CN114181984B - Biological preparation method of (S) -1- (4-pyridyl) -1, 3-propanediol (I) - Google Patents

Abstract

The application provides a biological preparation method of (S) -1- (4-pyridyl) -1, 3-propanediol (I). Specifically, the method of the application comprises the following steps: (a) In a reaction system, a compound of a formula IV is used as a substrate, and under the catalysis of carbonyl reductase in the presence of coenzyme, an asymmetric reduction reaction is carried out, so that a compound of a formula V is formed; (b) The compound of formula V is directly or stepwise reduced to obtain the compound of formula I.

Description

Biological preparation method of (S) -1- (4-pyridyl) -1, 3-propanediol (I)

Technical Field

The application belongs to the technical field of medicines, and particularly relates to a biological preparation method of (S) -1- (4-pyridyl) -1, 3-propanediol (I).

Background

(S) -1- (4-pyridyl) -1, 3-propanediol (I) is a key intermediate used in liver-targeted drugs and is used in a number of preclinical research drugs. Such as MB-07133, which has been given the FDA designation "orphan" in 2007, is currently being developed as a potential drug for treating liver cancer. MB-07133 is a liver-targeting prodrug of cytarabine, a nucleoside analogue that is clinically used in the treatment of acute myeloid leukemia. As with many other nucleoside drugs, one of the limitations of cytarabine is its poor in vivo phosphorylation activation, failing to produce a sufficient amount of the active metabolite cytarabine triphosphate, whereas elevated dosage levels produce myelosuppression, as the active metabolite cytarabine triphosphate is produced in healthy bone marrow cells. Compared to cytarabine, the compound MB-07133 produced the active metabolite cytarabine triphosphate in the liver at levels 12-fold and 19-fold higher than bone marrow and plasma, respectively.

The existing preparation method comprises the following steps:

WO2019119832/CN109929005 to Parthenocissi et al or J.Med.chem.49, 7711-7720 to Boyer et al

The scheme uses n-butyllithium and lithium aluminum hydride, and is purified by column chromatography for multiple times, so that the process is complex, the yield is low, and the scheme is obviously unsuitable for amplification reaction.

Erion, mark D et al WO 2007022073/AU2006279720

Ru-Cl catalysts (S, S) -Ts-DPEN-Ru-Cl- (p-cymene) are required. The scheme uses noble metal asymmetric catalysis to construct chiral alcohol, but the optical purity is insufficient, and the catalyst dosage is large (s/c=500:1) and the cost is high.

In view of the foregoing, there is a great need in the art to develop a novel process for the preparation of (S) -1- (4-pyridyl) -1, 3-propanediol (I).

Disclosure of Invention

The application aims to provide a novel preparation method of (S) -1- (4-pyridyl) -1, 3-propanediol (I).

In a first aspect of the present application there is provided a process for the preparation of a compound of formula v comprising the steps of:

(a) In a reaction system, taking a compound of formula IV as a substrate, and carrying out asymmetric reduction reaction on the substrate in the presence of carbonyl reductase and coenzyme to obtain a compound of formula V;

wherein R is C _1-8 An alkyl group;

and wherein the carbonyl reductase is one or more carbonyl reductase selected from the group consisting of:

(i) Carbonyl reductase EA with the amino acid sequence shown as SEQ ID NO. 2;

(ii) Mutants of carbonyl reductase EA; the mutant is mutated at a carbonyl reductase EA corresponding to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) amino acids selected from the group consisting of SEQ ID No.: bit 62 (a), bit 72 (E), bit 78 (D), bit 82 (L), bit 98 (E), bit 121 (a), bit 127 (I), bit 128 (E), bit 138 (L), bit 190 (V), bit 191 (D), bit 193 (a), and bit 206 (H); preferably, the mutant of the carbonyl reductase EA is shown as SEQ ID NO.5, 6, 7, 8, 9 or 10;

(iii) Performing one or more amino acid substitutions, deletions, alterations, insertions or additions to the amino acid sequence of the carbonyl reductase EA or mutant thereof within a range that retains enzymatic activity, the resulting amino acid sequence; wherein the amino acid sequence of the carbonyl reductase EA is shown as SEQ ID NO.2, and the mutant is the mutant of the carbonyl reductase EA as defined in (i); and

(iv) Performing insertion of one or more amino acids at the N-terminal or C-terminal of the sequence within the range of maintaining the enzymatic activity of the amino acid sequence of the carbonyl reductase EA or a mutant thereof, wherein the number of inserted amino acid residues comprises 1 to 30; wherein the amino acid sequence of the carbonyl reductase EA is shown in SEQ ID NO.2, and the mutant is the mutant of the carbonyl reductase EA as defined in (i).

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

in another preferred embodiment, the reaction system is a liquid reaction system.

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

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 50-1000 g/L.

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 60-700 g/L.

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 80-600 g/L.

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 20 to 500g/L.

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 50-200 g/L.

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 100 to 150g/L.

In another preferred example, in the reaction system, the mass ratio of the carbonyl reductase to the substrate is 40-60%; preferably 50.+ -. 5%.

In another preferred embodiment, the carbonyl reductase EA is derived from Exiguobacterium sp.F42.

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

(a) As set forth in SEQ ID No.:1, a sequence shown in seq id no;

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

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

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

In another preferred embodiment, the mutant of carbonyl reductase EA is mutated by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) mutations selected from the group consisting of: a62S, E D, D6272W, E E, L W, E D, A121V, I127V, E128D, L138A, V190A, D191N, A193S and H206K.

In another preferred embodiment, the amino acid sequence of the mutant of carbonyl reductase EA is shown in SEQ ID NO.5, 6, 7, 8, 9 or 10.

