CN109207531B - Biological preparation method of thiamphenicol and florfenicol key intermediate - Google Patents

Abstract

The invention provides a biological preparation method of a thiamphenicol and florfenicol key 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

Biological preparation method of thiamphenicol and florfenicol key intermediate

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method of key chiral intermediates of amino alcohol antibacterial drugs thiamphenicol and florfenicol.

Background

Thiamphenicol antibiotics include thiamphenicol (1) and florfenicol (2) which is a fluoro-compound thereof. They are similar in chemical structure to the obsolete antibiotic chloramphenicol (3), and are beta-aminoalcohol antibiotics^[1]。

The chemical structure of the amino alcohol medicine is as follows:

thiamphenicol is artificially synthesized for the first time in 1952, has the characteristics of strong antibacterial activity, quick absorption, lasting drug effect and low toxicity, is mainly used for treating typhoid fever, dysentery, respiratory tract infection, urinary tract infection, hepatobiliary system infection, intestinal tract infection, surgical infection, brucellosis, meningitis and other diseases clinically, and has no reports of fatal aplastic anemia and gray infant syndrome. The veterinary drug has obvious effect on preventing and treating epidemic diseases of livestock and poultry, is mainly used for controlling respiratory tract and gastrointestinal tract infection of cattle and poultry, and is used for treating various infectious diseases of pigs, sheep and fishes.

Florfenicol, also known as florfenicol, is a drug developed by the Prolin-Baoya company in the United states in the 70 s for finding better chloramphenicol derivatives, is sold in 20 countries such as Japan, Korea, Norway, France and the like in sequence, mainly meets the requirements of animal health care markets, and is produced in large quantities in China at present. Florfenicol passed the U.S. FDA registration in 1996 and became a new generation of feed antibiotics substituting for chloramphenicol and thiamphenicol. Florfenicol is a broad-spectrum antibiotic, has the characteristics of wide antibacterial spectrum, good absorption, wide in vivo distribution, safety, high efficiency and the like, is a first choice drug for treating bacterial animal diseases caused by typhoid bacillus, paratyphoid bacillus, salmonella and the like, has antibacterial activity superior to that of chloramphenicol and thiamphenicol (the minimum inhibitory concentration MIC is about 10 times lower), and also has obvious effects on large intestine bacteria, staphylococcus aureus, Klebsiella and the like which are resistant to chloramphenicol and thiamphenicol.

Thiamphenicol and florfenicol have been on the market for nearly 30 years, and on one hand, due to good antibacterial effect and structural characteristics of amino alcohol, the thiamphenicol and florfenicol attract research interest of synthetic pharmacologists in various countries all the time; on the other hand, the chemical structures of the two are only different from that of a fluorine atom, and in the existing industrial production route, the two share a key intermediate 6, and the structural formula is as follows:

the prior art reports about 20 routes for synthesizing thiamphenicol and florfenicol.

Route one: preparation of Compound 6 in existing Industrial production route

The structures of thiamphenicol and florfenicol both contain two chiral centers, so the key of the synthetic route is the construction method of the chiral centers. The existing production route is reported in patents DE1938513, US3927054 and US5382673, and is constructed by adopting a traditional resolution method. First, methyl sulfone benzaldehyde is used as an initial raw material, under the alkaline condition, copper sulfate is used for catalyzing and is condensed with glycine to obtain copper salt of a threo-type compound 3, and the erythroid content is less than 1%. 3, forming ester with ethanol, splitting by L- (+) -tartaric acid to obtain a compound 5, and reducing the compound 5 by sodium borohydride to obtain an amino alcohol key chiral intermediate 6. The reaction steps are shown as formula I:

reagents and reaction conditions: (a) glycine, NH₃H₂O,CuSO₄(b)EtOH,SOCl₂,H₂S(c)L-(+)-TA,MeOH(d)NaBH₄。

The prior production line has three disadvantages: (1) in the reaction, a large amount of copper sulfate is used, the color of the copper sulfate seriously pollutes water, and the copper sulfide formed by the copper sulfate and hydrogen sulfide generates solid waste pollution, so that the use of copper salt brings severe environmental protection pressure; (2) the theoretical yield of the traditional resolution is 50 percent; (3) the resolving agent needs to be recycled and reused.

