CN102732579A - Method for preparing (3S)-3-(tertbutyloxycarbonyl)amino-1-chlorin-4-phenyl-(2R)-butanol by microbial transformation - Google Patents

Abstract

The present invention provides a method for the preparation of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol by microbial conversion: (3S)-3-(tert- butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate, using enzyme-containing bacterial cells obtained from Saccharomycescerevisiae CGMCC No.2266 fermentation as a biocatalyst, and performing a conversion reaction to obtain the product. The bacterial strain produced by the present invention is safe and non-toxic, and the microbial thalline is easy to cultivate on a large scale, and the cost is lower than chemical catalysts and catalytic enzymes; and the reaction conditions are mild, the environment is friendly, the molar conversion rate is high, and it is easy to realize large-scale industrial production, which is industrial production ( A green process for 3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol.

Description

A method for preparing (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol by microbial transformation

technical field

The invention relates to the field of microbial transformation methods, in particular to a method for biocatalytically preparing (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol.

Background technique

(3S)-3-(tert-Butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol, English name: ((3S)-3-(tert-Butoxycarbonyl)amino-1-chloro -4-phenyl-(2R)-butanol), CAS number: 162536-40-5, molecular formula C ₁₅ H ₂₂ NClO ₃ , molecular weight 299.79. (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol is a key intermediate in the preparation of the anti-AIDS drug atazanavir. Atazanavir is a new type of azapeptide protease inhibitor (PI), which is designed according to the X-ray diffraction study of the enzyme-azapeptide complex and has a C-2 symmetrical chemical structure. It is a highly selective and efficient inhibitor of HIV-1 protease. It inhibits the production of viral structural proteins, reverse transcriptase, integrase and protease by blocking the cleavage of viral gap and gap-pol precursor polyproteins, HIV-1-infected cells release non-infectious immature virus particles. This product has stronger inhibitory activity against multiple strains of HIV-1 virus than other PIs. The half effective concentration (EC50) in the in vitro medium is 2.6-5.3 nmol/L, and the 90% effective concentration (EC90) is 9-15 nmol. /L.

The racemate (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-butanol can be resolved to obtain (3S)-3-(tert-butoxycarbonyl)amino-1- Chloro-4-phenyl-(2R)-butanol, but the maximum resolution efficiency is only 50%, and the production efficiency is not high. With (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as the substrate, the carbonyl group in the substrate can be asymmetrically reduced by chemical and microbial methods to obtain (3S )-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol. However, the chemical asymmetric reduction requires the preparation of a chiral chemical catalyst, which is expensive, cumbersome in the preparation process, and usually causes greater environmental pollution.

Contents of the invention

The object of the present invention is to provide a method for the preparation of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol by microbial transformation, the method has mild reaction conditions and is easy to operate , environment-friendly, high product conversion rate, easy industrial production.               

The technical scheme adopted in the present invention is:

A method for preparing (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol by microbial transformation, the method is based on (3S)-3-(tert- butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as the substrate, and the enzyme-containing bacterial cells obtained from the fermentation of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) CGMCC No. 2266 as the biocatalyst, and the conversion reaction The (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol was obtained.

The Saccharomyces cerevisiae CGMCC No.2266 has been protected as a new strain in the previously authorized patent 200810059686, and is preserved in the General Microorganism Collection Center of the China Microbial Culture Collection Management Committee, located in the Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, The deposit number is CGMCC No.2266, and the deposit date is November 26, 2007. the

The colony characteristics of the Saccharomyces cerevisiae CGMCC No.2266: on the agar medium, it presents a milky white, shiny, flat, neat edge, moist, smooth surface, and uniform colony morphology.

Further, the method for preparing (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol by microbial transformation of the present invention is as follows: Phosphate buffer was used as the reaction solvent, (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone was used as the substrate, and Saccharomyces cerevisiae CGMCC No. The enzyme-containing bacterial cells obtained by 2266 fermentation are used as biocatalysts, and the transformation reaction is carried out at 25~45°C for 12~72 hours. After the reaction, the transformation liquid is separated and purified to obtain the (3S)-3-(tert-butoxycarbonyl) Amino-1-chloro-4-phenyl-(2R)-butanol.

The initial concentration of the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone in the phosphate buffer is 0.1-1 mmol/L.