In another preferred example, the carbonyl reductase is one or more carbonyl reductase with amino acid sequences shown as SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

In another preferred embodiment, the coenzyme is selected from the group consisting of: reduced coenzyme, oxidized coenzyme, or a combination thereof.

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

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

In another preferred embodiment, the concentration of co-substrate in the reaction system is in the range of 5 to 30wt%.

In another preferred embodiment, the reaction system further comprises an enzyme for coenzyme regeneration.

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; preferably, the enzyme for coenzyme regeneration is Glucose Dehydrogenase (GDH).

In another preferred embodiment, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO. 4.

In another preferred embodiment, the coding gene sequence of the glucose dehydrogenase is shown in SEQ ID NO. 3.

In another preferred embodiment, in step (a), the reaction temperature is from 10℃to 50℃and preferably from 20℃to 40℃and more preferably from 25℃to 35 ℃.

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

In another preferred embodiment, the reaction time is the time taken for 90% or more (more preferably 95% or more; most preferably 99% or more) of the substrate to be converted.

In another preferred embodiment, in step (a), the reaction time is less than or equal to 10 hours (e.g., less than or equal to 5 hours), and the reaction time is the time taken for 90% or more (more preferably, 95% or more; most preferably, 99% or more) of the substrate to be converted.

In another preferred embodiment, in step (a), the pH is from 5 to-9, preferably from 5.5 to 7.5, more preferably from 5.8 to 7.3; most preferably 6.5 to 7.0.

In another preferred embodiment, the carbonyl reductase is an enzyme in a free form, an immobilized enzyme, or an enzyme in a bacterial form in the reaction system.

In another preferred example, in the reaction system, the concentration of the carbonyl reductase is in a bacterial form, and the mass ratio of the carbonyl reductase to the substrate is 10-100%; preferably, 40-60%; more preferably 50.+ -. 5%.

In another preferred example, the concentration of the carbonyl reductase in the reaction system is in the form of a bacterial cell, and the concentration of the carbonyl reductase in the reaction system is 10-100 g/L; preferably 50.+ -.10 g/L.

In a further preferred embodiment, the preparation process further comprises a work-up step for isolating and/or purifying the compound of formula V from the reaction system.

In another preferred embodiment, the post-processing step includes the steps of: the organic layer was extracted with ethyl acetate, dried and concentrated.

In a further preferred embodiment, the preparation gives compounds of the formula V with an ee value of 90% or more, preferably 95% or more, more preferably 99% or more.

In another preferred embodiment, the yield of the compound of formula V of the preparation method is not less than 80%; preferably not less than 85%, more preferably not less than 90%.

In a further preferred embodiment, more than or equal to 80% (preferably more than or equal to 85%, more preferably more than or equal to 90%, most preferably more than or equal to 95%) of the compound of formula VI is converted into the compound of formula V in the reaction system after the reaction.

In another preferred embodiment, in step (b), the concentration of the compound of formula V in the reaction system after the reaction is 50 to 1000g/L.

In a second aspect of the present application, there is provided a reaction system comprising:

(i) An aqueous solvent;

(ii) A substrate which is a compound of formula iv;

wherein R is as defined in the first aspect;

(iii) A coenzyme;

(iv) Carbonyl reductase;

(v) A co-substrate; and

(vi) Optionally an enzyme for coenzyme regeneration;

wherein the carbonyl reductase is as defined in the first aspect.

In another preferred embodiment, the coenzyme, co-substrate and enzyme for coenzyme regeneration are as defined in the first aspect.

In a third aspect of the application, there is provided a process for preparing a compound of formula v comprising the steps of: using the reaction system according to the second aspect of the present application, an enzymatic reaction is carried out to produce a compound of formula v:

wherein R is as defined in claim 1.

In another preferred embodiment, the reaction is carried out under conditions suitable for enzymatic catalysis.

In another preferred embodiment, the concentration of the compound of formula IV in the reaction system is 50-1000 g/L; preferably, 80-600 g/L; more preferably, it is 100 to 150g/L.

In a fourth aspect of the present application, there is provided a process for the preparation of a compound of formula I comprising the steps of:

allowing an ester reduction reaction of the compound of formula V to give a compound of formula I;

wherein, the liquid crystal display device comprises a liquid crystal display device,

r is as defined in the first aspect; and is also provided with

The compound of formula v is prepared by the preparation method as in the first aspect or the method as in the third aspect.

In another preferred embodiment, the ester reduction is carried out in the presence of a reducing agent.

In another preferred embodiment, the reducing agent is a combination of borohydride and lewis acid.

In another preferred embodiment, the borohydride is selected from the group consisting of: sodium borohydride, potassium borohydride, or a combination thereof.

In another preferred embodiment, the lewis acid is selected from the group consisting of: magnesium chloride, zinc chloride, aluminum trichloride, ferric chloride, cobalt chloride, or combinations thereof.

In a fifth aspect of the application, a process for the preparation of a compound of formula I comprises the steps of:

wherein R is C _1-8 An alkyl group;

(a) In a reaction system, taking a compound of formula IV as a substrate, and enabling the substrate to perform asymmetric reduction reaction in the presence of carbonyl reductase and coenzyme so as to obtain a compound of formula V; and

(b) Allowing the compound of formula V to undergo an ester reduction reaction to obtain a compound of formula I.

In another preferred embodiment, step (a) is as defined in the first aspect.