And a second route: preparation of compound 6 by metal ligand catalytic asymmetric synthesis

Route two was reported by the aged Final academy team in tetrahedron.2016,72:1787-1793 in my country. In the method, methylsulfonylbenzoic acid is used as an initial raw material, acyl chloride is formed by the methylsulfonylbenzoic acid and thionyl chloride, amide 10 is formed by glycine ethyl ester, a compound 12 is obtained by molecular rearrangement after nitrogen atoms are protected by di-tert-butyl carbonate, the compound 12 is transferred and hydrogenated under the participation of a metal ligand, two chiral centers are constructed by one-step reaction, a researcher synthesizes and screens the ligand, unfortunately, the configuration of hydroxyl in a product 13 obtained by reduction is an unnecessary configuration, so that the required hydroxyl configuration 17 is obtained by subsequent activation of methylsulfonyl chloride, turning over of acetic acid negative ions, hydrolysis, and deprotection, and the amino alcohol key intermediate 6 is obtained.

Compared with the existing production route, the second route has two advantages: firstly, the introduction of copper sulfate is avoided from the aspect of route design; secondly, a dynamic kinetic resolution method involving metal ligands is skillfully designed in the route, two chiral centers are constructed through one-step reaction, the theoretical yield is 100%, and the limitation of 50% of the traditional resolution is broken through. Unfortunately, the configuration of the hydroxyl group in the resulting reduction product is not required and requires inversion to achieve the desired configuration. The reaction steps are shown as formula III:

reagents and reaction conditions (a) SOCl₂,cat.DMF,CH₂Cl₂Refluxing for 2 h; (b) glycine ethyl ester hydrochloride, Na₂CO₃,EtOAc/H₂O,rt,1h；(c)Boc₂O,cat.DMAP,CH₃CN,rt,3h；(d)t-BuOK,THF,10℃,1h,71％；(e)RuCl₂(arene)₂,ligand,HCOONa,Tween 20,25℃；(f)MsCl,Et₃N,CH₂Cl₂,0-10℃,1h,94％；(g)LiBH₄,THF,rt,3h,81％；(h)DBU/AcOH(1:2.2),toluene,90℃,15h,74％；(i)K₂CO₃,MeOH,rt,2h,90％；(j)TFA,CH₂Cl₂,rt,2h,92％。

And a third route: resolution of amidohydrolase to prepare compound 6

Bernard et al in Organic Process Research & Development 1998,2:10-17 reported a method of preparing key intermediate 6 using amidohydrolase resolution. In the method, methylsulfonyl benzaldehyde is used as an initial raw material, aldol condensation is carried out on methylsulfonyl benzaldehyde and glycinamide to prepare a threo-type main product 22, 22 is deiminated under an acidic condition to obtain an enzyme hydrolysis substrate, researchers carry out a large amount of screening work on amidohydrolase, finally screen out an Ochrobactrum anthracroi NCIMB 40321, a single configuration 24(ee is more than 99%) is obtained with high stereoselectivity, and racemization can be realized by adding equivalent methylsulfonyl benzaldehyde into an unhydrolyzed substrate under an alkaline condition to obtain 22. 24 key intermediates 6 and 6 are prepared by sodium borohydride reduction, and thiamphenicol and florfenicol can be synthesized by the known process. The disadvantage of this route is that the theoretical yield of resolution is 50%, and then two equivalents of methylsulfonylbenzaldehyde are used, increasing the cost of the raw materials. The reaction step is shown as formula IV:

reagents and reaction conditions: (a) glycinamide hydrochloride, NaOH, H₂O-MeOH.；(b).HCl.；(c).NCIMB 40321amidase.；(d).NaBH₄,H₂SO₄。

The existing production process has the three problems, so that the environmental challenge is increased along with the increase of the environmental protection pressure. Researchers in various countries have conducted extensive research on the novel synthetic route, particularly the new synthetic route continuously reported by the team of old Fender institutions in China. Although the second route is reasonable and ingenious in design, a proper metal ligand is not screened, and the hydroxyl configuration in the transfer hydrogenation reduction product does not need the configuration, and complicated three-step reactions of activation, turnover and hydrolysis are required. In conclusion, the synthetic route of thiamphenicol and florfenicol with industrial application prospects is urgently needed to be developed.

