Preparation method of (S)-1-(3-bromopyridine-2-yl) ethanol

Abstract

The invention discloses a preparation method of (S)-1-(3-bromopyridine-2-yl) ethanol. Specifically, the method comprises the following steps: in a liquid reaction system, taking 1-(3-bromopyridine-2-yl) ethanone as shown in a formula I as a substrate, and carrying out enzymatic reaction by using carbonyl reductase CnKRED or a mutant thereof, so as to prepare (S)-1-(3-bromopyridine-2-yl) ethanol as shown in a formula II. The preparation method disclosed by the invention has the advantages of simple process operation, low cost, high yield, high purity, easiness in large-scale production and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to a method for synthesizing (S)-1-(3-bromopyridin-2-yl)ethanol. Background Art

[0002] Chiral alcohols contain a chiral hydroxyl functional group and are widely used in the fields of pharmaceutical intermediates, fine chemicals, etc. However, traditional chiral alcohol synthesis methods often require expensive chiral precious metal catalysts and harsh reaction conditions, and have problems such as low chiral purity of products, prominent pollution problems, and high production costs. Ketoredutase (KRED) can catalyze the reduction of carbonyl compounds to chiral alcohols under mild conditions, and has the advantages of high conversion rate and high chiral purity of products, so it plays an important role in the field of synthesis.

[0003] KRAS is one of the most common mutated proteins in cancer. Currently, two KRAS-G12C targeted inhibitors have been approved by the FDA for marketing, and more than a dozen follow-up drugs have entered the clinical trial stage.

[0004]

[0005] RMC-6236 is a non-covalent pan-RAS inhibitor developed by Revolution Medicine, and is expected to become a natural product molecule for KRAS selective molecular glue. (S)-1-(3-bromopyridin-2-yl)ethanol is an important chiral building block in the synthesis of RMC-6236. In 2021, Xing Xiangyou and others used ferrocene derivatives to catalyze the reduction of potential chiral ketones to obtain (S)-1-(3-bromopyridin-2-yl)ethanol with an ee value of 97%. In 2023, Xu Xingjun and others used a new chelated ruthenium catalyst to catalyze the reduction of 1-(3-bromopyridin-2-yl)ethanone to obtain (S)-1-(3-bromopyridin-2-yl)ethanol with a yield of 90% and an ee value of 97%. However, these methods use new metal catalysts with higher costs and are not suitable for industrial production. In 2000, Kaoru Nakamura et al. used G. candidum IFO 4597 and hydrophobic polymer AmberliteXAD 7 to reduce 1-(3-bromopyridin-2-yl)ethanone to obtain (S)-1-(3-bromopyridin-2-yl)ethanol with a yield of 99% and an ee value of 99%. However, the substrate used was only 0.08 mmol, making it difficult to achieve large-scale production.

[0006] Therefore, there is an urgent need to develop a method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol with high purity, high yield and scalable production. Summary of the invention

[0007] In view of the problems existing in the existing preparation methods, the present invention provides a preparation method of (S)-1-(3-bromopyridin-2-yl)ethanol with high purity, high yield and large-scale production.

[0008] In a first aspect of the present invention, a method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol is provided, comprising the steps of: in a liquid reaction system, using 1-(3-bromopyridin-2-yl)ethanone as a substrate, and using carbonyl reductase CnKRED or a mutant thereof to carry out an enzymatic reaction, thereby preparing (S)-1-(3-bromopyridin-2-yl)ethanol as shown in formula II;

[0009]

[0010] In another preferred embodiment, in the liquid reaction system, the concentration of 1-(3-bromopyridin-2-yl)ethanone is 10 to 300 g / L.

[0011] In another preferred embodiment, in the liquid reaction system, the concentration of 1-(3-bromopyridin-2-yl)ethanone is 10 to 200 g / L.

[0012] In another preferred embodiment, in the liquid reaction system, the concentration of 1-(3-bromopyridin-2-yl)ethanone is 10 to 150 g / L.

