

FIG. 7 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 10 of the present invention. A is racemic 2-methylsulfonyl-1- (3-chlorphenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (3-chlorophenyl) ethanol.

FIG. 8 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 11 of the present invention. A is racemic 2-methylsulfonyl-1- (4-chlorphenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (4-chlorophenyl) ethanol.

FIG. 9 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 12 of the present invention. A is racemic 2-methylsulfonyl-1- (4-bromophenyl) ethanol standard control; b is R-2-methylsulfonyl-1- (4-bromophenyl) ethanol.

FIG. 10 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 13 of the present invention. A is racemic 2-benzenesulfonyl-1-phenyl ethanol standard control; b is R-2-benzenesulfonyl-1-phenyl ethanol.

FIG. 11 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 14 of the present invention. A is racemic 2-benzenesulfonyl-1- (4-methylphenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (4-methylphenyl) ethanol.

FIG. 12 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 15 of the present invention. A is racemic 2-benzenesulfonyl-1- (2-methoxyphenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (2-methoxyphenyl) ethanol.

FIG. 13 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 16 of the present invention. A is racemic 2-benzenesulfonyl-1- (3-methoxyphenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (3-methoxyphenyl) ethanol.

FIG. 14 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 17 of the present invention. A is racemic 2-benzenesulfonyl-1- (4-methoxyphenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (4-methoxyphenyl) ethanol.

FIG. 15 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 18 of the present invention. A is racemic 2-benzenesulfonyl-1- (4-fluorophenyl) ethanol standard substance reference; b is R-2-benzenesulfonyl-1- (4-fluorophenyl) ethanol.

FIG. 16 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 19 of the present invention. A is racemic 2-benzenesulfonyl-1- (3-chlorphenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (3-chlorphenyl) ethanol.

FIG. 17 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 20 of the present invention. A is racemic 2-benzenesulfonyl-1- (4-chlorphenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (4-chlorophenyl) ethanol.

FIG. 18 is a chiral HPLC chromatogram of the R-2-methanesulfonyl-1-phenylethanol derivative synthesized in example 21 of the present invention. A is racemic 2-benzenesulfonyl-1- (4-bromophenyl) ethanol standard control; b is R-2-benzenesulfonyl-1- (4-bromophenyl) ethanol.

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.

The compound R-2-methylsulfonyl-1-phenyl ethanol derivative of the formula B is obtained by asymmetrically reducing the compound of the formula A through ketoreductase. And circulating the accessory factor NADPH under the action of glucose dehydrogenase and glucose:

wherein R is₁Is mono-or poly-substituted C1-C4 alkyl, C1-C4 alkoxy, or halogen;

R₂is C1-C4 alkyl or phenyl.

LB culture medium: 0.5% yeast extract, 1% peptone and 1% sodium chloride (for example, preparing solid medium, adding 1.5% agar before sterilization), and autoclaving at 115 deg.C for 30 min.

As an optimum implementation process, only the ketoreductase whose amino acid sequence is shown in SEQ ID No.1 is exemplified for specific implementation below.

Example 1 specific cultural preparation of ketoreductases:

adding 1mL of recombinant Escherichia coli BL21(DE3) overnight culture solution containing ketoreductase gene into 100mL of LB culture medium, and culturing at 30 ℃ and 220rpm until OD600 is 0.8-1; the ketoreductase was then expressed by induction with the addition of the inducer IPTG (final concentration 0.1mM) and induced at 220rpm for 12h at 18 ℃. Finally, after centrifugation at 3000rpm for 10min, the supernatant was decanted and the wet bacterial pellet containing ketoreductase was collected. The amino acid sequence of the ketoreductase is shown as SEQ ID NO. 1.

Example 2 specific culture preparation of glucose dehydrogenase:

1mL of an overnight culture of recombinant Escherichia coli BL21(DE3) containing a glucose dehydrogenase gene (NCBI database No. WP _044161863.1) was added to 100mL of LB medium and cultured at 30 ℃ and 220rpm until OD600 became 0.8 to 1; the glucose dehydrogenase was then induced to express by the addition of the inducer IPTG (final concentration 0.1mM) and induced at 220rpm at 18 ℃ for 12 h. Finally, after centrifugation at 3000rpm for 10min, the supernatant was decanted and the wet pellet containing glucose dehydrogenase was collected.