Disclosure of Invention

The invention aims to provide a biological preparation method of a thiamphenicol and florfenicol key intermediate W (compound 6).

The method relates to a dynamic reduction kinetic resolution technology involving carbonyl reductase, and two chiral centers are constructed through one-step 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,

R₁and R₂Each independently selected from: H.

R₃selected from the group consisting of:

R₄selected from the group consisting of:

a first aspect of the present invention provides 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;

in the formula A, the reaction solution is prepared,

R₁and R₂Each independently selected from: H.

R₃selected from the group consisting of:

R₄selected from the group consisting of:

in another preferred embodiment, R is₁＝H,

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 step (a), the temperature is from 10 ℃ to 50 ℃, preferably from 20 ℃ to 40 ℃, more preferably from 25 ℃ to 35 ℃.

In another preferred embodiment, in step (a), 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 step (a), the pH is from 6 to 10, preferably from 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 a combination 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 13) 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 90%, preferably greater than or equal to 95%, more preferably greater than or equal to 99%.

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 12) into compound Y (e.g., compound 13).

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 second aspect the present invention provides 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, R₁、R₂、R₃And R₄As defined above.

In another preferred embodiment, in the compound X, R₁＝H,

In another preferred embodiment, in the compound X, R₁＝H,

In a third aspect, the present invention provides 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:

in the formula A:

R₁、R₂、R₃and R₄As defined above.

In a fourth aspect, the present invention provides a process for preparing intermediate W, comprising the steps of:

(1) the compound Y, wherein R is prepared by the process according to the first aspect of the invention or the third aspect of the invention₁，R₂As defined above, the above-mentioned,

R₅is methyl, ethyl, isopropyl or tert-butyl;

(2) intermediate W (Compound 6) was prepared using Compound Y as a substrate.

In another preferred embodiment, in step (2), the carbonyl reductase is reduced to obtain carbonyl reductaseR of Compound Y₄To a hydroxyl group, thereby producing compound Z:

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), compound Z is deprived of R₂(protecting group), thereby producing intermediate W (Compound 6):

in another preferred embodiment, the elimination of R is performed₂(protecting group) protection is selected from: palladium carbon catalyzes hydrogen to carry out deprotection, protonic acid catalyzes deprotection and alkaline deprotection.

The fifth aspect of the invention provides a preparation method of thiamphenicol, which comprises the following steps:

(1) the compound Y, wherein R is prepared by the process according to the first aspect of the invention or the third aspect of the invention₁＝H，R₂As defined above, the above-mentioned,

R₅is methyl, ethyl, isopropyl or tert-butyl;

(2) preparing an intermediate W (compound 6) by using the compound Y as a substrate;

(3) under proper conditions, the compound 6 reacts with methyl dichloroacetate to prepare thiamphenicol, and the reaction formula is as follows:

in another preferred embodiment, in the preparation method, the synthetic route of thiamphenicol is as follows:

wherein R is₁＝H，R₂As defined above, the above-mentioned,

R₅is methyl, ethyl, isopropyl or tert-butyl.

The sixth aspect of the invention provides a preparation method of florfenicol, which comprises the following steps:

(1) preparing compound Y by a process according to the first or third aspect of the invention; wherein R is₁＝H，R₂As defined above, the above-mentioned,

R₅is methyl, ethyl, isopropyl or tert-butyl;

(2) preparing an intermediate W (compound 6) by using the compound Y as a substrate;

(3) under appropriate conditions, compound 6 is protected, substituted by fluorine, and methyl dichloroacetate is converted into amide, and the reaction formula is as follows:

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 liquid phase diagram of the four isomers of the mesomeric compound Y in example 3.