[0013] In another preferred embodiment, in the liquid reaction system, the concentration of 1-(3-bromopyridin-2-yl)ethanone is 10 to 100 g / L.

[0014] In another preferred embodiment, in the liquid reaction system, the concentration of 1-(3-bromopyridin-2-yl)ethanone is 20 to 90 g / L.

[0015] In another preferred embodiment, the liquid reaction system is a phosphate buffer system.

[0016] In another preferred embodiment, the pH of the liquid reaction system is 6 to 8; preferably, the pH is 6.5 to 7.5; more preferably, the pH is 7.0 to 7.5.

[0017] In another preferred embodiment, the reaction temperature is 15°C to 65°C; preferably, 20°C to 55°C.

[0018] In another preferred embodiment, the reaction time is 1-24 hours; preferably 1-12 hours; more preferably 2-8 hours.

[0019] In another preferred embodiment, in the liquid reaction system, the carbonyl reductase or its mutant is a free enzyme, an immobilized enzyme, or an enzyme in bacterial form.

[0020] In another preferred embodiment, a co-substrate also exists in the liquid reaction system.

[0021] In another preferred embodiment, the co-substrate is selected from the group consisting of isopropanol, glucose, or a combination thereof.

[0022] In another preferred embodiment, when the co-substrate is isopropanol, the concentration of the co-substrate is 10-80% (v / v); preferably 10-70% (v / v); more preferably 20-50% (v / v).

[0023] In another preferred embodiment, when the co-substrate is glucose, the concentration of the co-substrate is 1-300 g / L; preferably 10-200 g / L; more preferably 20-160 g / L.

[0024] In another preferred embodiment, (S)-1-(3-bromopyridin-2-yl)ethanol is separated from the reaction system after the enzymatic reaction.

[0025] In another preferred embodiment, the separation comprises: centrifugation, taking the supernatant, extracting the aqueous layer with ethyl acetate or dichloromethane, combining the organic layers, washing with saturated brine, drying with anhydrous sodium sulfate, and filtering.

[0026] In another preferred embodiment, in the reaction system after the separation reaction, the ee value of the (S)-1-(3-bromopyridin-2-yl)ethanol is ≥99%; preferably, the ee value is >99.9%; more preferably, the ee value is >99.99%.

[0027] In another preferred example, in the reaction system after the separation reaction, the concentration of the (S)-1-(3-bromopyridin-2-yl)ethanol is 1 to 300 g / L; preferably; 10 to 200 g / L; more preferably 10 to 90 g / L.

[0028] In another preferred embodiment, the carbonyl reductase CnKRED is selected from the following group:

[0029] (i) a polypeptide with an amino acid sequence as shown in SEQ ID NO.2;

[0030] (ii) One or more amino acids are substituted, deleted, altered, inserted or added to the amino acid sequence shown in SEQ ID NO.2, and the resulting amino acid sequence is a carbonyl reductase CnKRED mutant having carbonyl reductase CnKRED activity.

[0031] In a preferred embodiment, the carbonyl reductase CnKRED mutant is selected from the following group:

[0032] (a) Carbonyl reductase CnKRED mutant with E49G, E64K, V78G, A98T, I158V, A222T, D238N, T240A;

[0033] (b) carbonyl reductase CnKRED mutants with E49G, D62N, V67A, T72V, I73V, S75N, V78G, A178S, R182I, A222T;

[0034] (c) carbonyl reductase CnKRED mutants with S22A, S46K, E49G, D62N, V67A, T72V, I73V, S75N, V78G, N118R, S136V, A222T;

[0035] Wherein, the amino acid numbering is based on the amino acid numbering of the carbonyl reductase CnKRED shown in SEQ ID No.2.

[0036] In a preferred embodiment, the coding gene sequence of the carbonyl reductase CnKRED is selected from the following group:

[0037] (a) the sequence shown in SEQ ID NO.1;

[0038] (b) a polynucleotide complementary to the sequence defined in (a); or

[0039] (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%) sequence identity with the sequence defined in (a).