Further, ethanol with a final concentration of 5-20% can also be added as an auxiliary substrate in the reaction system of the present invention. Microbial cells contain a large amount of alcohol dehydrogenase, which converts ethanol to acetaldehyde, and at the same time transfers hydrogen to nicotinamide adenine dinucleotide (NAD) to form the reduced state of nicotinamide adenine dinucleotide ( Reduced coenzyme Ⅰ, NADH), the addition of ethanol is conducive to the in situ regeneration of coenzyme NADH, providing a large amount of hydrogen donors for the reaction process, thereby increasing the conversion rate of the reaction. At the same time, the addition of ethanol can appropriately enhance the permeability of the cells, promote more substrates to enter the microbial cells for biotransformation, and increase the conversion rate.

Described enzyme-containing bacterium cell dosage is 1～20 g/g substrate by cell dry weight, and the amount of substrate described here is (3S)-3-(tert-butoxycarbonyl) amino-1-chloro - The mass of 4-phenyl-2-butanone. the

The dry weight of the enzyme-containing bacterial cells refers to the weight of the obtained dry cells after centrifuging the fermentation broth, discarding the supernatant, and drying the obtained wet cells at 120° C. for 48 hours to constant weight. The method for determining the amount of fermented liquid containing enzyme-containing bacterial cells required for quantifying the dry weight of cells is: take part of the fermented liquid and centrifuge, discard the supernatant, dry the wet cells at 120°C for 48 hours to constant weight, Measure the dry weight of the obtained cells, calculate the ratio of dry cells in the fermented broth of the unit enzyme-containing bacterium cells, and then calculate the amount of fermented liquid containing the enzyme-containing bacterium cells required for the quantitative dry weight of the cells.

The enzyme-containing bacterial cells are prepared according to the following method: Saccharomyces cerevisiae CGMCC No. 2266 is inoculated into the fermentation medium, the shaker speed is 150-200 r/min, and cultured at 26-35°C for 18-30 h, the The fermented liquid is centrifuged to obtain enzyme-containing bacterial cells. the

Further, the method for obtaining the pure product of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol is as follows: Centrifuge for 20 minutes, discard the precipitated bacteria, extract the supernatant with an equal volume of ethyl acetate for 3 times, combine the ethyl acetate extracts, add anhydrous sodium sulfate to the ethyl acetate extracts to remove water, filter with suction, Take the filtrate and distill off the ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol.

The preparation method of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol of the present invention is recommended to be carried out according to the following steps:

(1) Slant culture: Inoculate Saccharomyces cerevisiae CGMCC No. 2266 into the slant medium, and culture it at 26-35°C for 4-6 days to obtain the slant; the final concentration of the slant medium is composed of: wort juice 5-15 g /L, yeast powder 2~4 g/L, peptone 4~6 g/L, glucose 7~12 g/L, agar 15~25 g/L, natural pH value, solvent is water;

(2) Seed culture: take an inoculation loop from the slant of the bacterium and transfer it to the seed medium, the rotation speed of the shaker is 150-200 r/min, and cultivate at 26-35°C for 18-26 hours to obtain the seed liquid; The final concentration of the seed medium is composed of: glucose 26~32 g/L, yeast powder 2~4 g/L, ammonium sulfate 3~6 g/L, anhydrous MgSO ₄ 0.2~0.4 g/L, K ₂ HPO ₄ 3H ₂ O 0. 5~1.5 g/L, KH ₂ PO ₄ 0. 6~1.5 g/L, use NaOH or HCl solution to adjust the pH of the liquid medium to 7.0, and the solvent is water;

(3) Fermentation culture: Take the seed liquid, inoculate it into the fermentation medium with an inoculum volume fraction of 10-20%, and incubate at 26-35°C for 18-30 h at a shaker speed of 150-200 r/min. The enzyme-containing bacterial cells were obtained by centrifugation of liquid; the final concentration of the fermentation medium was composed of: glucose 26-32 g/L, yeast powder 2-4 g/L, ammonium sulfate 3-6 g/L, anhydrous MgSO ₄ 0.2~0.4 g/L, K ₂ HPO ₄ 3H ₂ O 0.5~1.5 g/L, KH ₂ PO ₄ 0.6~1.5 g/L, adjust liquid medium with NaOH or HCl solution The pH value is 7.0, and the solvent is water;

(4) Biotransformation: Add ethanol at a final concentration of 5 to 20% as an auxiliary substrate in a phosphate buffer solution with a pH of 5.0 to 8.0, and add (3S)-3- at a final concentration of 0.1 to 1 mmol/L (tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4- Phenyl-2-butanone 1~20 times of enzyme-containing bacterial cells, transformation reaction at 25~45°C for 12~72 hours, to obtain transformation solution;

(5) The method to obtain the pure product of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol after the reaction is finished is as follows: Centrifuge at 4000 r/min for 20 minutes, discard the bacterial precipitate, extract the supernatant with an equal volume of ethyl acetate for 3 times, combine the ethyl acetate extract, add anhydrous sodium sulfate to the ethyl acetate extract to remove water , filtered with suction, and the filtrate was distilled to remove ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol.

(3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol of the present invention is enantiomeric excess value (ee%) and substrate conversion rate Confirmation: during the reaction process, 5 mL of sample was taken every 1-11 h, the sample was centrifuged at 4000 r/min for 20 minutes, the precipitate was discarded, the supernatant was extracted with 5 mL of ethyl acetate, and 5 μL of the ethyl acetate extract was absorbed into liquid chromatography for analysis. The liquid chromatography model is Agilent 1200, the chromatographic column is chiral column CHIRACEL OD-H (4.6 mm×250 mm, 5 um), and the buffer solution is n-hexane-tert-butyl methyl ether-trifluoroacetic acid (800:200: 2), the mobile phase was prepared from the above buffer solution and ethanol at a volume ratio of 99:1, the flow rate of the mobile phase was 0.5 mL/min, and the injection volume was 5 ul. Under the analytical conditions, the substrate (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone, (3S)-3-(tert-butoxycarbonyl)amino -1-chloro-4-phenyl-(2R)-butanol and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2S)-butanol are completely separated, thereby further The molar conversion of the substrate and the enantiomeric excess of the product (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol were calculated.

The (3S)-3-(tert-butoxycarbonyl) amino-1-chloro-4-phenyl-(2R)-butanol pure product can be detected by liquid chromatography-mass spectrometry to determine the purity and molecular weight.

In the present invention, microbial cells are used to asymmetrically reduce (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone to prepare (3S)-3-(tert-butoxycarbonyl)amino -1-Chloro-4-phenyl-(2R)-butanol can obtain products with high enantiomeric excess value, and adding 5-20% ethanol as an auxiliary substrate is beneficial to improve the molar conversion rate of the substrate.

The present invention adopts microbial conversion method to prepare (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol, which has the following advantages compared with chemical synthesis method and enzyme catalysis method: (1) The production strain is safe and non-toxic, and the microbial cells are easy to cultivate on a large scale, and a large amount of biocatalysts can be obtained, which is cheaper than chemical catalysts and catalytic enzymes; (2) The reaction conditions are mild, environmentally friendly, and the molar conversion rate is high, which is easy to realize Large-scale industrial production is a green process for the industrial production of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol.

(1) Specific implementation methods

The present invention will be further described below in conjunction with specific examples, but the scope of the present invention is not limited thereto.

Slant medium preparation: wort juice 10 g/L, yeast powder 3 g/L, peptone 5 g/L, glucose 10 g/L, agar 20 g/L, natural pH value, solvent is water; sterilized at 121 ℃ for 20 min, sterilized and cooled to make a slope.

Preparation of seeds and fermentation medium: glucose 30 g/L, yeast powder 3 g/L, ammonium sulfate 5 g/L, anhydrous MgSO ₄ 0.25 g/L, K ₂ HPO ₄ 3H ₂ O 1 g/L, KH ₂ PO ₄ 1 g/L, the solvent is water, the pH of the liquid medium is adjusted to 7.0 with NaOH or HCl solution, and sterilized at 121 °C for 20 min.

Example 1

Saccharomyces cerevisiae CGMCC No. 2266 was inoculated into the slant medium, and cultured at 30°C for 6 days to prepare the slant. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and cultivate it at 30 °C and 180 r/min for 24 h to obtain a seed solution. Inoculate 10 mL of seed solution (the inoculation amount is 10% of the volume of the liquid medium) into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and culture it at 30°C and 180 r/min for 24 hours to obtain a fermentation broth. The solution was centrifuged to obtain enzyme-containing bacterial cells.

After determination, the dry weight of enzyme-containing bacterium cells contained in each liter of fermented liquid of the present embodiment is 50 grams.

Add 200 mL of the fermented liquid obtained above to ten portions of Erlenmeyer flasks containing 100 mL of pH7.0 phosphate buffer solution, which contains 10 g of the dry weight of enzyme-containing bacterial cells, and add (3S)-3-(tert- Butoxycarbonyl) amino-1-chloro-4-phenyl-2-butanone, so that the terminal of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone Concentrations are 0.1 mmol/L, 0.2 mmol/L, 0.3 mmol/L, 0.4 mmol/L, 0.5 mmol/L, 0.6 mmol/L, 0.7 mmol/L, 0.8 mmol/L, 0.9 mmol/L, 1.0 mmol /L, were placed in a shaker at 30 °C and 180 r/min for 36 h. After the reaction, the transformation solution was centrifuged at 4000 r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extract was combined, and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate to remove ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)- Butanol, initial substrate concentration versus molar conversion and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) The impact can be seen in Table 1.

Table 1 Effect of initial substrate concentration on conversion and enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

Substrate concentration (mmol/L) Conversion rate(%) Enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) 0.1 100 100 0.2 92.2 100 0.3 80.5 100 0.4 77.3 95.6 0.5 68.8 83.2 0.6 52.1 70.3 0.7 43.2 68.2 0.8 35.9 62.1 0.9 28.2 58.5 1.0 19.5 51.2

It can be seen from Table 1 that the conversion rate gradually decreases with the increase of the substrate concentration. The conversion rate was 100% when the substrate concentration was 0.1 mmol/L. The substrate concentration has a greater impact on the enantiomeric excess of the product (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol, (3S)- The enantiomeric excess of 3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol decreases with increasing substrate concentration. The enantiomeric excess of the product (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol is 100% at substrate concentrations below 0.3 mmol/L .

Example 2

Saccharomyces cerevisiae CGMCC No. 2266 was inoculated into the slant medium and cultured at 30°C for 6 days to obtain the slant. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and cultivate it at 30 °C and 180 r/min for 24 h to obtain a seed solution. Inoculate 10 mL of seed solution (the inoculation amount is 10% of the volume of the liquid medium) into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and culture it at 30 °C and 180 r/min for 24 h to obtain a fermentation broth. The fermented liquid is centrifuged to obtain enzyme-containing bacterium cells, and the dry weight of the enzyme-containing bacterium cells in every liter of fermented liquid is 50 grams.

In seven Erlenmeyer flasks containing 100 mL of pH7.0 phosphate buffer solution, 200 mL of the above-mentioned fermented liquid was added, in which the dry weight of enzyme-containing bacterial cells was 10 g, and (3S)-3-(tert- Butoxycarbonyl) amino-1-chloro-4-phenyl-2-butanone makes (3S)-3-(tert-butoxycarbonyl) amino-1-chloro-4-phenyl-2-butanone final concentration of 0.3 mmol/L, respectively add auxiliary substrate ethanol so that the volume percentage of ethanol in the reaction system (v/v) is 0%, 5%, 10%, 15%, 20%, 25% and 30%, respectively, set React in a shaker at 30°C and 180 r/min for 36 h. After the reaction, centrifuge the transformation liquid at 4000 r/min for 20 minutes, discard the precipitated bacteria, and extract the supernatant continuously with an equal volume of ethyl acetate for 3 Next, combine the ethyl acetate extracts, add anhydrous sodium sulfate to the ethyl acetate extracts to remove a small amount of water, filter with suction, and distill the filtrate to remove ethyl acetate to obtain the (3S)-3-(tert-butoxy Carbonyl) amino-1-chloro-4-phenyl-(2R)-butanol, auxiliary substrate ethanol concentration versus molar conversion and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4- The effect of the enantiomer excess (ee%) of phenyl-(2R)-butanol is shown in Table 2.

Table 2: Effect of ethanol addition on conversion and enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

Ethanol (%) Conversion rate(%) Enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) 0 80.5 100 5 92.8 100 10 100 100 15 98.9 100 20 96.2 100 25 90.5 100 30 82.7 100

It can be seen from Table 2 that the addition of an appropriate amount of ethanol is conducive to improving the molar conversion rate of the substrate. When the amount of ethanol added is 10% to 15%, the effect is better, and the higher concentration of ethanol will reduce the biotransformation ability of microorganisms. will gradually decrease. The enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol did not change with the amount of ethanol added and remained at 100%.

Example 3

Saccharomyces cerevisiae CGMCC No. 2266 was inoculated into the slant medium and cultured at 30°C for 6 days to obtain the slant. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and cultivate it at 30 °C and 180 r/min for 24 h to obtain a seed solution. The seed liquid was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium at an inoculation amount of 10%, and cultured at 30°C and 180 r/min for 24 hours to obtain a fermentation liquid, and the fermentation liquid was centrifuged to obtain enzyme-containing bacteria For somatic cells, the dry weight of enzyme-containing bacterial somatic cells per liter of fermentation broth is 50 grams.

Add 200 mL of the fermented liquid obtained above to five parts of Erlenmeyer flasks containing 100 mL of pH7.0 phosphate buffer solution, which contains 10 g of enzyme-containing bacterium cells in dry weight, and add (3S)-3-(tert- Butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone to make the final concentration of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone 0.3 mmol/L, the auxiliary substrate ethanol was added to make the final concentration of ethanol 10%, and the conversion reaction was carried out at 25°C, 30°C, 35°C, 40°C, and 45°C for 36 h in a shaker at 180 r/min. After the reaction, the transformation solution was centrifuged at 4000 r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extract was combined, and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate to remove ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)- Butanol, reaction temperature versus substrate molar conversion and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) The impact can be seen in Table 3.

Table 3 Effect of reaction temperature on conversion and enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

Conversion temperature (°C) Conversion rate(%) Enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) 25 89.7 100 30 100 100 35 86.2 100 40 72.5 100 45 63.1 100

It can be seen from Table 3 that the reaction temperature has a great influence on the conversion rate. Excessively high reaction temperature can lead to partial inactivation of the enzyme, thus lowering the conversion rate. The preferred conversion temperature is about 30 °C, and the enantiomeric excess value of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol does not change with the conversion temperature While changing, all remain at 100%.

Example 4:

Saccharomyces cerevisiae CGMCC No. 2266 was inoculated into the slant medium and cultured at 30°C for 6 days to obtain the slant. Use an inoculation needle to take an inoculation loop from the slant of the bacterium and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and cultivate it at 30 °C and 180 r/min for 24 h to obtain a seed solution. The seed liquid was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium at an inoculation amount of 10%, and cultured at 30°C and 180 r/min for 24 hours to obtain a fermentation liquid, and the fermentation liquid was centrifuged to obtain enzyme-containing bacteria For somatic cells, the dry weight of enzyme-containing bacterial somatic cells per liter of fermentation broth is 50 grams.

Add 200 mL of the above-mentioned fermented liquid to five Erlenmeyer flasks containing 100 mL of pH7.0 phosphate buffer solution, which contains 10 g of enzyme-containing bacterium cells in dry weight, and add (3S)-3-(tert-butyl Oxycarbonyl) amino-1-chloro-4-phenyl-2-butanone, so that the final concentration of (3S)-3-(tert-butoxycarbonyl) amino-1-chloro-4-phenyl-2-butanone is 0.3 mmol/L, the auxiliary substrate ethanol was added to make the final ethanol concentration 10%, and placed in a shaker at 30 °C and 180 r/min to react for 12 h, 24 h, 36 h, 48 h, and 72 h, respectively. After the reaction, the transformation solution was centrifuged at 4000 r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extract was combined, and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, remove ethyl acetate by distillation from the filtrate, and obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)- Butanol, reaction time versus substrate molar conversion and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) The impact can be seen in Table 4.

Table 4: Effect of reaction time on conversion and enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

Conversion time (h) Conversion rate(%) Enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) 12 35.0 100 twenty four 80.2 100 36 100 100 48 100 100 72 100 100

It can be seen from Table 4 that the conversion rate gradually increases with the prolongation of the conversion time. After 36 hours of reaction, the conversion rate can basically reach 100%, so the optimum conversion time is 36 hours. The enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol remained at 100% without changing with the conversion time.

Example 5:

Saccharomyces cerevisiae CGMCC No. 2266 was inoculated into the slant medium, and cultured at 30°C for 6 days to obtain the slant. A ring of bacteria was taken from the slant of the bacteria with an inoculation needle and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, and cultured at 30 °C and 180 r/min for 24 h to obtain a seed liquid. The seed solution was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of liquid medium at an inoculum amount of 10%, cultured at 30°C and 180 r/min for 24 hours to obtain a fermentation liquid, and the fermentation liquid was centrifuged to obtain enzyme-containing bacteria For somatic cells, the dry weight of enzyme-containing bacterial somatic cells per liter of fermentation broth is 50 grams.

Add respectively 40 milliliters, 100 milliliters, 200 milliliters, 300 milliliters and 400 milliliters of the above-mentioned gained fermentation broth in five parts of Erlenmeyer flasks containing 100 mLpH7. 2g, 5g, 10g, 15g and 20g. Add (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone to the above five conical flasks to make (3S)-3-(tert-butoxycarbonyl) The final concentration of amino-1-chloro-4-phenyl-2-butanone is 0.3 mmol/L, and then the auxiliary substrate ethanol is added to make the final concentration of ethanol 10%, and placed in a shaking table at 30°C and 180 r/min React in medium for 36 h. After the reaction, the transformation solution was centrifuged at 4000 r/min for 20 minutes, the bacterial precipitate was discarded, and the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extract was combined, and added to the ethyl acetate extract. Add anhydrous sodium sulfate to remove a small amount of water, filter with suction, and distill the filtrate to remove ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)- Butanol, bacterial cell mass to substrate molar conversion rate and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol corresponding excess value (ee %) can be seen in Table 5.

Table 5 Effects of bacterial mass on conversion rate and enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

Table 5 Effect of bacterial mass on conversion rate and enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol

Bacterial volume (dry weight) (g) Conversion rate(%) Enantiomeric excess of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol (ee%) 2 32.1 100 5 52.6 100 10 100 100 15 100 100 20 100 100

It can be seen from Table 5 that the conversion rate gradually increases with the increase of the bacterial cell dosage. The increase in the amount of bacteria not only increases the amount of enzyme, but also increases the amount of coenzyme NADH, so it is beneficial to improve the conversion rate of the reaction. The enantiomeric excess value of (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol does not change with the amount of bacteria added, and remains at 100% .

Claims (9)