FIG. 2 shows a liquid phase diagram of chiral purity of compound Y prepared by the enzymatic conversion reaction in example 3.

Detailed Description

The present inventors have made extensive and intensive studies and, for the first time, have unexpectedly found a process for the biological production of a compound Y (e.g., compound 13). Specifically, the biological preparation method of the invention takes a compound X (such as a compound 12) as a raw material, takes carbonyl reductase as a biocatalyst, efficiently prepares a compound Y (such as a compound 13) 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 97%) in the presence of coenzyme, and constructs two chiral centers through one-step reaction, thereby greatly improving the production efficiency and reducing the production cost. In addition, the method greatly simplifies the subsequent treatment, and compared with the existing production route, the method eliminates the use of copper sulfate, and obviously reduces or eliminates the use of various polluting chemicals, thereby obviously reducing the risk of environmental pollution.

The method only needs extraction operation, is simple to operate, low in cost, green and environment-friendly, and is more suitable for industrial production of the key intermediate W, namely the compound 6 with high chemical purity and high optical purity, so that the method is used for preparing medicines such as thiamphenicol, florfenicol and the like.

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

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) ≥ 80%.

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 80% 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 an amino acid sequence shown in SEQ ID No.1, and a coding gene shown in SEQ ID No. 2.

SEQ ID No.：1

MKYTVITGASSGIGYETAKLLAGKGKSLVLVARRTSELEKLRDEVKQISPDSDVILKSVDLADNQNVHDLYEGLKELDIETLINNAGFGDFDLVQDIELGKIEKMLRLNIEALTILSSLFARDHHDIEGTTLVNISSLGGYRIVPNAVTYCATKFYVSAYTEGLAQELQKGGAKLRAKVLAPAATETEFVDRARGEAGFDYSKNVHKYHTAAEMAGFLHQLIESDAIVGIVDGETYEFELRGPLFNYAG

SEQ ID No.：2

aagtacacggtcattacaggagcaagttcaggaattggatatgagacagcaaaactactcgcaggaaaaggaaaatcactcgtcctcgtcgcacggcggacgtctgagctcgaaaaacttcgggatgaagtcaaacaaatctcaccagatagtgatgtcatcctcaagtcggtcgatctcgcagataaccaaaatgtccatgatttatatgagggactaaaggaactcgacatcgagacgctcatcaacaatgctggattcggcgattttgatctcgtccaggacattgagctcgggaaaatcgagaaaatgctccgcttgaacatcgaggcgctgacgattctatcgagtctgttcgcacgcgatcatcatgacatcgaaggaacgacactcgtcaatatctcgtcactcggtggctaccggatcgttccgaacgcggtcacgtattgcgcgacgaagttctatgtcagtgcctatacggaagggctagcgcaagaactgcaaaaaggcggggcaaaactccgggcgaaagtactggcaccagctgcgactgagacagagtttgtcgatcgtgcacgcggcgaagcagggttcgactacagcaagaacgtccataagtaccatacggcggctgagatggcaggcttcttgcatcagttgatcgaaagtgacgcgatcgtcggcatcgtcgacggtgagacgtatgagttcgaattgcgtggtccgttgttcaactacgcaggataa

Due to the degeneracy of codons, the base sequence encoding the amino acid sequence shown in SEQ ID NO.1 is not limited to only 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 homologue of the polynucleotide of the present invention can be produced by substituting, deleting or adding one or more bases of the base sequence SEQ ID NO.2 within a range in which the activity of the enzyme is maintained.

The carbonyl reductases of the present invention also include a catalytic activity 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 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 solution is 6.0-10.

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 12 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 cosolvent is dimethyl sulfoxide, methanol, ethanol, isopropanol, acetonitrile, toluene, acetone, concentration is preferably 5-30% (v/v), and dimethyl sulfoxide, methanol, ethanol, isopropanol are preferably selected.

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.

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.

Intermediate W (Compound 6) and its use in the Synthesis of downstream products

After the intermediate 6 is synthesized, thiamphenicol and florfenicol can be synthesized by a known process.