[0040] In a preferred embodiment, the reaction is carried out in the presence of a coenzyme.

[0041] In a preferred embodiment, the coenzyme is selected from the following group: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.

[0042] In another preferred embodiment, the reducing coenzyme is selected from NADH, NADPH, or a combination thereof.

[0043] In another preferred embodiment, the oxidative coenzyme is selected from NAD + 、NAD(P) + , or a combination thereof.

[0044] In another preferred embodiment, the concentration of the coenzyme is 0.05-1 g / L; preferably 0.1-1 g / L.

[0045] In another preferred embodiment, the coenzyme is NAD(P) +, its concentration is 0.05-1g / L; preferably 0.1-1g / L; more preferably 0.2-0.8g / L.

[0046] In a preferred embodiment, the reaction system also contains an enzyme for regenerating the coenzyme.

[0047] In a preferred example, the enzyme used for coenzyme regeneration is selected from the following group: glucose dehydrogenase, isopropanol dehydrogenase, formate dehydrogenase.

[0048] In another preferred embodiment, the concentration of the dehydrogenase is 1-100 g / L; preferably, 20-80 g / L; more preferably, 30-60 g / L.

[0049] The second aspect of the present invention provides an isolated or purified carbonyl reductase CnKRED mutant, wherein the carbonyl reductase CnKRED mutant is selected from the following group:

[0050] (a) carbonyl reductase mutants with E49G, E64K, V78G, A98T, I158V, A222T, D238N, T240A;

[0051] (b) carbonyl reductase mutants having E49G, D62N, V67A, T72V, I73V, S75N, V78G, A178S, R182I, A222T;

[0052] (c) carbonyl reductase mutants having S22A, S46K, E49G, D62N, V67A, T72V, I73V, S75N, V78G, N118R, S136V, A222T;

[0053] Wherein, the amino acid numbering is based on the amino acid numbering of the wild-type carbonyl reductase shown in SEQ ID No.2.

[0054] In another preferred embodiment, the carbonyl reductase CnKRED is derived from Chryseobacterium nepalense.

[0055] In another preferred embodiment, the gene of the carbonyl reductase or its mutant is constructed on an expression vector.

[0056] In another preferred example, the mass ratio of 1-(3-bromopyridin-2-yl)ethanone to carbonyl reductase or its mutant is 1:(0.01-15); preferably 1:(0.1-10); more preferably 1:(0.5-8).

[0057] The third aspect of the present invention provides a reaction system, comprising:

[0058] (i) an aqueous solvent;

[0059] (ii) a substrate, wherein the substrate is 1-(3-bromopyridin-2-yl)ethanone represented by formula I;

[0060]

[0061] and (iii) carbonyl reductase CnKRED or a mutant thereof.

[0062] In another preferred embodiment, the reaction system further includes a coenzyme.

[0063] In another preferred embodiment, the reaction system further comprises a co-substrate.

[0064] In another preferred embodiment, the reaction system further comprises an enzyme for regenerating the coenzyme.

[0065] The fourth aspect of the present invention is a method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol, characterized in that it comprises the steps of: using the reaction system described in the third aspect of the present invention, under enzyme catalysis conditions, to carry out an enzymatic reaction to obtain (S)-1-(3-bromopyridin-2-yl)ethanol.

[0066] The fifth aspect of the present invention provides a use of the preparation method according to the first aspect of the present invention for preparing the KRAS-G12C targeted inhibitor RMC-6236.

[0067] The sixth aspect of the present invention provides an isolated polynucleotide encoding the carbonyl reductase CnKRED mutant shown in the second aspect of the present invention.

[0068] The seventh aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the sixth aspect of the present invention.

[0069] The eighth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector described in the seventh aspect of the present invention, or the polynucleotide described in the sixth aspect of the present invention is integrated into its genome.

[0070] The ninth aspect of the present invention provides a use of the carbonyl reductase CnKRED mutant described in the second aspect of the present invention for catalytic reaction or for preparing a catalyst for catalytic reaction.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 The chiral HPLC spectrum of (S)-1-(3-bromopyridin-2-yl)ethanol prepared by carbonyl reductase CnKRED catalyzed reduction is shown.