In a sixth aspect of the application, there is provided a carbonyl reductase selected from the group consisting of:

(i) A mutant of carbonyl reductase EA that is mutated at a carbonyl reductase EA corresponding to one or more amino acids selected from the group consisting of SEQ ID No.: bit 62 (a), bit 72 (E), bit 78 (D), bit 82 (L), bit 98 (E), bit 121 (a), bit 127 (I), bit 128 (E), bit 138 (L), bit 190 (V), bit 191 (D), bit 193 (a), and bit 206 (H);

(ii) The amino acid sequence obtained by substituting, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence of carbonyl reductase EA or its mutant in the range of maintaining the enzymatic activity; wherein the amino acid sequence of the carbonyl reductase EA is shown as SEQ ID NO.2, and the mutant is the mutant of the carbonyl reductase EA as defined in (i); and

(iii) Performing insertion of one or more amino acids at the N-terminal or C-terminal of the sequence within the range of maintaining the enzymatic activity of the amino acid sequence of the carbonyl reductase EA or a mutant thereof, wherein the number of inserted amino acid residues comprises 1 to 30; wherein the amino acid sequence of the carbonyl reductase EA is shown in SEQ ID NO.2, and the mutant is the mutant of the carbonyl reductase EA as defined in (i).

In another preferred embodiment, the mutant of carbonyl reductase EA is mutated to one or more mutations selected from the group consisting of: a62S, E D, D6272W, E E, L W, E D, A121V, I127V, E128D, L138A, V190A, D191N, A193S and H206K.

In another preferred embodiment, the amino acid sequence of the mutant of carbonyl reductase EA is shown in SEQ ID NO.5, 6, 7, 8, 9 or 10.

In a seventh aspect of the application there is provided the use of a carbonyl reductase as described in the sixth aspect for the preparation of a compound of formula V or for the preparation of a compound of formula I using a compound of formula IV as a substrate

Wherein R is as defined above.

In another preferred embodiment, the reaction system is as defined in the first aspect.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows a chiral pattern of the racemate of the compound of formula v.

FIG. 2 shows the ee values of the compounds of formula V obtained in example 4 by carbonyl reductase catalysis.

Figure 3 shows a chiral pattern of racemates of the compounds of formula I.

Figure 4 shows the ee values of the compounds of formula I finally obtained in example 6.

Detailed Description

The inventors have conducted extensive and intensive studies. Surprisingly, it has been found that substrates of the formula IV are particularly suitable for efficient asymmetric chiral reduction to alcohols of the formula V under the catalysis of a carbonyl reductase EA, in particular a carbonyl reductase EA having the amino acid sequence SEQ ID NO.2 and/or mutants thereof, thus providing a novel process for the preparation of the compound of the formula V and the important intermediate (S) -1- (4-pyridinyl) -1, 3-propanediol (I) which is time-consuming, simple, high-yielding and low-cost. Based on this, the inventors completed the present application.

Typically, the present application uses carbonyl reductase to efficiently prepare key chiral alcohols in the existing processes for preparing (S) -1- (4-pyridyl) -1, 3-propanediol (I), i.e., stereoselective reduction of compounds of formula IV to compounds of formula V in the presence of carbonyl reductase and coenzyme (even at high concentrations of substrate, e.g., 50-400 g/L (e.g., >100 g/L), can produce compounds of formula V having a stereochemical configuration very efficiently). Then taking the compound of the formula V as a substrate to carry out subsequent reaction. The application only needs extraction operation, has simple operation and low cost, is environment-friendly, is more suitable for industrialized production of the compound of the formula V with high chemical purity and high optical purity, and then further carries out subsequent reaction to produce the key intermediate (S) -1- (4-pyridyl) -1, 3-propanediol (I) for preparing MB-07133.

Terminology

As used herein, the term "C _1-8 Alkyl "refers to a straight or branched chain alkyl group having the indicated number of carbon atoms, including but not limited to: methyl, ethyl, propyl, butyl, pentyl, hexyl.

Herein, as A ₁ NA ₂ The mutation represented by E72D, wherein A1 and A2 each represent a different amino acid, and N is a number, means that the amino acid at the N-th position in the original sequence (e.g., SEQ ID NO. 2) is represented by A ₁ Mutation to A ₂ . For example, a62S indicates that alanine (a) at position 62 is mutated to serine (S), E72D indicates that glutamic acid (E) at position 72 is mutated to aspartic acid (D), D78E indicates that aspartic acid (D) at position 78 is mutated to glutamic acid (E), L82W indicates that leucine (L) at position 82 is mutated to tryptophan (W), E98D indicates that glutamic acid (E) at position 98 is mutated to aspartic acid (D), a121V indicates that alanine (a) at position 121 is mutated to valine (V), I127V indicates that isoleucine (I) at position 127 is mutated to valine (V), E128D indicates that aspartic acid (D) at position 128 is mutated to glutamic acid (E), L138A indicates that leucine (L) at position 138 is mutated to alanine (a), V190A indicates that valine (V) at position 190 is mutated to alanine (a), D191N indicates that aspartic acid (D) at position 191 is mutated to asparagine (N), a193S indicates that alanine (a) at position 193 is mutated to serine (S), and H206K indicates that histidine (H) at position 206 is mutated to lysine (K).

As used herein, enantiomeric excess (ee, enantiomeric excess), i.e., ee value: typically used to characterize the excess value of one enantiomer relative to another in a chiral molecule.

Unless otherwise indicated, each abbreviation has the meaning well known to those skilled in the art, e.g., me=methyl, et=ethyl.

Carbonyl reductase

In the present application, the "carbonyl reductase" is an enzyme capable of stereoselectively catalyzing asymmetric reduction of a potentially chiral ketone to obtain a chiral alcohol.

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

Carbonyl reductases useful in the present application may be from different species. For example, carbonyl reductase from the genus Microbacterium, more preferably from the genus Microbacterium F42 (Exiguobacterium sp.F42). In addition, enzymes (including enzymes from other species) having similar activities or homology (e.g., 80% or more, preferably 90% or more, more preferably 95% or more) to the carbonyl reductase described above are also within the scope of the application. A typical carbonyl reductase has the amino acid sequence shown as SEQ ID NO.2 and the encoding gene shown as SEQ ID NO.1.