Typically, intermediate 6 is reacted with methyl dichloroacetate to form the amide to form thiamphenicol.

Typically, the florfenicol in the existing production route shares a key intermediate 6 with thiamphenicol, the compound 6 reacts with dichloroacetonitrile to form oxazole rings 7-1 and 7-2, the compound 7-2 can be converted into 7-1, 7-1 under the alkaline condition, fluorine atoms are introduced through a fluorine reagent, and then the florfenicol is directly obtained through ring opening. The reaction step is shown as formula II:

reagents and reaction conditions (a) Cl₂CHCN,H₂SO₄i-PrOH,70 ℃,1.5 h; (b) saturated NH₃95 percent of/i-PrOH at 60-80 ℃ for 20 h; (c) ishiawa reagent, CH₂Cl₂,100℃,2h；(d)AcOK,i-PrOH,H2O,pH＝3.5-4.0,rt,10h,85.7％.

The synthesis of thiamphenicol and florfenicol can also be found in DE1938513, US3927054, US 5382673.

The main advantages of the invention are:

(1) provides a biological preparation method of a key intermediate W, namely a compound 6, 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 chiral de value is more than 97 percent.

(2) After breakthrough is made in the synthesis of the key intermediate, a new synthesis route of thiamphenicol and florfenicol 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 copper salt and water pollution existing in the prior 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, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: 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 by weight. The experimental materials referred to in the present invention are commercially available without specific reference.

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 sequence of the gene coding the carbonyl reductase 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 to remove 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 (VI) in the system is 10-200g/L, the reaction temperature is 20-40 ℃, the rotating speed is 200rpm/min, the reaction time is about 3-10h, the conversion condition of the raw materials is monitored according to the change of the substrate concentration or by HPLC, and the reaction is stopped when the residual of the raw materials is less than 2 percent.

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

HPLC conditions: daicel IC-3 (250X 4.6mm, 3 μm); the flow rate is 0.8 ml/min; mobile phase: n-hexane, i-propanol, 80: 20; ultraviolet detection wavelength is 260 nm; the column temperature is 25 ℃; 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: 260 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％TFA(％)	ACN-0.1％TFA(％)
			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, 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:

raw material	Mass content (%)
		Yeast extract	2.4
Soybean peptone	1.2
		Sodium chloride	0.3
Glycerol	0.5
		Dipotassium hydrogen phosphate	0.2
Magnesium sulfate heptahydrate	0.05

Example 3 biocatalytic preparation of Compound Y (configuration (R, S))

Wherein R is₁Is H; r₂Is composed of

R₃Is composed of

R₄Is composed of

0.1M phosphate buffer (100ml) was added with NADP⁺(0.1g), adding glucose 25g, adding the above fermented thallus EA (5g), adjusting pH to 7.3-8.0 with 1M sodium hydroxide aqueous solution, adding DMSO (30ml) solution of compound X (10g) in portions with vigorous stirring, shaking table reacting at 25 deg.C and 220rpm, adjusting pH at intervals of 0.5h, and monitoring reaction conversion rate by HPLC>At 98%, the reaction was terminated.

Centrifuging, removing bacteria, collecting supernatant, adding isopropanol/methyl tert-ether (v/v-1/3, 100ml) for extraction, extracting water layer with isopropanol/methyl tert-ether, combining organic layers, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain light yellow oily substance with product configuration of (R, S) (compound Y).

In order to obtain carbonyl reductase with high stereoselectivity and high reaction conversion rate, the inventors screened carbonyl reductase.

When QNR and KETONE carbonyl reductase catalyze a substrate, the conversion rate is less than 30%, and the chiral purity is difficult to measure, and when CK03 carbonyl reductase catalyzes a substrate, the conversion rate is 95%, ee: 99.2% > 80%, but de: 20.3%, which does not satisfy the (R, S) -carbonyl reductase defined in the present invention, and thus does not belong to the (R, S) -carbonyl reductase.