[0073] Figure 2 The HPLC spectrum of the racemate of 1-(3-bromopyridin-2-yl)ethanol is shown. DETAILED DESCRIPTION

[0074] After long and in-depth research, the inventors unexpectedly discovered a method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol for the first time. Specifically, in a liquid reaction system, 1-(3-bromopyridin-2-yl)ethanone shown in formula I is used as a substrate, and carbonyl reductase CnKRED or its mutant is used to perform an enzymatic reaction to obtain (S)-1-(3-bromopyridin-2-yl)ethanol as shown in formula II. The preparation method of the present invention has the advantages of simple process operation, low cost, high yield, high purity, and easy large-scale production. Based on this, the inventors have completed the present invention.

[0075]

[0076] the term

[0077] As used herein, "ee value" is the abbreviation of enantiomeric excess, which refers to the enantiomeric excess, and is defined as the percentage of the excess of one isomer a over the other isomer b in an enantiomeric mixture.

[0078] As used herein, “one or more” is generally 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10.

[0079] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0080] Carbonyl reductase

[0081] In the present invention, "carbonyl reductase" is an enzyme that can stereoselectively catalyze the asymmetric reduction of prochiral ketones to chiral alcohols.

[0082] In the present invention, the carbonyl reductase may be wild type or mutant, and may be isolated or recombinant.

[0083] Carbonyl reductases useful in the present invention may be derived from different species. For example, from the genus Chryseobacterium, preferably from Chryseobacterium nepalense. In addition, enzymes having similar activity or homology (such as ≥80%, preferably ≥90%, more preferably ≥95%) to the above-mentioned carbonyl reductase (including enzymes from other species) are also within the scope of the present invention.

[0084] In the present invention, a representative carbonyl reductase is a carbonyl reductase derived from Chryseobacterium nepalense.

[0085] The amino acid sequence of a typical carbonyl reductase is shown in SEQ ID No.2, and its encoding gene is shown in SEQ ID No.1.

[0086] Due to the degeneracy of codons, the base sequence encoding the amino acid sequence shown in SEQ ID NO.2 is not limited to SEQ ID NO.1. Those skilled in the art can obtain homologues of the base sequence by introducing appropriate substitutions, deletions, changes, insertions or additions, and the present invention covers these homologues as long as the recombinant enzyme expressed by them maintains the catalytic reduction activity for the substrate compound 2. The homologues of the polynucleotide in the present invention can be obtained by replacing, deleting or adding one or more bases of the base sequence SEQ ID NO.1 within the range of maintaining the enzyme activity.

[0087] The carbonyl reductase of the present invention also includes a polypeptide represented by the amino acid sequence obtained by replacing, deleting, changing, inserting or adding one or more amino acids to the amino acid sequence represented by SEQ ID NO. 2 within the range of maintaining enzyme activity.

[0088] According to the common sense in the art, the reaction system can use the above-mentioned recombinant enzyme resting cells, wet bacteria, crude enzyme solution, pure enzyme or crude enzyme powder constructed by the carbonyl reductase. In order to obtain a higher conversion efficiency, it is preferred to use a crude enzyme solution. 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 resting cell mass to the substrate mass is 10-100%.

[0089] Coenzyme

[0090] In the present invention, "coenzyme" refers to a coenzyme that can achieve electron transfer in a redox reaction.

[0091] Typically, the coenzyme of the present invention is a reducing coenzyme NADH, NADPH or an oxidizing coenzyme NAD + 、NAD(P) + Since the cost of reducing coenzymes is high, it is preferred to choose oxidative coenzyme NAD+ 、NAD(P) + .

[0092] When an oxidative coenzyme is selected, a method for achieving coenzyme regeneration needs to be selected, which mainly includes three types: (1) glucose dehydrogenase and co-substrate glucose; (2) alcohol dehydrogenase and co-substrate isopropanol; (3) formate dehydrogenase and co-substrate ammonium formate.

[0093] In a preferred embodiment, the coenzyme is NAD(P) + , the coenzyme regeneration system is glucose dehydrogenase, and the present invention preferably uses glucose dehydrogenase and cosubstrate glucose.

[0094] In another preferred embodiment of the present invention, the concentration of the oxidative coenzyme NAD+ is 0.05-1 g / L, preferably 0.1-1 g / L.

[0095] Typically, the buffer has a pH of 6-8; preferably, the pH is 7.0-7.3.

[0096] Typically, the coenzyme of the present invention can be regenerated by using glucose dehydrogenase and glucose.

[0097] In another preferred embodiment, the carbonyl reductase of the present invention can also achieve coenzyme regeneration by adding isopropanol and catalyzing the conversion of isopropanol into acetone by itself.

[0098] Preparation method

[0099] The present invention provides a method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol, comprising the steps of: in a liquid reaction system, using 1-(3-bromopyridin-2-yl)ethanone as a substrate, and using carbonyl reductase CnKRED or a mutant thereof to perform an enzymatic reaction, thereby preparing (S)-1-(3-bromopyridin-2-yl)ethanol as shown in formula II;

[0100]

[0101] In the present invention, the above reaction may be coupled with or not coupled with the coenzyme regeneration system.

[0102] Preferably, the above reaction is coupled with a coenzyme regeneration system in the same system, thereby further improving production efficiency, reducing production costs and increasing substrate tolerance.

[0103] Reaction system

[0104] The present invention also provides a reaction system for the preparation method of the present invention.

[0105] A typical reaction system includes:

[0106] (i) an aqueous solvent;

[0107] (ii) a substrate, wherein the substrate is 1-(3-bromopyridin-2-yl)ethanone represented by formula I;

[0108]

[0109] and (iii) carbonyl reductase CnKRED or a mutant thereof.

[0110] In another preferred embodiment, the reaction system further includes a coenzyme.

[0111] In another preferred embodiment, the reaction system further comprises a co-substrate.

[0112] In another preferred embodiment, the reaction system also includes an enzyme for regenerating the coenzyme.

[0113] The main advantages of the present invention are:

[0114] 1. The present invention provides a method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol by catalysis of carbonyl reductase CnKRED. The whole process is simple to operate, the reagents are cheap, and it is easy to scale up and reproduce in industrial production, which effectively reduces the cost of use.

[0115] 2. The present invention uses carbonyl reductase CnKRED as a biocatalyst to carry out biocatalytic preparation of (S)-1-(3-bromopyridin-2-yl)ethanol, the substrate concentration can reach up to 50 g / L, the conversion rate can reach 100%, the ee value is greater than 99.99%, and the product yield and purity are high.

[0116] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0117] Example 1 Construction and expression of carbonyl reductase CnKRED

[0118] The carbonyl reductase CnKRED gene (accession number WP_248392211.1) from Chryseobacterium nepalense was synthesized after codon optimization and cloned into the pET22b(+) vector to obtain the plasmid pET22b(+)-CnKRED. The plasmid was transformed into Escherichia coli BL21(DE3) competent cells, spread on ampicillin-resistant plates (50μg / mL), and cultured at 37°C overnight. Pick the positive transformation single colony, culture it in Luria-Bertani (LB) medium, extract the plasmid and sequence it. Finally, a recombinant genetic engineering bacterium expressing carbonyl reductase CnKRED was obtained.