Due to the degeneracy of the codons, the base sequence encoding the amino acid sequence shown in SEQ ID NO.2 is not limited to SEQ ID NO.1 only. Homologs of the base sequence can be obtained by a person skilled in the art by appropriate introduction of substitutions, deletions, alterations, insertions or additions, which are covered by the present application, as long as the expressed recombinant enzyme retains catalytic reduction activity on the compound of formula IV. A homolog of the polynucleotide of the present application may be obtained by substituting, deleting or adding one or more bases of the base sequence SEQ ID NO.1 within a range in which the enzymatic activity is maintained.

The carbonyl reductase of the application also comprises an amino acid sequence obtained by substituting, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2 within the range of maintaining the enzymatic activity.

In the present application, mutants of carbonyl reductase having the amino acid sequence shown as SEQ ID NO.5, 6, 7, 8, 9 or 10 and having the amino acid sequence shown as SEQ ID NO.2 are provided.

As a reaction system, wet cells, crude enzyme solution, pure enzyme, crude enzyme powder, or the like of the above carbonyl reductase can be used according to common general knowledge in the art. In order to obtain a high transformation efficiency, it is preferable to use wet cells. The ratio of the amount of carbonyl reductase to the amount of substrate is preferably 1% to 6% (w/w), or the ratio of the mass of cells of carbonyl reductase such as resting cell cells to the mass of substrate is 10% to 100%.

Coenzyme A

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

Typically, the coenzyme of the application is the reduced coenzyme NADH, NADPH or the oxidized coenzyme NAD ⁺ 、NADP ⁺ . Preferably the oxidative coenzyme NAD is selected ⁺ 、NADP ⁺ 。

When selecting an oxidative coenzyme, a method for achieving coenzyme regeneration is required, which mainly comprises three of (1) glucose dehydrogenase and co-substrate glucose; (2) an alcohol dehydrogenase and a co-substrate isopropanol; (3) formate dehydrogenase with co-substrate ammonium formate.

In another preferred embodiment, the carbonyl reductase may also effect regeneration of the coenzyme by adding isopropanol, by autocatalyzing the isopropanol to acetone.

In a preferred embodiment, the coenzyme is NADP ⁺ The coenzyme regeneration system is glucose dehydrogenase, preferably glucose dehydrogenase and co-substrate glucose, preferably, the coding gene sequence of the glucose dehydrogenase derived from bacillus megaterium (Bacillus megaterium) is shown as SEQ ID NO.3, and the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO. 4.

In a preferred embodiment, the oxidative coenzyme NADP ⁺ The ratio of the dosage to the dosage of the substrate is 0.01-0.5% (w/w), and the dosage of the glucose is 0.5-2 (such as 1 to 2)1.1 mol/L buffer. In another preferred embodiment, the buffer system is 0.1mol/L phosphate buffer salt. The pH of the buffer is 6.9-7.3.

Process for producing enzyme

The genes of glucose dehydrogenase and carbonyl reductase for realizing coenzyme regeneration are respectively constructed on pET28a (+) vectors by the conventional technology in the art, and then introduced into an expression host escherichia coli, and the expression is induced to obtain thalli containing glucose dehydrogenase and thalli containing carbonyl reductase EA or mutants thereof. The method can directly use centrifugation to obtain thalli, or break walls to obtain crude enzyme liquid, and the crude enzyme powder is used for subsequent bioconversion reaction.

Biocatalytic reduction process

The application provides a method for preparing a compound (V) by catalyzing and reducing the compound (IV) by carbonyl reductase. The reaction formula is as follows:

wherein R is C _1-8 An alkyl group; preferably, R is selected from the group consisting of:

preferably, the biocatalytic system comprises a carbonyl reductase, and a coenzyme.

In another preferred example, the concentration of the compound of formula IV in the reaction system is 50-1000 g/L; preferably, the concentration is 60-700 g/L; more preferably, 80 to 600g/L; still more preferably, 20 to 500g/L; still more preferably, 50 to 200g/L; and most preferably from 100 to 150g/L.

In a specific embodiment, the gene sequence encoding the carbonyl reductase described in the present application is SEQ ID NO.1, and/or the carbonyl reductase is derived from Exiguobacterium sp.F42, and the amino acid sequence of the carbonyl reductase is SEQ ID NO.2. The above carbonyl reductase gene can be obtained by commercial total gene synthesis according to common general knowledge in the art.

In another embodiment, the carbonyl reductase described in the present application is a mutant carbonyl reductase having the amino acid sequence of any one or more of SEQ ID NOS.5-10.

According to the preferred system, the preparation method is carried out as follows: adding substrate and co-substrate (such as glucose) into phosphate buffer solution, stirring, adding carbonyl reductase (in form of thallus (wet thallus), crude enzyme solution, crude enzyme powder or pure enzyme), adding coenzyme such as NADP ⁺ And glucose dehydrogenase GDH, maintained at 20℃to 40℃and optionally monitored by HPLC until the starting material remains<1%, terminate the reaction. Optionally, the reaction solution is extracted with an organic solvent, preferably one or more of methyl tertiary butyl ether, toluene, ethyl acetate, isopropyl acetate, methylene chloride, 2-methyltetrahydrofuran and n-butanol (more preferably ethyl acetate), optionally extracting the aqueous layer with an organic solvent for 2-3 times, and combining the organic phases; washing with saturated saline solution for 2-3 times, drying and concentrating to obtain the compound of the formula V.

Preferably, the final concentration of the substrate compound (IV) in the system is 50-200g/L, more preferably 100-150g/L, meeting the industrial requirements (substrate concentration >100 g/L). Preferably, the reaction temperature is 20-40℃and preferably the rotational speed is 200rpm. The reaction time varies depending on the substrate concentration, preferably about 0.5 to 20 hours, or the conversion of the starting material is monitored by HPLC, and the reaction is terminated when the starting material remains <1% (typically, it takes 1 to 5 hours).