Example 3 cell disruption preparation of crude enzyme solution:

4g of ketoreductase wet thallus precipitate (example 1) and 0.8g of glucose dehydrogenase wet thallus precipitate (example 2) are respectively taken, 20mL of 100mM sodium phosphate buffer solution with pH 7.0 is added to resuspend the thallus, ultrasonic disruption is carried out in an ice water bath under the conditions of power of 55 percent and ultrasonic 5s interval of 5s, after ultrasonic 10min, centrifugation is carried out at 12000rpm for 5min, and the supernatant is taken to be crude enzyme mixed liquor containing glucose dehydrogenase and ketoreductase.

EXAMPLE 4 biocatalytic preparation of the Apremilast chiral alcohol intermediate (R) -1- (3-ethoxy-4-methoxyphenyl) -2- (methylsulfonyl) ethanol

Taking 0.01mmol of substrate ketone (compound 1a), adding 50 mu L of dimethyl sulfoxide to fully dissolve the substrate; the crude enzyme mixture obtained in example 3 was added to make the reaction volume 950. mu.L; finally, 4mg of NADPH (reduced nicotinamide adenine dinucleotide phosphate) and 8mg of glucose were added, and the reaction was carried out at 30 ℃. After reaction for 12h, samples were taken for detection of substrate and product until complete reaction. After extraction three times with ethyl acetate and distillation under reduced pressure, the solvent was removed, and the conversion of the product alcohol compound 1b was > 99% by HPLC, with an ee value of 99.2%.

Example 5

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1-acetophenone (compound 2a) to give compound 2b with a conversion of > 99% and an ee value of 94%.

Example 6

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (4-methylphenyl) ethanone (compound 3a) prepared to give compound 3b with > 99% conversion and 96.7% ee.

Example 7

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (3-methoxyphenyl) ethanone (compound 4a) to afford compound 4b with a conversion > 99% and an ee value of 99.7%.

Example 8

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (4-methoxyphenyl) ethanone (compound 5a) to give compound 5b with 80% conversion and 99.7% ee.

Example 9

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (4-fluorophenyl) ethanone (compound 6a) to afford compound 6b with a conversion of 80% and an ee of 99.7%.

Example 10

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (3-chlorophenyl) ethanone (compound 7a) and compound 7b was prepared with a conversion of 99% and an ee value of 98.5%.

Example 11

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (4-chlorophenyl) ethanone (compound 8a) and compound 8b was prepared with a conversion of 99% and an ee value of 99.9%.

Example 12

Essentially the same as example 4, except that: the substrate ketone used was 2-methanesulfonyl-1- (4-bromophenyl) ethanone (compound 9a) to afford compound 9b with a 99% conversion and an ee of 99.9%.

Example 13

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1-acetophenone (compound 10a) and compound 10b was prepared with a conversion of 97.8% and an ee value of 94.3%.

Example 14

Essentially the same as in example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (4-methylphenyl) ethanone (compound 11a) and compound 11b was prepared with a conversion of 99.4% and an ee value of 98.7%.

Example 15

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (2-methoxyphenyl) ethanone (compound 12a) and compound 12b was prepared with a conversion of 12.7% and an ee value of 91.2%.

Example 16

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (3-methoxyphenyl) ethanone (compound 13a), and compound 13b was prepared with a conversion of 98.6% and an ee value of 93.0%.

Example 17

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (4-methoxyphenyl) ethanone (compound 14a), and compound 14b was prepared with a conversion of 73.9% and an ee value of 94.3%.

Example 18

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (4-fluorophenyl) ethanone (compound 15a) and compound 15b was prepared with a conversion of 99% and an ee value of 96.0%.

Example 19

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (3-chlorophenyl) ethanone (compound 16a) and compound 16b was prepared with a conversion of 99% and an ee value of 78.9%.

Example 20

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (4-chlorophenyl) ethanone (compound 17a) and compound 17b was prepared with a conversion of 97.7% and an ee value of 95.1%.