[0119] The genetically engineered bacteria expressing carbonyl reductase CnKRED constructed above were inoculated into LB liquid culture medium containing a final concentration of 50 μg / mL ampicillin resistance, cultured at 37°C, 220rpm for 12 hours, and then inoculated into fresh fermentation liquid culture medium containing a final concentration of 50 μg / mL ampicillin resistance at an inoculum amount of 1.5% (v / v), cultured at 37°C, 220rpm until the bacterial OD600>0.8, added with isopropylthiogalactoside (IPTG) at a final concentration of 0.5mM, induced cultured at 28°C for 12 hours, centrifuged at 4°C, 8,000rpm for 10 minutes, discarded the supernatant, and collected the precipitate to obtain carbonyl reductase K18 wet bacteria. The fermentation medium formula is as follows: yeast extract (2.4%), soy peptone (1.2%), sodium chloride (0.3%), glycerol (0.5%), dipotassium hydrogen phosphate (0.2%), magnesium sulfate heptahydrate (0.05%).

[0120] Example 2: Preliminary screening of carbonyl reductase

[0121] In this example, a variety of carbonyl reductases were preliminarily screened, among which:

[0122] CnKRED is derived from Chryseobacterium nepalense, and its Genbank sequence number is WP_248392211.1;

[0123] G4 was derived from Empedobacter brevis ZJUY-1401;

[0124] CRS is derived from Lentilactobacillus rapi DSM 19907;

[0125] B12 was derived from K. marxianus ZJB14056;

[0126] F4 is derived from Glabrata CBS138;

[0127] A10 is derived from Streptomyces cyaneogriseus.

[0128] The screening results are shown in Table 1, where the specific reaction conditions are shown in the corresponding notes.

[0129] Table 1 Screening results of carbonyl reductase

[0130]

[0131] a indicates the reaction conditions: 2 mL reaction system (1): 20 g / L substrate, 40 g / L glucose, 1 mM NAD(P), 10 g / L glucose dehydrogenase (GDH), phosphate buffer (0.1 M, pH 7.0), 28°C, 220 rpm, 2 h.

[0132] b indicates the reaction conditions: 2 mL reaction system (2): 20 g / L substrate, 20% isopropanol, 1 mM NAD(P), phosphate buffer (0.1 M, pH 7.0), 28° C., 220 rpm, 2 h.

[0133] The liquid chromatography column used to detect chiral purity was Daicel CHIRALPAK IF. Mobile phase: n-hexane / isopropanol = 90 / 10, flow rate 1 mL / min, detection wavelength 214 nm, column temperature: 25°C. The elution time of the S-configuration product was about 9.2 minutes, and the elution time of the R-configuration isomer was about 7.2 minutes.

[0134] As shown in Table 1, the reduction effects of carbonyl reductases from different sources on the substrate 1-(3-bromopyridin-2-yl)ethanone in the present invention are significantly different, and the stereo configurations of the products are also different. Carbonyl reductase CnKRED has advantages over other carbonyl reductases in both conversion rate and stereoselectivity.

[0135] Among them, the chiral HPLC spectrum of (S)-1-(3-bromopyridin-2-yl)ethanol prepared by carbonyl reductase CnKRED catalytic reduction is as follows Figure 1 The HPLC spectrum of the 1-(3-bromopyridin-2-yl)ethanol racemate prepared by carbonyl reductase A10b is shown in Figure 2 shown.

[0136] Example 3: Construction and evaluation of carbonyl reductase CnKRED homologous enzyme

[0137] Using the wild-type carbonyl reductase CnKRED as a template, three homologous enzymes were constructed based on sequence homology alignment and PROSS tools (https: / / pross.weizmann.ac.il / step / pross-terms), and constructed and expressed according to Example 1. The homologous enzyme evaluation reaction system is as follows: Take 100 mM phosphate buffer (10 mL) at pH 7.0, add NAD(P) + (0.005g), glucose (0.6g), carbonyl reductase CnKRED or homologous enzyme (0.2g), glucose dehydrogenase (0.1g), compound 1-(3-bromopyridin-2-yl)ethanone (0.3g), react at 45°C for 15min, and monitor the reaction conversion rate by HPLC. The relative activities of each homologous enzyme are shown in Table 2. The results show that homologous enzyme 3 has the best activity.

[0138] Table 2 Comparison of the activities of CnKRED and three homologous enzymes

[0139]

[0140] Please note: Compared with Example 2, the reaction time here is shorter (15 min) and the corresponding conversion rate is lower.