Example 4 biocatalytic preparation of Compound Y' (configuration (R, R))

Wherein R is₁Is H; r₂Is composed of

R₃Is composed of

R₄Is composed of

0.1M phosphate buffer (25ml) was taken and NAD was added⁺(0.1g), adding 7.5ml of isopropanol, adding cell LK (1g) obtained by the above fermentation, adjusting pH to 7.3-8.0 with 1M aqueous sodium hydroxide solution, adding DMSO (3ml) solution of compound X (2g) in portions with vigorous stirring, 25 ℃, 220rpm, shaking table reaction, and monitoring reaction conversion rate by HPLC>At 98%, the reaction was terminated.

Centrifuging, removing bacteria, taking supernatant, adding isopropanol/methyl tert-ether (v/v-1/3, 100ml) for extraction, extracting an aqueous layer by isopropanol/methyl tert-ether, combining organic layers, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain a light yellow oily product with the configuration of (R, R) (compound Y'), the ee value of 99.7% and the de value of 98.2%.

Example 5 biocatalytic preparation of Compound Y (configuration (R, S))

Wherein R is₁Is H; r₂Is composed of

R₃Is composed of

R₄Is composed of

0.1M phosphate buffer (25ml) was added with NADP⁺(0.01g), glucose 5g, bacterial strain EA (1g) obtained by the above fermentation and 1M aqueous sodium hydroxide solution were added to adjust pH to 7.3-8.0, a DMSO (30ml) solution of Compound X (2g) was added in portions with vigorous stirring, the mixture was subjected to shaking reaction at 25 ℃ and 220rpm, the pH was adjusted every 0.5h, and the reaction conversion was monitored by HPLC>At 98%, the reaction was terminated.

Centrifuging, removing bacteria, taking supernatant, adding isopropanol/methyl tert-ether (v/v-1/3, 100ml) for extraction, extracting an aqueous layer by isopropanol/methyl tert-ether, combining organic layers, washing with saturated saline, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain light yellow oily matter, wherein the product configuration is (R, S) (compound Y), the ee value is 99.4%, and the de value is 98.5%.

EXAMPLE 6 Synthesis of Compound 17

In ice bath, compound 13(5.0) was dissolved in methanol (40ml), temperature was controlled to 5 ℃, sodium borohydride (0.5g) was added in portions and then raised to 50 ℃, LCMS was used to monitor the reaction until the conversion of the starting material was complete, water was added to quench, methanol was partially concentrated, n-butanol was extracted 3 times, water was washed, anhydrous magnesium sulfate was dried, filtered and concentrated to give 4.18g of a pale yellow solid (compound 17).

EXAMPLE 7 Synthesis of Compound 6

The light yellow solid (compound 17) was dissolved in methanol, saturated methanolic hydrogen chloride solution (10ml) was added, heated to 40 ℃, LCMS monitored reaction until complete conversion of starting material, and concentrated to dryness to give a light yellow solid (compound 6).

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 can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Shanghai institute for pharmaceutical industry

China Pharmaceutical Industry Research Institute

Biological preparation method of <120> thiamphenicol and florfenicol key intermediate

<130> P2017-0095

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 249

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

85 90 95

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

100 105 110

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

<210> 2

<211> 747

<212> DNA

<213> Artificial sequence

<400> 2

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 (26)

1. A process for the preparation of compound Y, characterized in that: the method comprises the following steps:

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;

in the formula A, the reaction solution is prepared,

wherein the carbonyl reductase is derived from carbonyl reductase EA, and the amino acid sequence of the carbonyl reductase is shown in SEQ ID No.:1 is shown.

2. The method of claim 1, wherein the coenzyme is selected from the group consisting of: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.

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

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

5. The method according to claim 3, wherein the weight ratio of the amount of NADH, NADPH, NAD, or NADP to the amount of the substrate is 0.01 to 0.5%.

6. The production method according to any one of claims 1 or 2, wherein an enzyme for coenzyme regeneration is further present in the reaction system.

7. The method according to claim 6, wherein the enzyme for coenzyme regeneration is selected from the group consisting of: alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, or a combination thereof.

8. The method according to claim 6, wherein a co-substrate for coenzyme regeneration is further present in the reaction system.

9. The method of claim 8, wherein the co-substrate is selected from the group consisting of: isopropanol, glucose, ammonium formate, or a combination thereof.

10. The method according to claim 8, wherein the concentration of the cosubstrate in the reaction system is 5 to 30%.

11. The method of any one of claims 1 or 2, further comprising: and separating the compound Y from the reaction system after the reaction in the previous step.

12. The preparation method according to claim 11, wherein the ee value of the compound Y in the reaction system after the reaction is not less than 90%; the de value is more than or equal to 90 percent.

13. The preparation method according to claim 11, wherein the reaction system after the reaction has a conversion rate of the compound X to the compound Y of 80% or more.

14. The preparation method according to claim 11, wherein the de value of the compound Y in the reaction system after the reaction is not less than 95%.

15. The process according to any one of claims 12 to 14, wherein the ee value of compound Y in the reaction system after the reaction is 99% or more.

16. The process according to claim 11, wherein the conversion of compound X to compound Y is 85% or more.

17. The process according to claim 11, wherein the conversion of compound X into compound Y is 95% or more.

18. The method according to claim 1, wherein the gene sequence encoding carbonyl reductase EA is as set forth in SEQ ID No.:2, or a pharmaceutically acceptable salt thereof.

19. 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) -carbonyl reductase derived from carbonyl reductase EA having an amino acid sequence as set forth in SEQ ID No.:1 is shown in the specification;

(v) a co-substrate; the co-substrate is selected from the group consisting of: isopropanol, glucose, ammonium formate, or a combination thereof; and

(vi) an enzyme for coenzyme regeneration; in the compound X, R₁、R₂、R₃And R₄As defined in claim 1.

20. A process for the preparation of compound Y, comprising the steps of: performing the reaction of formula a using the reaction system of claim 19 under conditions catalyzed by (R, S) -carbonyl reductase enzymes to produce compound Y:

in the formula A:

R₁、R₂、R₃and R₄As defined in claim 1.

21. A process for preparing intermediate W, comprising the steps of:

(1) the preparation of compound Y by the process of claim 1 or 20, wherein R₁，R₂As defined in claim 1, wherein the first and second groups are,

R₅is methyl or ethyl;

(2) taking a compound Y as a substrate to prepare an intermediate W, namely a compound 6, wherein the intermediate W is

22. The method of claim 21, wherein in step (2), the carbonyl reductase is reduced to obtain R of compound Y₄To a hydroxyl group, thereby producing compound Z:

wherein R is₁、R₂、R₃And R₄And R₅As defined in claim 21.

23. The method of claim 22, wherein R is₁In step (2), compound Z is deprived of R₂To obtain intermediate W:

wherein R is₂As defined in claim 1.

24. A preparation method of thiamphenicol is characterized by comprising the following steps:

(1) the preparation of compound Y by the process of claim 1 or 20, wherein R₁＝H，R₂As defined in claim 1, wherein the first and second groups are,

R₅is methyl or ethyl;

(2) taking the compound Y as a substrate to prepare an intermediate W, namely a compound 6;

(3) under proper conditions, the compound 6 reacts with methyl dichloroacetate to prepare thiamphenicol, and the reaction formula is as follows:

25. the method as claimed in claim 24, wherein in the preparation method, the synthetic route of thiamphenicol is as follows:

wherein R is₁＝H，R₂As defined in claim 1, wherein the first and second groups are,

R₅is methyl or ethyl.

26. A preparation method of florfenicol is characterized by comprising the following steps:

(1) preparing compound Y using the process of claim 1 or 20; wherein R is₁＝H，R₂As defined in claim 1, wherein the first and second groups are,

R₅is methyl or ethyl;

(2) taking a compound Y as a substrate to prepare an intermediate W, namely a compound 6, wherein the intermediate W is

(3) Under appropriate conditions, compound 6 is protected, substituted by fluorine, and methyl dichloroacetate is converted into amide, and the reaction formula is as follows:

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