Example 21

Essentially the same as example 4, except that: the substrate ketone used was 2-benzenesulfonyl-1- (4-bromophenyl) ethanone (compound 18a) and compound 18b was prepared with 98.6% conversion and 94.2% ee.

SEQUENCE LISTING

<110> Shenyang university of pharmacy

<120> preparation method of R-2-sulfonyl-1-phenylethanol derivative

<130> 2022

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 247

<212> PRT

<213> ketoreductase (R-KRED)

<400> 1

Met Gly Gln Phe Asp Asn Lys Val Ala Leu Val Thr Gly Gly Thr Lys

1 5 10 15

Gly Ile Gly Leu Ala Ile Ala Glu Leu Phe Leu Lys Glu Gly Ala Lys

20 25 30

Gly Val Ala Phe Thr Gly Arg His Glu Asp Glu Gly Lys Ala Val Gln

35 40 45

Glu Arg Leu Gly Glu Arg Ser Leu Phe Ile Thr Gln Asp Val Ser Lys

50 55 60

Glu Glu Asp Trp Gln Asn Ala Thr Lys Ala Val Val Glu Lys Phe Gly

65 70 75 80

Gln Leu Asp Ala Ile Val Asn Asn Ala Gly Ile Gly Thr Pro Leu Gly

85 90 95

Ile Glu Glu Met Thr Leu Asp His Trp Asn Arg Glu Ile Ala Ile Asp

100 105 110

Leu Thr Gly Thr Met Leu Gly Cys Lys Tyr Gly Val Lys Ala Met Lys

115 120 125

Glu His Gly Gly Ala Ile Val Asn Ile Ser Ser Ile Ile Gly Met Ile

130 135 140

Gly Asp Pro Thr Val Pro Ala Tyr Asn Ala Ala Lys Gly Gly Val Arg

145 150 155 160

Leu Leu Thr Lys Ser Val Ala Leu Glu Cys Ala Glu Lys Gly Tyr Ala

165 170 175

Ile Arg Val Asn Ser Ile Tyr Pro Gly Ala Ile Ala Thr Pro Leu Ile

180 185 190

Asp His Leu Asp Asp Ala Thr Lys Gln Phe Tyr Ile Asp Lys His Pro

195 200 205

Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Lys Met Ala Val Phe

210 215 220

Val Ala Ser Asp Gly Ala Ser Phe Ser Thr Gly Ser Glu Phe Val Val

225 230 235 240

Asp Gly Gly Tyr Thr Ala Gln

245

Claims

1. A biocatalyst comprising a ketoreductase, a glucose dehydrogenase, and an NADP⁺Or NAD⁺And glucose, wherein the amino acid sequence of the ketoreductase is shown as SEQ ID NO. 1.

2. The biocatalyst of claim 1, wherein said ketoreductase enzyme: glucose dehydrogenase: NADP⁺Or NAD⁺: the glucose is 0.02-40 g/L: 0.02-40 g/L: 0-0.5 mmol: 6-200 g/L.

3. The use of the biocatalyst of claim 1 or 2 for the catalytic preparation of R-2-sulfonyl-1-phenylethanol derivatives, characterized in that 2-sulfonyl-1-phenylethanone derivatives are used as starting substrates, and under the action of the biocatalyst, the substrate carbonyl is asymmetrically reduced to hydroxyl groups, yielding R-2-sulfonyl-1-phenylethanol derivatives.

4. The use of claim 3, wherein the structural formula of the 2-sulfonyl-1-phenylethanone derivative is shown as formula A, and the structural formula of the R-2-sulfonyl-1-phenylethanol derivative is shown as formula B;

5. Use according to claim 3, wherein the catalytic reaction has a pH of 5.5 to 8.

6. Use according to claim 3, wherein the temperature of the catalytic reaction is between 28 and 45 ℃.

7. Use according to claim 3, wherein the substrate is present at a concentration of 0.5 to 200 g/L.

8. Use according to claim 3, wherein the 2-sulfonyl-1-phenylethanone derivative is selected from any one of the following: