CN111808893B - Novel biological preparation method of amino alcohol drug intermediate - Google Patents

Abstract

The invention provides a biological preparation method of an amino alcohol drug intermediate. Specifically, the method comprises the following steps: (a) in a liquid reaction system, taking a compound X as a substrate, and carrying out a reaction shown as a formula A under the catalysis of carbonyl reductase in the presence of coenzyme to form a compound Y; and (b) optionally separating the compound Y from the reaction system after the reaction of the above step. The present invention also provides a reaction system comprising: (i) an aqueous solvent; (ii) a substrate, said substrate being compound X; (iii) a coenzyme; (iv) a carbonyl reductase; (v) a co-substrate; and (vi) an enzyme for coenzyme regeneration.

Description

Novel biological preparation method of amino alcohol drug intermediate

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a key chiral intermediate of an amino alcohol antibacterial drug.

Background

Chloramphenicol was a natural antibiotic that was successfully isolated by David Goteli from S.renilaginis, south America, earliest in 1947. The researchers in 1949 found that it has broad-spectrum antibacterial activity, and its molecular structure contains chlorine atom, so it is named as chloramphenicol. Since the first separation was successful, researchers around the world began to develop multiple chemical synthesis routes. The successful preparation of chloramphenicol by a chemical synthesis method provides possibility for wide commercial application of chloramphenicol. However, it was later found that it has serious adverse effects, especially on the hematopoietic system, and it produces symptoms such as granulocytopenia and thrombocytopenia, aplastic anemia, and hemolysis. It has some unique clinical advantages: the antibacterial spectrum is wide, and the effect on gram-negative bacteria is particularly strong; the second is the first choice medicine for treating typhoid fever and paratyphoid fever, and is also one of specific medicines for treating anaerobic bacterial infection. It is also used for treating meningitis patients with poor curative effect of other medicines. Thirdly, the chloramphenicol can be prepared into external preparations such as eye drops, eye ointments, ear drops, suppositories and the like, and can be widely applied to various fields of dermatology, ophthalmology, gynecology and the like. Except for human use, chloramphenicol is widely used in poultry and livestock industry at present due to its low cost.

The chloramphenicol is initially developed in 1951 in China, the racemic synmycin is firstly synthesized by Shenyang northeast pharmaceutical factories in 1954 and is gradually eliminated in the future due to great toxic and side effects, and a research team leading in Shenxiang in 1958 develops a route suitable for industrial production in 1958, and the route is continuously optimized for many years and is used up to now. The existing production process route is as follows:

route-production process route of chloramphenicol

The method uses ethylbenzene as a starting material, prepares a key intermediate 4-nitroacetophenone (3) through nitration and oxidation, prepares a compound 5 through bromination and ammonolysis, prepares a compound 7 through acetic anhydride protection and hydroxymethylation, prepares a compound 8 through aluminum isopropoxide and aluminum trichloride catalytic stereoselective reduction, deprotects the compound 8, prepares racemic 9 through dissociation, prepares (R, R) -9 through preferential crystallization and resolution, and finally prepares chloramphenicol by grafting a side chain dichloroacetyl group. The existing production process route has the following three defects:

(1) the construction of the chiral center needs to be completed through two steps of reactions of reduction and resolution, the theoretical yield of the resolution is only 50%, and aluminum trichloride and aluminum isopropoxide used in the reduction step contain a large amount of aluminum-containing wastewater in post-treatment wastewater;

(2) the resolved isomer (S, S) -9 is not effectively utilized;

(3) the whole route has longer reaction steps and lower productivity.

Therefore, under the existing environment-friendly situation, a highly efficient and environment-friendly chloramphenicol synthesis route more suitable for industrial application is urgently needed in the industry.

Disclosure of Invention

The invention aims to provide a biological preparation method of a key intermediate Y of chloramphenicol. The method relates to a dynamic reduction kinetic resolution technology involving carbonyl reductase, namely two chiral centers are generated through one-step reduction reaction. The key steps of the dynamic reduction kinetic resolution process are shown as a formula A:

in the formula A, the reaction solution is prepared,

R¹selected from:

R²selected from:

in a first aspect of the present invention, there is provided a process for the preparation of compound Y, comprising the steps of:

in a liquid reaction system, taking a compound X as a substrate, and carrying out a reaction shown as a formula A under the catalysis of (R, S) -carbonyl reductase in the presence of coenzyme to form a compound Y;

wherein the content of the first and second substances,

R¹selected from:

R²selected from:

in another preferred embodiment, R is¹Is composed of

R²Is composed of

In another preferred embodiment, the coenzyme is selected from the group consisting of: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.

In another preferred embodiment, the coenzyme is selected from the group consisting of: NADH, NADPH, NAD, NADP, or a combination thereof.

In another preferred embodiment, the ratio of the amount of NADH, NADPH, NAD or NADP to the amount of substrate is 0.01-1.0% (w/w), preferably 0.01-0.5% (w/w).

In another preferred embodiment, an enzyme for coenzyme regeneration is also present in the reaction system.

In another preferred embodiment, the enzyme for coenzyme regeneration is selected from the group consisting of: alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof.

In another preferred embodiment, the reaction system further comprises a co-substrate for coenzyme regeneration.

In another preferred embodiment, the co-substrate is selected from the group consisting of: isopropanol, glucose, ammonium formate, or a combination thereof.

In another preferred embodiment, the concentration of the cosubstrate in the reaction system is 5-30%.

In another preferred embodiment, in said step, the temperature is from 10 ℃ to 50 ℃, preferably from 20 ℃ to 40 ℃, more preferably from 25 ℃ to 35 ℃.

In another preferred embodiment, in said step, the time is 0.1 to 240 hours, preferably 0.5 to 120 hours, more preferably 1 to 72 hours, still more preferably 3 to 10 hours.

In another preferred embodiment, in said step, the pH is 6 to 10, preferably 7.0 to 9.0.

In another preferred example, in the reaction system, the (R, S) -carbonyl reductase is an enzyme in a free form, an immobilized enzyme, or an enzyme in the form of bacterial cells.

In another preferred embodiment, the reaction system is an aqueous system.

In another preferred embodiment, the reaction system is a phosphate buffer system.

In another preferred embodiment, the reaction system further comprises a cosolvent.

In another preferred embodiment, the cosolvent is selected from the following group: dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, or combinations thereof.

In another preferred embodiment, the concentration of the cosolvent is 5-30%.

In another preferred example, the method further comprises: and separating the compound Y from the reaction system after the reaction in the previous step.

In another preferred embodiment, the separation comprises: adding isopropanol, centrifuging, partially concentrating, extracting with methyl tert-ether or ethyl acetate, and concentrating the organic layer.

In another preferred embodiment, the ee value of compound Y (e.g. compound 14) in the reaction system after the reaction is greater than or equal to 90%, preferably greater than or equal to 95%, and more preferably greater than or equal to 99%; the de value is greater than or equal to 70%, preferably greater than or equal to 90%, more preferably greater than or equal to 95%.

In another preferred embodiment, the reaction system after the reaction has a conversion rate of 80% or more, preferably 85% or more, and more preferably 95% or more, for converting compound X (e.g., compound 13) into compound Y (e.g., compound 14).

In another preferred embodiment, the carbonyl reductase is selected from the group consisting of:

(i) derived from carbonyl reductase EA, the amino acid sequence of which is shown in SEQ ID NO: 1 is shown in the specification;

(ii) for SEQ ID NO: 1, and (b) the amino acid sequence obtained by performing substitution, deletion, change, insertion or addition of one or more amino acids within the range of keeping the enzyme activity.

In another preferred embodiment, the gene sequence encoding carbonyl reductase EA is selected from the group consisting of:

(a) SEQ ID NO: 2;

(b) a polynucleotide complementary to the sequence defined in (a); or

(c) Any polynucleotide or complementary sequence having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more sequence identity to the sequence defined in (a).

In another preferred embodiment, the carbonyl reductase gene is constructed on an expression vector.

In a third aspect of the present invention, there is provided a reaction system comprising:

(i) an aqueous solvent;

(ii) a substrate, said substrate being compound X;

(iii) a coenzyme;

(iv) (R, S) -carbonyl reductase;

(v) a co-substrate; and

(vi) an enzyme for coenzyme regeneration;

in the compound X, the compound X is a compound,

R¹selected from:

R²selected from:

in another preferred embodiment, R is¹Is composed of

R2 is

In a third aspect of the present invention, there is provided a process for the preparation of compound Y, comprising the steps of: carrying out the reaction of formula a using the reaction system according to the second aspect of the present invention under the catalytic conditions of (R, S) -carbonyl reductase enzyme to produce compound Y;

wherein the content of the first and second substances,

R¹selected from:

R²selected from:

in a fourth aspect of the present invention, there is provided a process for preparing an intermediate W comprising the steps of:

(1) preparing compound Y by a process according to the first or third aspect of the invention;

(2) and (3) preparing an intermediate compound W by using the compound Y as a substrate.

In another preferred embodiment, the step (2) includes a reaction shown as formula B, and the carbonyl reductase is reduced to obtain R of the compound Y²The ester bond in (b) is reduced to a hydroxyl group, thereby producing a compound Z; wherein R is³Is methyl, ethyl, isopropyl or tert-butyl;

in another preferred embodiment, the reducing agent used in step (2) is sodium borohydride and/or potassium borohydride or a combination thereof with magnesium chloride, zinc chloride and/or calcium chloride.

In another preferred example, the reaction solvent used in the step (2) is a common organic solvent.

In another preferred embodiment, the organic solvent is selected from methanol, ethanol, tetrahydrofuran, dichloromethane, methyl tertiary ether, or a combination thereof.

In another preferred embodiment, R¹In step (2), the compound Z is subjected to a reaction shown as formula C to remove R¹(protecting group), thereby producing intermediate W;

in another preferred embodiment, the elimination of R is performed¹The protection is selected from: palladium carbon catalyzes hydrogen to carry out deprotection, protonic acid catalyzes deprotection and alkaline deprotection.

In a fifth aspect of the present invention, there is provided a process for the preparation of chloramphenicol, comprising the steps of:

(I) preparing compound W using the process of the fourth aspect of the invention;

(II) preparation of chloramphenicol using the compound W as a substrate.

In another preferred example, the method includes a route shown as route two:

route two

Wherein compound X is compound 13 of route two, compound Y is compound 14 of route two, and compound W is compound 9 of route two.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 shows a spectrum of chiral purity of compound 14 obtained in example 3.

Detailed Description

The present inventors have made extensive and intensive studies and, as a result of extensive screening, have unexpectedly developed for the first time a process for the biological preparation of compound Y (e.g., compound 14). Specifically, the biological preparation method of the invention takes a compound X (such as a compound 13) as a raw material, takes carbonyl reductase as a biocatalyst, efficiently prepares a compound Y (such as a compound 14) with a three-dimensional conformation (the reduction yield is more than 98%, the chiral ee value is more than 99%, and the chiral de value is more than 70%) in the presence of coenzyme, and constructs two chiral centers through one-step reaction, thereby replacing the method of wasting invalid enantiomers through reduction resolution in the existing synthesis process, greatly shortening the process route, improving the production efficiency and reducing the production cost. In addition, the method of the invention eliminates the water body pollution of aluminum salt caused by using aluminum trichloride and aluminum isopropoxide in the existing production route, thereby obviously reducing the risk of environmental pollution. On the basis of this, the present invention has been completed.

Term(s) for

Enantiomeric excess (ee, enantiomeric excess): are commonly used to characterize the excess of one enantiomer relative to the other in chiral molecules.

Diastereomeric excess (de, diasteromeric processes): are commonly used to characterize the excess of one diastereomer over another in molecules with two or more chiral centers.

(R, S) -carbonyl reductases

As used herein, "carbonyl reductase" of the invention "," R, S) -carbonyl reductase "are used interchangeably and refer to carbonyl reductases that catalyze the reaction of formula A described herein.

In the present invention, "stereoselective carbonyl reductase" refers to an enzyme capable of stereoselective asymmetric catalytic reduction of a prochiral ketone to a chiral alcohol.

Typically, in the present invention, the stereoselective carbonyl reductase is preferably (R, S) -carbonyl reductase, and stereoselectivity is defined as enantiomeric excess (ee) ≥ 80%, diastereomeric excess (de) ≥ 70%.

In the same way, when the enzyme is an (R, R) -carbonyl reductase, stereoselectivity is defined as enantiomeric excess (ee) of 80% or more, diastereomeric excess (de) of 70% or more, and so on.

In the present invention, the configuration is defined by referring to compound Y, wherein the configuration of hydroxyl is R, the configuration of amino is S, and any carbonyl reductase capable of stereoselectively recognizing the S-configuration of amino in compound X and reducing the carbonyl in compound X to R-configuration hydroxyl is defined as (R, S) -carbonyl reductase in the present invention.

In the present invention, the carbonyl reductase may be wild-type or mutant. Furthermore, they may be isolated or recombinant.

Carbonyl reductases useful in the present invention may be from different species. A typical carbonyl reductase has the amino acid sequence shown in SEQ ID No: 1 and the coding gene is shown as SEQ ID No. 2.

SEQ ID No：1

MKYTVITGASSGIGYETAKLLAGKGKSLVLVARRTSELEKLRDEVKQISPDSDVILKSVDLADNQNVHDLYEGLKELDIETLINNAGFGDFDLVQDIELGKIEKMLRLNIEALTILSSLFARDHHDIEGTTLVNISSLGGYRIVPNAVTYCATKFYVSAYTEGLAQELQKGGAKLRAKVLAPAATETEFVDRARGEAGFDYSKNVHKYHTAAEMAGFLHQLIESDAIVGIVDGETYEFELRGPLFNYAG

SEQ ID No：2

aagtacacggtcattacaggagcaagttcaggaattggatatgagacagcaaaactactcgcaggaaaaggaaaatcactcgtcctcgtcgcacggcggacgtctgagctcgaaaaacttcgggatgaagtcaaacaaatctcaccagatagtgatgtcatcctcaagtcggtcgatctcgcagataaccaaaatgtccatgatttatatgagggactaaaggaactcgacatcgagacgctcatcaacaatgctggattcggcgattttgatctcgtccaggacattgagctcgggaaaatcgagaaaatgctccgcttgaacatcgaggcgctgacgattctatcgagtctgttcgcacgcgatcatcatgacatcgaaggaacgacactcgtcaatatctcgtcactcggtggctaccggatcgttccgaacgcggtcacgtattgcgcgacgaagttctatgtcagtgcctatacggaagggctagcgcaagaactgcaaaaaggcggggcaaaactccgggcgaaagtactggcaccagctgcgactgagacagagtttgtcgatcgtgcacgcggcgaagcagggttcgactacagcaagaacgtccataagtaccatacggcggctgagatggcaggcttcttgcatcagttgatcgaaagtgacgcgatcgtcggcatcgtcgacggtgagacgtatgagttcgaattgcgtggtccgttgttcaactacgcaggataa

Due to codon degeneracy, the nucleotide sequence encoding SEQ ID NO: 1 is not limited to the nucleotide sequence of SEQ ID NO: 2. the homologues of this base sequence may be obtained by those skilled in the art by appropriately introducing substitutions, deletions, alterations, insertions or additions, and the present invention encompasses these homologues as long as the recombinant enzyme expressed therefrom retains the catalytic reduction activity for compound X. The polynucleotide of the present invention may be homologous by aligning the nucleotide sequences of SEQ ID NO:2 by substitution, deletion or addition of one or more bases within a range where the enzyme activity is maintained.

The carbonyl reductases of the present invention also include the enzymes of SEQ ID NOs: 1, and (b) the amino acid sequence obtained by performing substitution, deletion, change, insertion or addition of one or more amino acids within the range of keeping the enzyme activity.

In the present invention, the (R, S) -carbonyl reductase may be used in various forms. For example, resting cells or wet cells expressing the carbonyl reductase of the present invention may be used, various forms such as crude enzyme solution, pure enzyme or crude enzyme powder may be used, or immobilized enzyme may be used.

Preferably, crude enzyme solutions are preferably used for higher conversion efficiency and cost reduction.

The ratio of the amount of carbonyl reductase to the amount of substrate is preferably 0.1 to 20%, preferably 1 to 6% (w/w) (based on the mass of the enzyme and the mass of the substrate), or the ratio of the mass of resting cells to the mass of substrate is 1 to 200%, preferably 10 to 100%.

Coenzyme

In the present invention, "coenzyme" means a coenzyme capable of effecting electron transfer in a redox reaction.

Typically, the coenzyme of the invention is a reducing coenzyme NADH, NADPH or an oxidizing coenzyme NAD⁺、NADP⁺. Since the reducing coenzyme is expensive, the oxidizing coenzyme NAD is preferred⁺、NADP⁺。

When the oxidative coenzyme is selected, a method for realizing coenzyme regeneration needs to be selected, and the method mainly comprises three types of (1) glucose dehydrogenase and cosubstrate glucose; (2) alcohol dehydrogenase and co-substrate isopropanol; (3) formate dehydrogenase co-substrate ammonium formate.

In a preferred embodiment, the coenzyme is NADP⁺The coenzyme regenerating system is glucose dehydrogenase or oxidative coenzyme NADP⁺The ratio of the dosage to the substrate dosage is 0.01 percent to 0.5 percent (w/w), and the buffer system is 0.1mol/L phosphate buffer salt. The pH of the buffer is 6.0-10, preferably 7.0-9.0.

Cosolvent

In the present invention, a co-solvent may be added or not added to the reaction system.

As used herein, the term "co-solvent" refers to a sparingly soluble substance that forms a soluble intermolecular complex, association, double salt, or the like with an added third substance in a solvent to increase the solubility of the sparingly soluble substance in the solvent. This third material is referred to as a co-solvent.

In the present invention, the substrate compound X is hardly soluble in water, and when the substrate concentration is increased, the reaction conversion rate is seriously affected. Therefore, it is necessary to improve the substrate solubility by adding a cosolvent to improve the reaction conversion. Optional co-solvents are dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene and acetone, preferably at concentrations of 5-30% (v/v), preferably dimethyl sulfoxide, methanol, ethanol and isopropanol.

Principle of dynamic reduction kinetic resolution reaction

The carbonyl reductase stereoselectively reduces the prochiral ketone with a single configuration (such as R-configuration), and simultaneously, the prochiral ketone with another configuration (such as S-configuration) interconverts through enol of carbonyl, so that racemization of alpha-chiral configuration is realized, and reduction and racemization are carried out under the same reaction condition, and the purpose of efficiently constructing secondary alcohol containing two chiral centers is realized.

Note: EWG is an electron withdrawing group

Typically, in the invention, carbonyl reductase only recognizing X-R (R configuration compound X) is obtained by screening, and chiral hydroxyl is obtained by stereoselectively reducing carbonyl, while unidentified X-S (S configuration compound X) is converted into X-R by racemization, and racemization is carried out in conjunction with the reduction process, so that the conversion can theoretically obtain 100% of the required chiral product. The method has the advantages that the carbonyl reductase is used for reducing the latent chiral carbonyl substrate, meanwhile, two chiral centers are constructed efficiently and economically through enol tautomerism and ingenious combination of enol tautomerism and enol tautomerism, and good application and development prospects are expressed.

The main advantages of the invention are:

(1) provides a biological preparation method of a key intermediate 14, which relates to the dynamic reduction kinetic resolution mediated by carbonyl reductase, wherein the reduction yield is more than 98 percent, the chiral ee value is more than 99 percent, and the de value is more than 70 percent.

(2) After breakthrough is made in the synthesis of the key intermediate, a new process route for synthesizing chloramphenicol is provided.

(3) The biological preparation method has the characteristics of environmental protection and economy, is obviously improved compared with the prior art, and provides a solution with a prospect for solving the problems of a large amount of aluminum salt, long reaction operation steps and the like in the existing production process.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Material

The complete synthesis of the gene is completed by Shanghai Baili lattice.

The coding gene is obtained by commercial total gene synthesis, then the coding gene is constructed into an expression vector and is introduced into host bacteria to obtain carbonyl reductase through induced expression.

The enzyme-reduced substrate compound X was prepared as described in tetrahedron.2016,72: 1787-1793.

Method

1. Process for producing enzyme

The glucose dehydrogenase for coenzyme regeneration and the target gene are constructed on the same plasmid pET28a (+) vector by the conventional technology in the field, and then are introduced into an expression host escherichia coli to obtain the thallus containing the target double enzymes by induction expression. The bacteria can be obtained directly by centrifugation, or crude enzyme solution can be obtained by breaking the walls of the bacteria, and the crude enzyme powder is used for subsequent biotransformation reaction.

2. Method for preparing compound Y by biocatalytically reducing compound X

The invention provides a method for preparing a compound Y by catalyzing and reducing a compound X by carbonyl reductase. The reaction formula is as follows:

wherein the biological catalytic system comprises carbonyl reductase and coenzyme. The carbonyl reductase coding gene sequence disclosed by the invention is SEQ ID NO:2, the amino acid sequence of the carbonyl reductase is SEQ ID NO: 1. the carbonyl reductase gene can be obtained by a commercial total gene synthesis according to the general knowledge in the art.

According to the preferred system, the preparation method is implemented as follows: dissolving substrate in cosolvent such as dimethyl sulfoxide or isopropanol, adding into phosphate buffer solution, stirring, adding thallus, crude enzyme solution, crude enzyme powder or pure enzyme, adding coenzyme NADP⁺Co-substrate glucose, maintained at 20-40 ℃ and monitored by TLC or HPLC until the starting material remains<2%, the reaction was terminated. Adding isopropanol into the reaction solution, centrifuging or passing through ceramic membrane, removing thallus, collecting supernatant, and extracting the supernatant with organic solvent such as methyl tert-butyl ether, toluene, ethyl acetate, isopropyl acetate, dichloromethane, 2-methyltetrahydrofuran, and n-butanol. Extracting the water layer for 2-3 times, and mixing the organic phases; washing with saturated saline water for 2-3 times, and concentrating to obtain light yellow oily matter.

The final concentration of the substrate compound in the system is 10-200g/L, the reaction temperature is 20-40 ℃, the rotation speed is 200rpm/min, the reaction time is about 3-10h, the conversion condition of the raw materials is monitored according to the substrate concentration or by HPLC, and the reaction is stopped when the residual of the raw materials is less than 2%.

3. The chiral normal phase monitoring method of the compound Y comprises the following steps:

HPLC conditions: daicel IB-3 (250X 4.6mm, 3 μm); the flow rate is 0.8 ml/min; mobile phase: n-hexane to isopropanol 95: 5; the ultraviolet detection wavelength is 224 nm; the column temperature is 30 ℃; the sample is dissolved in methanol with the concentration of 10 mg/ml; the injection volume was 2. mu.l.

4. Reverse phase monitoring method for compound Y:

HPLC conditions: phenomenex Gemini 5u C18110A, 250 × 4.6mm,5 μm; flow rate: 1 ml/min; mobile phase gradients are as follows; ultraviolet detection wavelength: 224 nm; column temperature: 30 ℃; sample concentration: 10 mg/ml; the injection volume was 10. mu.l.

Gradient of mobile phase:

time (min)	H2O-0.1％HCOOH(％)	ACN(％)
			0	80	20
15	20	80
			35	20	80
35.1	80	20
			40	20	80

Example 1: construction of carbonyl reductase engineering bacteria

The EA carbonyl reductase target gene and the glucose dehydrogenase target gene are entrusted to a commercial company for whole-gene synthesis, cloned into a pET28a (+) vector, transferred into escherichia coli DH5 alpha competent cells, subjected to plate culture, a single colony of a positive transformant is selected, extracted and determined by plasmid sequencing, a recombinant plasmid is extracted, introduced into a BL21(DE3) strain, and subjected to LB culture to obtain the genetically engineered bacteria capable of inducing and expressing the recombinant carbonyl reductase and the alcohol dehydrogenase.

Example 2: preparation of recombinant carbonyl reductase and glucose dehydrogenase

Inoculating the genetically engineered bacteria preserved in glycerol in the previous step into LB liquid culture medium containing kanamycin, culturing at 37 deg.C and 220rpm for 13h to obtain seed culture medium, inoculating the seed culture solution to liquid culture medium containing 50ug/ml kanamycin resistance according to a proportion of 1.5%, and culturing at 37 deg.C and 220rmp to OD₆₀₀Value of>2.0, adding lactose with the final concentration of 1.0%, cooling to 25 ℃, continuing to culture for 3h, adding lactose with the final concentration of 0.5%, culturing for 20h, canning, and centrifuging to obtain thalli, thus preparing for biological transformation.

The fermentation formula is as follows:

example 3: biocatalytic preparation of Compound Y (Compound 14)

Wherein R is¹Is composed of

R²Is composed of

0.1M phosphate buffer (100ml) was added with NADP⁺(0.1g) addingAdding glucose 25g, adding thallus EA (5g) obtained by the above fermentation, adjusting pH to 7.3-8.0 with 1M sodium hydroxide aqueous solution, adding compound 13(10g) in DMSO (30ml) by batch under vigorous stirring, adjusting pH at 25 deg.C and 220rpm, performing shake reaction at interval of 0.5h, and monitoring reaction conversion rate by HPLC>At 98%, the reaction was terminated.

Centrifuging, sterilizing, taking supernatant, adding isopropanol/methyl tertiary ether (v/v ═ 1/3, 100ml) for extraction, extracting an aqueous layer by isopropanol/methyl tertiary ether, combining organic layers, washing with saturated common salt water, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain light yellow oily matter 4.8g, wherein the product configuration is (R, S) -14 (compound 14), the chiral ee value of the product is 99.9%, and the chiral ee value of the product is de 70%. The nuclear magnetic data are:¹H NMR(600MHz,CDCl₃)δ8.17(t,J＝12.8Hz,2H),7.54(t,J＝12.8Hz,2H),5.36(t,J＝12.7Hz,2H),4.57(d,J＝8.1Hz,1H),3.78(s,3H),3.53(s,1H),1.26(d,J＝17.0Hz,9H).¹³C NMR(151MHz,CDCl₃)δ170.82,147.54,147.31,127.05,123.45,80.56,77.28,77.07,76.85,73.06,59.16,52.87,28.26,28.08.

EXAMPLE 4 Synthesis of Compound W

Adding methanol into compound 14(2g) to partially dissolve, stirring in a suspension at the temperature of more than 5 ℃ in an ice bath, adding potassium borohydride (0.64g) in batches, gradually dissolving a solid, changing the color from yellow to yellow and then changing to light yellow, continuing to react for 1h in the ice bath, adding 5ml of concentrated hydrochloric acid when TLC shows that the raw materials are basically reacted, separating out a large amount of salt, heating to 60 ℃, reacting for 2h, TLC (CH2Cl2: MeOH-2; 1, Et3N 2d) shows that the raw materials are basically disappeared, adjusting the pH to 7-8 with NaOH, concentrating methanol and water to dry solid, adding a methanol-soluble product, filtering the salt, concentrating to dry to obtain compound 9, and then preparing for later use.

EXAMPLE 5 Synthesis of Chloramphenicol (1)

And (3) adding methyl dichloroacetate into the crude product obtained in the previous step, and carrying out reflux reaction in methanol to obtain a crude chloramphenicol product.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai institute for pharmaceutical industry

China Pharmaceutical Industry Research Institute

<120> novel biological preparation method of amino alcohol drug intermediate

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Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu

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Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala

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

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Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn

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Gln Asn Val His Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu

65 70 75 80

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

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Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Ala

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Leu Thr Ile Leu Ser Ser Leu Phe Ala Arg Asp His His Asp Ile Glu

115 120 125

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

130 135 140

Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr

145 150 155 160

Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg

165 170 175

Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Val Asp Arg

180 185 190

Ala Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val His Lys Tyr

195 200 205

His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser

210 215 220

Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu

225 230 235 240

Arg Gly Pro Leu Phe Asn Tyr Ala Gly

245

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aagtacacgg tcattacagg agcaagttca ggaattggat atgagacagc aaaactactc 60

gcaggaaaag gaaaatcact cgtcctcgtc gcacggcgga cgtctgagct cgaaaaactt 120

cgggatgaag tcaaacaaat ctcaccagat agtgatgtca tcctcaagtc ggtcgatctc 180

gcagataacc aaaatgtcca tgatttatat gagggactaa aggaactcga catcgagacg 240

ctcatcaaca atgctggatt cggcgatttt gatctcgtcc aggacattga gctcgggaaa 300

atcgagaaaa tgctccgctt gaacatcgag gcgctgacga ttctatcgag tctgttcgca 360

cgcgatcatc atgacatcga aggaacgaca ctcgtcaata tctcgtcact cggtggctac 420

cggatcgttc cgaacgcggt cacgtattgc gcgacgaagt tctatgtcag tgcctatacg 480

gaagggctag cgcaagaact gcaaaaaggc ggggcaaaac tccgggcgaa agtactggca 540

ccagctgcga ctgagacaga gtttgtcgat cgtgcacgcg gcgaagcagg gttcgactac 600

agcaagaacg tccataagta ccatacggcg gctgagatgg caggcttctt gcatcagttg 660

atcgaaagtg acgcgatcgt cggcatcgtc gacggtgaga cgtatgagtt cgaattgcgt 720

ggtccgttgt tcaactacgc aggataa 747

Claims (10)

1. A process for the preparation of chloramphenicol, comprising the steps of: (1) in a liquid reaction system, a compound X is used as a substrate, and in the presence of coenzyme, the reaction is carried out in the presence ofR,S) -carbonyl reductase, thereby forming compound Y;

formula A

Wherein the carbonyl reductase is carbonyl reductase EA, and the amino acid sequence of the carbonyl reductase is shown in SEQ ID NO: 1 is shown in the specification;

(2) r of compound Y obtained by reduction of carbonyl reductase²The ester bond in (b) is reduced to a hydroxyl group, thereby producing a compound Z;

；

(3) removing R from compound Z¹Thereby preparing an intermediate W;

；

and step (4), preparing chloramphenicol from the compound W;

wherein R is¹Is composed of

；

R²Is composed of

；

R³Is methyl.

2. The method of claim 1, wherein the coenzyme is selected from the group consisting of: NADH, NADPH, NAD, NADP, or a combination thereof.

3. The method according to claim 2, wherein the ratio of the amount of NADH, NADPH, NAD, or NADP to the amount of the substrate is 0.01 to 1.0 w/w%.

4. The method according to claim 1, wherein the ratio of the amount of NADH, NADPH, NAD, or NADP to the amount of the substrate is 0.01 to 0.5 w/w%.

5. The process according to claim 1, wherein the ee value of the compound Y is 90% or more and the de value is 70% or more in the reaction system after the reaction.

6. The process according to claim 1, wherein the ee value of the compound Y is 95% or more and the de value is 90% or more in the reaction system after the reaction.

7. The method according to claim 1, wherein the reaction system after the reaction has a conversion rate of 80% or more of compound X to compound Y.

8. A reaction system, characterized in that the reaction system comprises:

(i) an aqueous solvent;

(ii) a substrate, said substrate being compound X;

(iii) a coenzyme;

(iv) (R,S) -a carbonyl reductase having the sequence shown as SEQ ID number 1;

(v) a co-substrate; and

(vi) an enzyme for coenzyme regeneration;

in the compound X, the compound X is a compound,

R¹selected from:

；

R²selected from:

。

9. a process for the preparation of compound Y, comprising the steps of: use of the reaction system as claimed in claim 8 in (A), (B), (C) and C)R,S) -carrying out the reaction of formula a under carbonyl reductase enzymatic conditions, thereby obtaining compound Y;

wherein the content of the first and second substances,

R¹selected from:

；

R²selected from:

。

10. the method of claim 1, wherein the method comprises a route shown as route two: route two

Wherein compound X is compound 13 of route two, compound Y is compound 14 of route two, and compound W is compound 9 of route two.

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