1. A method for preparing (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol by microbial transformation, characterized in that, the method is based on (3S )-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as the substrate, and the enzyme-containing bacterial cells obtained from the fermentation of Saccharomyces cerevisiae CGMCC No. 2266 as the organism Catalyst, carry out conversion reaction to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol. 2. the preparation method as claimed in claim 1, is characterized in that, described method is: with the phosphate buffer saline solution of pH 5.0～8.0 as reaction solvent, with (3S)-3-(tert-butoxycarbonyl)amino -1-Chloro-4-phenyl-2-butanone as the substrate, using the enzyme-containing bacterial cells obtained from the fermentation of Saccharomyces cerevisiae (Saccharomyces cerevisiae ) CGMCC No. 2266 as the biocatalyst, and transforming the reaction at 25~45 °C12 After ~72 hours, after the reaction, the conversion solution was separated and purified to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol. 3. preparation method as claimed in claim 1, is characterized in that, the initial concentration of described substrate in reaction system is 0.1～1 mmol/L. 4. the preparation method as claimed in claim 1, is characterized in that, can also be added with the ethanol that final concentration is 5～20% in described reaction system. 5. the preparation method as claimed in claim 1, is characterized in that, described enzyme-containing bacterium cell dosage is 1～20 g/g substrate in terms of dry cell weight. 6. The preparation method according to claim 1, wherein the enzyme-containing bacterial cells are prepared according to the following method: Saccharomyces cerevisiae CGMCC No. 2266 is inoculated into the fermentation medium, and the shaking table speed is 150 ~ 200 r/min, culture at 26-35°C for 18-30 h, and centrifuge the fermentation broth to obtain enzyme-containing bacterial cells. 7. The preparation method according to claim 2, characterized in that, said (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-(2R)-butanol is separated and purified The method is as follows: after the reaction, the transformation solution was centrifuged at 4000 r/min for 20 minutes, the bacterial precipitate was discarded, the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, the ethyl acetate extracts were combined, and the Anhydrous sodium sulfate was added to the extract to remove water, suction filtered, and the filtrate was distilled to remove ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-phenyl-( 2R)-butanol. 8. preparation method as claimed in claim 1, is characterized in that, described method is carried out according to the following steps: (1) Slant culture: Inoculate Saccharomyces cerevisiae CGMCC No. 2266 into the slant medium, and culture it at 26-35°C for 4-6 days to obtain the slant surface; (2) Seed culture: Take an inoculation loop from the slant of the thallus and transfer it to the seed medium, the rotation speed of the shaker is 150-200 r/min, and cultivate at 26-35°C for 18-26 hours to obtain the seed liquid; (3) Fermentation culture: Take the seed liquid, inoculate it into the fermentation medium with an inoculum volume fraction of 10-20%, and incubate at 26-35°C for 18-30 hours at a shaking table speed of 150-200 r/min. centrifugation to obtain the enzyme-containing bacterial cells; (4) Biotransformation: In the phosphate buffer solution with pH 5.0~8.0, add ethanol with a final concentration of 5~20% as an auxiliary substrate, and add (3S)-3- with a final concentration of 0.1~1 mmol/L (tert-butoxycarbonyl)amino-1-chloro-4-phenyl-2-butanone as a substrate, and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro -4-Phenyl-2-butanone 1~20 times of enzyme-containing bacterial cells, transformation reaction at 25~45°C for 12~72 hours, after the reaction is completed, the transformation solution is obtained; (5) Separation and purification: after the conversion reaction, centrifuge the conversion liquid at 4000 r/min for 20 minutes, discard the bacterial precipitate, extract the supernatant with an equal volume of ethyl acetate for 3 times, and combine the ethyl acetate extracts , adding anhydrous sodium sulfate to the ethyl acetate extract to remove water, suction filtration, and distilling the filtrate to remove ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4- Phenyl-(2R)-butanol. 9. The method according to claim 2, characterized in that the method is carried out as follows: (1) Slant culture: Inoculate Saccharomyces cerevisiae CGMCC No. 2266 into the slant medium, and culture it at 26-35°C for 4-6 days to obtain the slant; the final concentration of the slant medium is composed of: wort juice 5-15 g /L, yeast powder 2~4 g/L, peptone 4~6 g/L, glucose 7~12 g/L, agar 15~25 g/L, natural pH value, solvent is water; (2) Seed culture: Take an inoculation loop from the slant of the thallus and transfer it to the seed medium, at 26-35 ℃, with a shaker speed of 150-200 r/min, and cultivate for 18-26 h to obtain the seed liquid; The final concentration of the seed medium is as follows: glucose 26~32 g/L, yeast powder 2~4 g/L, ammonium sulfate 3~6 g/L, anhydrous MgSO ₄ 0.2~0.4 g/L, K ₂ HPO ₄ 3H ₂ O 0.5~1.5 g/L, KH ₂ PO ₄ 0.6~1.5 g/L, use NaOH or HCl solution to adjust the pH of the liquid medium to 7.0, and the solvent is water; (3) Fermentation culture: take the seed solution, inoculate the fermentation medium with an inoculum volume fraction of 10-20%, culture temperature is 26-35 ℃, shaker speed is 150-200 r/min, culture 18~ 30 h to obtain the fermented liquid containing enzyme-containing bacterium cells, centrifuged to obtain the enzyme-containing bacterium cells; the final concentration of the fermentation medium consists of: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L , ammonium sulfate 3~6 g/L, anhydrous MgSO ₄ 0.2~0.4 g/L, K ₂ HPO ₄ 3H ₂ O 0.5~1.5 g/L, KH ₂ PO ₄ 0.6~1.5 g/L, use NaOH or The HCl solution adjusts the pH value of the liquid medium to 7.0, and the solvent is water; (4) Biotransformation: Add (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4-benzene at a final concentration of 0.1-1 mmol/L to a phosphate buffer solution with a pH of 5.0-8.0 Base-2-butanone, add ethanol with a final concentration of 5~20% as an auxiliary substrate, and (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4 -Phenyl-2-butanone 1~20 times the enzyme-containing bacterial cells, transforming at 25~45°C for 12~72 hours to obtain the transformation solution; (5) Separation and purification: after the reaction, the transformation solution was centrifuged at 4000 r/min for 20 minutes, the bacterial precipitate was discarded, the supernatant was continuously extracted 3 times with an equal volume of ethyl acetate, and the ethyl acetate extracts were combined. Add anhydrous sodium sulfate to the ethyl acetate extract to remove water, filter with suction, take the filtrate and distill off ethyl acetate to obtain the (3S)-3-(tert-butoxycarbonyl)amino-1-chloro-4- Phenyl-(2R)-butanol.