[0141] Example 4 Preparation of (S)-1-(3-bromopyridin-2-yl)ethanol using carbonyl reductase CnKRED

[0142]

[0143] Take 100 mM phosphate buffer (10 mL) at pH 7.0 and add NAD(P) + (0.003g), glucose (0.38g), carbonyl reductase CnKRED (1.0g), glucose dehydrogenase (0.5g), compound 1-(3-bromopyridin-2-yl)ethanone (0.2g), react at 30°C, monitor the reaction conversion rate when >99% by HPLC, centrifuge, take the supernatant, extract the aqueous layer with ethyl acetate (10ml×2), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 0.17g of light yellow oil, with a yield of 85.0%, and an ee value of >99.99%.

[0144] Example 5 Preparation of (S)-1-(3-bromopyridin-2-yl)ethanol using carbonyl reductase CnKRED

[0145] Take 100 mM phosphate buffer (100 mL) at pH 7.0 and add NAD(P) +(0.03g), add glucose (9g), add carbonyl reductase CnKRED (10.0g), add glucose dehydrogenase (5.0g), add compound 1-(3-bromopyridin-2-yl)ethanone (5.0g), react at 30°C, monitor the reaction conversion rate when >99% by HPLC, centrifuge, take the supernatant, extract the aqueous layer with ethyl acetate (100ml×2), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 4.6g of light yellow oil, with a yield of 92.0%, and an ee value of >99.99%.

[0146] Example 6 Preparation of (S)-1-(3-bromopyridin-2-yl)ethanol using carbonyl reductase CnKRED

[0147] Take 100 mM phosphate buffer (14 mL) at pH 7.0 and add NAD(P) + (0.006g), add isopropanol (6mL), add carbonyl reductase CnKRED (2.0g), isopropanol dehydrogenase (1.0g), add compound 1-(3-bromopyridin-2-yl)ethanone (0.4g), react at 30°C, monitor the reaction conversion rate when >99% by HPLC, centrifuge, take the supernatant, extract the aqueous layer with ethyl acetate (20ml×2), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 0.34g of light yellow oil, with a yield of 83.3%, and ee value>99.99%.

[0148] Example 7 Preparation of (S)-1-(3-bromopyridin-2-yl)ethanol using carbonyl reductase CnKRED homologous enzyme (sequence number 3)

[0149] Take 100 mM phosphate buffer (90 mL) at pH 7.0 and add NAD(P) + (0.03g), glucose (13g), carbonyl reductase CnKRED homologous enzyme (serial number 3) (10.0g), glucose dehydrogenase (4.0g), 1-(3-bromopyridin-2-yl)ethanone (7.0g) dissolved in 10mL DMSO were added, and the reaction was carried out at 45°C. When the reaction conversion rate was >99% monitored by HPLC, the supernatant was taken, and the aqueous layer was extracted with dichloromethane (100ml×2). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 6.6g of a light yellow oil with a yield of 94.3% and an ee value of >99.99%.

[0150] Example 8 Preparation of (S)-1-(3-bromopyridin-2-yl)ethanol using carbonyl reductase CnKRED homologous enzyme (sequence number 3)

[0151] Take 100 mM phosphate buffer (70 mL) at pH 7.0 and add NAD(P) +(0.05g), add isopropanol (30mL), add carbonyl reductase CnKRED homologous enzyme (serial number 3) (10.0g), isopropanol dehydrogenase (4.0g), add compound 1-(3-bromopyridin-2-yl)ethanone (8.0g), react at 45°C, monitor the reaction conversion rate when >99% by HPLC, centrifuge, take the supernatant, extract the aqueous layer with dichloromethane (100ml×2), combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 7.6g of light yellow oil with a yield of 95.0% and an ee value of >99.99%.