Reaction system

The application also provides a reaction system, which comprises:

(i) An aqueous solvent (e.g., water or buffer);

(ii) A substrate which is a compound of formula iv;

r is as previously defined;

(iii) A coenzyme;

(iv) Carbonyl reductase (as defined previously);

(v) Cosubstrates (as defined previously); and

(vi) Enzymes (as defined above) for use in the regeneration of coenzymes.

In another preferred embodiment, the concentration of the compound of formula IV is 50-1000 g/L; preferably, 50-200 g/L; and most preferably from 100 to 150g/L.

Process for the preparation of (S) -1- (4-pyridinyl) -1, 3-propanediol (I)

Aiming at the problems of the existing preparation method, the application also provides a biocatalytic preparation method for preparing (S) -1- (4-pyridyl) -1, 3-propanediol (I) from isonicotinic acid, which comprises the following steps:

wherein R is as defined above, wherein the compound (V) is obtained by the aforementioned biocatalytic reduction method.

The main advantages of the application include:

(1) The application is suitable for industrial production of the compound of formula V with high chemical purity and high optical purity, and then further subsequent reaction is carried out to prepare (S) -1- (4-pyridyl) -1, 3-propanediol (I).

(2) The method has high reaction efficiency, and generally, more than 98 percent of the compound of the formula IV can be converted into the compound of the formula V only in 1-3 hours.

(3) The method and the catalytic reduction reaction system have stereoselectivity as high as 99 percent (such as ee value of 99.5 percent), tolerance to a substrate, high catalytic activity and substrate concentration of more than 100g/L, so that mass production can be carried out under higher substrate concentration.

(4) The application greatly improves the production efficiency and reduces the production cost.

(5) The method of the application greatly simplifies the subsequent treatment. The post-treatment only needs extraction operation, and the operation is simple.

(6) The method is green and environment-friendly. Compared with chemical synthesis, the application reduces or eliminates the use of various pollution chemicals, thereby reducing the risk of environmental pollution.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Raw material and detection method

Unless otherwise indicated, the starting materials used herein may be obtained commercially or synthesized or prepared according to the prior art. For example, the respective coding genes used in the examples can be obtained by commercial total gene synthesis. Wet cells or wet cells may be obtained by reference to the methods provided herein or according to conventional methods well known to those skilled in the art.

Racemates of the compounds of formula V and formula I and standard substances thereof

In the application, racemates of the compounds of the formula V and the formula I are obtained by reducing the formula IV according to a sodium borohydride method which is conventional in the art, and then racemic reference substances V and I are obtained through chromatographic separation.

Chiral monitoring method for compound of formula V

Large xylonite IC,5 μm,4.6 x 250mm; mobile phase: isopropanol: n-hexane=25:75; the flow rate is 1.0mL/min; run time: 20min; column temperature 25 ℃; detection wavelength: 254nm. S configuration 9.8min, R configuration 11.6min.

For example, fig. 1 and 2 provided by the present application are chiral chromatograms obtained according to the method.

4. Chiral monitoring method of (S) -1- (4-pyridyl) -1, 3-propanediol (I)

Chromatographic column: large xylonite IC,5 μm,4.6 x 250mm; mobile phase: ethanol: n-hexane=15:85; the flow rate is 1.0mL/min; run time: 20min; column temperature 25 ℃; detection wavelength: 254nm. S configuration 9.8min, R configuration 11.6min.

For example, fig. 3 and 4 provided by the present application are chiral chromatograms obtained according to the method.

EXAMPLE 1 screening of carbonyl reductase

In this example, a preliminary screening was performed for 16 carbonyl reductases, wherein,

lseah is derived from Leifsonia sp.strain S749, genbank accession number AB213459;

ck03 is derived from Chryseobacterium sp.CA49, genbank accession No. KC342003

See the description of other parts of the text for sources of EA and its sequence, etc.;

lk08 is derived from Lactobacillus kefir, see in particular Wei T Y, tang J W, ni gw, et al Development of an Enzymatic Process for the Synthesis of (S) -2-Chloro-1- (2, 4-dichlorophenyl) Ethanol [ J ]. Organic Process Research & Development 2019;

the other enzymes used in this screening were purchased from bioengineering limited in hong an, zhejiang, except Laadh, ck03, EA and Lk 08.

The screening results are shown in Table 1, wherein specific reaction conditions and test methods are identified by corresponding comments:

TABLE 1

In Table 1, the superscripts are described below

a represents the reaction conditions: 10g/L substrate (formula IV, R=Et), 20g/L wet cell of the corresponding enzyme in Table 1, 0.5g/L NAD ⁺ 2g/L GDH wet cells, 2.0 equivalents of glucose were shake-reacted in 2mL PBS buffer (100 mM, pH 7.0) at 25℃and 220rpm for 24h.

b represents the reaction conditions: 10g/L substrate (formula IV, R=Et), 20g/L wet cell of the corresponding enzyme in Table 1, 0.5g/L NADP ⁺ 2g/L GDH wet cells were shake-reacted with 2.0 equivalents of glucose in 2mL PBS buffer (100 mM, pH 7.0) at 25℃and 220rpm for 24h.

c represents the reaction conditions: 10g/L substrate (formula IV, R=Et), 20g/L wet cell of the corresponding enzyme in Table 1, 0.5g/L NAD ⁺ 3eq IPA (isopropanolmM, pH 7.0) was shake-reacted at 25℃and 220rpm for 24h.

d represents the reaction conditions: 10g/L substrate (formula IV, R=Et), 20g/L wet cell of the corresponding enzyme in Table 1, 0.5g/L NADP ⁺ 3eq IPA was shake-reacted in 2mL PBS buffer (100 mM, pH 7.0) at 25℃and 220rpm for 24h.

e represents conversion as measured by reverse phase HPLC.

f represents ee value as measured by chiral IC column on normal phase HPLC (detailed test conditions are as described in reaction conditions).

N.d. means not tested.

EXAMPLE 2,

(1) Construction of carbonyl reductase engineering bacteria and carbonyl reductase homologous mutation library

7 carbonyl reductase genes from Exiguobacterium and glucose dehydrogenase genes from Bacillus megaterium are cloned into pET28a (+) vector respectively, and then introduced into host escherichia coli BL21 (de 3), and recombinant genetically engineered bacteria of carbonyl reductase EA and recombinant genetically engineered bacteria of glucose dehydrogenase are obtained through induced expression.

(2) Preparation of recombinant carbonyl reductase and glucose dehydrogenase

Inoculating recombinant genetically engineered bacteria stored in glycerol in the previous step into LB liquid medium containing 100ug/ml of ammonia-gas-phase mycin, respectively, culturing at 37deg.C and 220rpm for 12-16 hr to obtain seed culture medium, inoculating the seed liquid into liquid culture medium containing 100ug/ml of ammonia-gas-phase mycin resistance at a ratio of 1.5%, and culturing at 37deg.C and 220rpm to OD ₆₀₀ Value of>2.0, adding lactose with the final concentration of 1.0%, cooling to 25 ℃, continuously culturing for 2 hours, adding lactose with the final concentration of 0.5%, culturing for 20 hours, placing in a tank, and centrifuging to obtain EA wet thalli and GDH wet thalli, wherein the EA wet thalli and the GDH wet thalli are used as a catalyst for bioconversion.

Example 3 screening of carbonyl reductase homologous mutant libraries

Carbonyl reductase EA and its mutants undergo chiral reduction of the substrate under the following reaction conditions, respectively: contains 10g/L substrate (formula IV, R=Et), 20g/L EA wet cell, 0.5g/L NADP ⁺ 2mL PB containing 2.0 eq glucose in 2g/L GDH wet cellS buffer (100 mM, pH 7.0) was shake-reacted at 25℃and 220rpm for 30min. The conversion was checked by HPLC.

The relative enzyme activity was calculated from the conversion results and the ee value of the obtained product was tested, and the screening results are shown in table 2:

TABLE 2

Example 4 biocatalytic preparation of Compound V (R=Et)

115.9g of Compound IV (R=Et) (0.6 mol) was weighed into a 3L four-necked flask, 1.15L of 100mmol/L phosphate buffer pH7.0 was added, 216g (1.2 mol) of glucose was added, and the mixture was stirred uniformly, 11.5g of GDH wet cell (prepared in example 2), 57.5g of EA wet cell (prepared in example 2, SEQ ID NO. 6) and NADP were added ⁺ 0.5g is placed in a 35 ℃ water bath and reacted under mechanical stirring at 220rpm, after half an hour, the pH value of the reaction solution is measured to be about 5.5, the pH value of the reaction solution is adjusted to be 7.3 by using a saturated sodium carbonate solution, after half an hour, the pH value of the reaction solution is measured to be about 6.3, the pH value of the reaction solution is adjusted to be 7.2 by using a saturated sodium carbonate solution, and then the pH value is continuously monitored and is kept basically unchanged, and the pH value is kept between 6.9 and 7.0. The reaction time 3h was taken to sample TLC for monitoring the substantial disappearance of the starting material spot, HPLC for monitoring the remaining 0.55% of the starting material, termination of the reaction, addition of 1.2L of ethyl acetate, stirring for about 10min, followed by separation of the aqueous layer, extraction with ethyl acetate, 500 mL. Times.2, combining of ethyl acetate, washing of 500 mL. Times.2 with saturated NaCl, drying of the organic phase over anhydrous sodium sulfate, concentration to give 107.8g of a pale yellow oil (Compound V) with a yield of 92%, HPLC purity of 99.5% and ee value of 99.8% (see FIG. 2).

Example 5 biocatalytic preparation of Compound V (R=Me)

107.5g of Compound IV (R=Me) (0.6 mol) was weighed into a 3L four-necked flask, 1.15L of 100mmol/L phosphate buffer pH7.0 was added, 216g (1.2 mol) of glucose was added, and the mixture was stirred uniformly, 11.5g of GDH wet cell (prepared in example 2), 57.5g of EA wet cell (prepared in example 2, SEQ ID NO. 6) and NADP were added ⁺ 0.5g is placed in a 35 ℃ water bath and reacted under mechanical stirring at 220rpm, after half an hour, the pH value of the reaction liquid is measured to be about 5.6, the pH value of the reaction liquid is adjusted to be 7.1 by saturated sodium carbonate solution, after half an hour, the pH value of the reaction liquid is measured to be about 6.1, the pH value of the reaction liquid is adjusted to be 7.2 by saturated sodium carbonate solution, and then the pH value is continuously monitored, and the reaction liquid is kept basically unchanged and is kept between 6.9 and 7.0. The reaction time 3h was taken to sample TLC to monitor the substantial disappearance of the starting material spot, HPLC to monitor the remaining 0.39% of the starting material, termination of the reaction, addition of 1.2L of ethyl acetate, stirring for about 10min, followed by separation of the aqueous layer, extraction with 500 mL. Times.2 ethyl acetate, combining of ethyl acetate, washing of 500 mL. Times.2 with saturated NaCl, drying of the organic phase over anhydrous sodium sulfate, concentration to give 101.1g of a pale yellow oil with a yield of 93%, HPLC purity of 99.0% and ee of 99.5%.

EXAMPLE 6 preparation of (S) -1- (4-pyridyl) -1, 3-propanediol (I)

5.1 esterification

147.7g isonicotinic acid (1.2 mol) is taken in a 2L four-neck flask, 1.11kg (24 mol) of absolute ethyl alcohol is added, mechanical stirring is started at 200rpm, 176.5g (1.8 mol) of concentrated sulfuric acid (98%) is weighed and placed in a constant pressure dropping funnel, the concentrated sulfuric acid is dripped, heating is started after dripping, reflux is carried out until the internal temperature reaches 80 ℃, and the reaction is terminated after 4 hours. 500mL of water was added, ethanol was concentrated, pH was adjusted to 9.0 with sodium carbonate, 300mL of ethyl acetate was added for extraction, and after drying, 163g of colorless liquid was obtained as ethyl isonicotinate in 89% yield.

5.2 ester condensation

224g (2 mol) of potassium tert-butoxide is taken in a 2L four-neck flask, 1L of tetrahydrofuran is added, ice bath cooling is carried out, the temperature is kept to 0 ℃, 151g (1 mol) of ethyl isonicotinate obtained in the previous step and 114.5g (1.3 mol) of ethyl acetate are taken, mixed and placed in a constant pressure dropping funnel for slow dropping, and the temperature is kept below 5 ℃. TLC was followed until the starting material point disappeared, after 1h of reaction, 500mL of water was added to quench the reaction, followed by pH adjustment to about 10.0 with concentrated hydrochloric acid to give a clear solution, after concentrating THF, the aqueous phase was stirred in an ice bath for crystallization to give 166g of ethyl isonicotinylacetate (0.86 mol) in 86% yield.

5.3 enzymatic reduction

I.e. example 4. 107.8g of compound V was obtained in 92% yield with an HPLC purity of 99.5% and an ee value of 99.8%.

5.4 ester reduction

Taking 97.6g (0.5 mol) of the product compound V of the enzyme reduction in the last step, adding 600mL of absolute ethyl alcohol, starting mechanical stirring, slowly adding 9.52g (0.1 mol) of anhydrous magnesium chloride, heating to 40 ℃, adding 28.3g (0.75 mol) of sodium borohydride in batches after dissolving, tracking and monitoring by TLC until the raw material point disappears, adding 200mL of water, adjusting pH to 0 by 60% sulfuric acid, stirring to be clear, and adjusting pH to 9 by sodium hydroxide solution after 1 h. After filtering the solid, concentrating the ethanol, adding 200mL of n-butanol for extraction, repeating the extraction twice, drying the organic phase, adding 500mL of ethyl acetate into the concentrated oil, heating, refluxing and dissolving, cooling and crystallizing to obtain 62.0g (0.405 mol) of (S) -1- (4-pyridyl) -1, 3-propanediol (I) as a product, wherein the yield is 81%, the purity is 99.5%, and the ee is 99.9% (see FIG. 4). As a white solid. The melting point is 98.1-99.5 ℃.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Shanghai pharmaceutical industry institute

China Pharmaceutical Industry Research Institute

Biological preparation method of <120> (S) -1- (4-pyridyl) -1, 3-propanediol (I)

<130> P2020-1144

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 750

<212> DNA

<213> Microbacterium genus F42 (Exiguobacterium sp.F42)

<400> 1

atgaaataca ccgttatcac cggtgcttct tctggtatcg gttacgaaac cgctaaactg 60

ctggctggta aaggtaaatc tctggttctg gttgctcgtc gtacctctga actggaaaaa 120

ctgcgtgacg aagttaaaca gatctctccg gactctgacg ttatcctgaa atctgttgac 180

ctggctgaca accagaacgt tcacgacctg tacgaaggtc tgaaagaact ggacatcgaa 240

accctgatca acaacgctgg tttcggtgac ttcgacctgg ttcaggacat cgaactgggt 300

aaaatcgaaa aaatgctgcg tctgaacatc gaagctctga ccatcctgtc ttctctgttc 360

gctcgtgacc accacgacat cgaaggtacc accctggtta acatctcttc tctgggtggt 420

taccgtatcg ttccgaacgc tgttacctac tgcgctacca aattctacgt ttctgcttac 480

accgaaggtc tggctcagga actgcagaaa ggtggtgcta aactgcgtgc taaagttctg 540

gctccggctg ctaccgaaac cgaattcgtt gaccgtgctc gtggtgaagc tggtttcgac 600

tactctaaaa acgttcacaa ataccacacc gctgctgaaa tggctggttt cctgcaccag 660

ctgatcgaat ctgacgctat cgttggtatc gttgacggtg aaacctacga attcgaactg 720

cgtggtccgc tgttcaacta cgctggttaa 750

<210> 2

<211> 249

<212> PRT

<213> Microbacterium genus F42 (Exiguobacterium sp.F42)

<400> 2

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> 3

<211> 786

<212> DNA

<213> Bacillus megaterium (Bacillus megaterium)

<400> 3

atgtatccgg atttaaaagg aaaagtagtt gtcataacag gttcatctac aggtttggga 60

aaatcaatgg cgattcgttt tgcgacagaa aaagccaaag tagttgtgaa ttatcgttcg 120

aaagaagacg aagctaacag cgtcttagaa gaaattaaaa aagttggcgg agaggcaatt 180

gccgtcaaag gtgatgtaac agttgagtct gacgttatca atttagttca atctgctatt 240

aaagaatttg gaaagctaga cattatgatt aacaacgcag ggttagaaaa tccggtttca 300

tctcatgaaa tgtctttaag tgactggaat aaagtcattg atacgaactt aacgggagca 360

ttcttaggca gccgtgaagc gattaaatat tttgtagaaa atgatgttaa gggaacagtt 420

attaacatgt cgagtgttca cgagaaaatt ccttggccat tatttgttca ttacgcagca 480

agtaaaggcg gtatgaagct catgactgaa acacttgcat tagaatacgc tccaaaaggc 540

attcgtgtaa ataacattgg accgggagcg attaatacac cgattaacgc tgagaaattt 600

gctgatcctg agcagcgtgc agatgtagaa agcatgattc caatgggata catcggagag 660

ccggaagaaa ttgcagcggt tgctgcatgg ctagcttctt cagaggcaag ttatgtaaca 720

gggattacgc tctttgctga cggcggtatg acacagtacc catcattcca ggcaggccgc 780

ggttaa 786

<210> 4

<211> 261

<212> PRT

<213> Bacillus megaterium (Bacillus megaterium)

<400> 4

Met Tyr Pro Asp Leu Lys Gly Lys Val Val Val Ile Thr Gly Ser Ser

1 5 10 15

Thr Gly Leu Gly Lys Ser Met Ala Ile Arg Phe Ala Thr Glu Lys Ala

20 25 30

Lys Val Val Val Asn Tyr Arg Ser Lys Glu Asp Glu Ala Asn Ser Val

35 40 45

Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly

50 55 60

Asp Val Thr Val Glu Ser Asp Val Ile Asn Leu Val Gln Ser Ala Ile

65 70 75 80

Lys Glu Phe Gly Lys Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu

85 90 95

Asn Pro Val Ser Ser His Glu Met Ser Leu Ser Asp Trp Asn Lys Val

100 105 110

Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile

115 120 125

Lys Tyr Phe Val Glu Asn Asp Val Lys Gly Thr Val Ile Asn Met Ser

130 135 140

Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala

145 150 155 160

Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr

165 170 175

Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn

180 185 190

Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Glu Gln Arg Ala Asp

195 200 205

Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile

210 215 220

Ala Ala Val Ala Ala Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr

225 230 235 240

Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe

245 250 255

Gln Ala Gly Arg Gly

260

<210> 5

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

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 Trp 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 Val Arg Asp His His Asp Val Glu

115 120 125

Gly Thr Thr Leu Val Asn Ile Ser Ser Ala 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 Ala Asp Arg

180 185 190

Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys 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> 6

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

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 Trp 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 Val Arg Asp His His Asp Val Asp

115 120 125

Gly Thr Thr Leu Val Asn Ile Ser Ser Ala 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 Ala Asp Arg

180 185 190

Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys 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> 7

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

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 Trp 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 Val Arg Asp His His Asp Val Glu

115 120 125

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

180 185 190

Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys 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> 8

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

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

85 90 95

Ile Asp 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 Val Arg Asp His His Asp Val Asp

115 120 125

Gly Thr Thr Leu Val Asn Ile Ser Ser Ala 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 Ala Asp Arg

180 185 190

Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys 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> 9

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

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 Asp Gly Leu Lys Glu Leu Asp Ile Glu

65 70 75 80

Thr Trp 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 Val Arg Asp His His Asp Val Asp

115 120 125

Gly Thr Thr Leu Val Asn Ile Ser Ser Ala 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 Ala Asp Arg

180 185 190

Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys 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> 10

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

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 Ser Asp Asn

50 55 60

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

65 70 75 80

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

85 90 95

Ile Asp 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 Val Arg Asp His His Asp Val Glu

115 120 125

Gly Thr Thr Leu Val Asn Ile Ser Ser Ala 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 Ala Asp Arg

180 185 190

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

Claims (8)

1. A process for the preparation of a compound of formula v comprising the steps of:

(a) In a reaction system, taking a compound of formula IV as a substrate, and carrying out asymmetric reduction reaction on the substrate in the presence of carbonyl reductase and coenzyme to obtain a compound of formula V;

wherein R is selected from the group consisting of:

and wherein the carbonyl reductase is one or more of carbonyl reductase with amino acid sequences shown as SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

2. The method of claim 1, wherein the carbonyl reductase is a carbonyl reductase having an amino acid sequence shown in SEQ ID NO.2.

3. The method of claim 1, further characterized by one or more of the following:

(1) In the step (a), the reaction temperature is 10-50 ℃;

(2) In step (a), the reaction time is 0.1 to 240 hours;

(3) In step (a), the pH is 5-9;

(4) In the reaction system, the carbonyl reductase is an enzyme in a free form, an immobilized enzyme or an enzyme in a bacterial form;

(5) In the reaction system, the concentration of the compound of the formula IV is 50-1000 g/L.

4. The method of claim 1, further characterized by one or more of the following:

(1) In the step (a), the reaction temperature is 20-40 ℃;

(2) In step (a), the reaction time is 0.5 to 120 hours;

(3) In step (a), the pH is from 5.5 to 7.5.

5. The method of claim 1, further characterized by one or more of the following:

(1) In the step (a), the reaction temperature is 25-35 ℃;

(2) In step (a), the reaction time is 3-10 hours;

(3) In step (a), the pH is 6.5 to 7.0.

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

(i) An aqueous solvent;

(ii) A substrate which is a compound of formula iv;

wherein R is as defined in claim 1;

(iii) A coenzyme;

(iv) Carbonyl reductase; the carbonyl reductase is as defined in claim 1;

(v) A co-substrate; and

(vi) Optionally an enzyme for coenzyme regeneration.

7. A process for preparing a compound of formula v comprising the steps of: the use of the reaction system according to claim 6 for enzymatic reactions to produce compounds of formula v:

wherein R is as defined in claim 1.

8. A process for the preparation of a compound of formula I comprising the steps of:

wherein R is as defined in claim 1;

(a) In a reaction system, taking a compound of formula IV as a substrate, and enabling the substrate to perform asymmetric reduction reaction in the presence of carbonyl reductase and coenzyme so as to obtain a compound of formula V; wherein the carbonyl reductase is as defined in claim 1; and

(b) Allowing the compound of formula V to undergo an ester reduction reaction to obtain a compound of formula I.