[0152] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0153] CnKRED nucleotide sequence (SEQ ID No. 1):

[0154] ATGAAAACTGTACTAATAACAGGAGCTAATAGATCGATTGGTCTGGAAACTGCGAAGCAACTGTCCGAGAAAGGCCTCTTCGTGTACCTGGGCTCTCGTAATCTGGCAAAGGGTGAAGCAACCGTTAAAGAATTATCGGAGAAGGAGTTCCAGAACATCAAGGCGATTGAGATCGACGTTACGGACCCGGAATCTATCGTTAAGGCCAAGAATACCATCGAGTCTGAACAAGTTAAACTGGACATCCTGATCAACAACGCGGGTATTTTGGGTGTCAACCCGCAAACCGCCGCGGAAACCTCCGTGAACGACATCCGCGAAGTTTTTGATACCAATTTCTTCGGCGTGATTAACGTGACGCAGGCATTTCTGGATCTGCTGAAAAAGAGCGATAGCCCGCGTATTTCCAACATTACCTCCGGTTTGGGCAGCCTGACCCTGCATACGGACCCGGAGTGGAAATATTACCACATCAAGACCGCGGCTTACGGCCCAAGCAAATCAGCGTTGAATGCGTACACCATTGCGCTGGCGTATGAATTGCGTGAGCTTCCGTTTAAAGTTAACGTGATCGATCCGGGTTATACCGCTACCGATTTTAACGGCCACAGCGGTCCGGGTAGCGTAGAGAGCGCAGCTAGCTTTATCATCAAACATACCATTGCGGACGAGAACGGCCCTACAGGTCAGTATTTCAGCAATGACATCGAGGATGAAACCGGCATCAGCCCGTGGTAA

[0155] Amino acid sequence of CnKRED (SEQ ID No. 2):

[0156] mktvlitganrsigletakqlsekglfvylgsrnlakgeatvkelsekefqnikaieidvtdpesivkakntieseqvkldilinnagilgvnpqtaaetsvndirevfdtnffgvinvtqafldllkksdsprisnitsglgsltlhtdpewkyyhiktaaygpsksalnaytialayelrelpfkvnvidpgytatdfnghsgpgsvesaasfiikhtiadengptgqyfsndiedetgispw.

Claims

1. A method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol, It is characterized in that The method comprises the following steps: in a liquid reaction system, using 1-(3-bromopyridin-2-yl)ethanone as a substrate and using carbonyl reductase CnKRED or a mutant thereof to carry out an enzymatic reaction, thereby preparing (S)-1-(3-bromopyridin-2-yl)ethanol as a substrate; 2. The preparation method according to claim 1, It is characterized in that The carbonyl reductase CnKRED mutant is selected from the following group: (a) Carbonyl reductase CnKRED mutant with E49G, E64K, V78G, A98T, I158V, A222T, D238N, T240A; (b) carbonyl reductase CnKRED mutants with E49G, D62N, V67A, T72V, I73V, S75N, V78G, A178S, R182I, A222T; (c) carbonyl reductase CnKRED mutants with S22A, S46K, E49G, D62N, V67A, T72V, I73V, S75N, V78G, N118R, S136V, A222T; Wherein, the amino acid numbering is based on the amino acid numbering of the carbonyl reductase CnKRED shown in SEQ ID No.

2.

3. The preparation method according to claim 1, It is characterized in that The coding gene sequence of the carbonyl reductase CnKRED is selected from the following group: (a) the sequence shown in SEQ ID NO.1; (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%) sequence identity with the sequence defined in (a).

4. The preparation method according to claim 1, It is characterized in that The reaction is carried out in the presence of a coenzyme.

5. The preparation method according to claim 4, It is characterized in that The coenzyme is selected from the following group: a reducing coenzyme, an oxidizing coenzyme, or a combination thereof.

6. The preparation method according to claim 1, It is characterized in that In the reaction system, there is also an enzyme for regenerating the coenzyme.

7. The preparation method according to claim 6, It is characterized in that The enzyme used for coenzyme regeneration is selected from the following group: glucose dehydrogenase, isopropanol dehydrogenase, formate dehydrogenase.

8. An isolated or purified carbonyl reductase CnKRED mutant, It is characterized in that The carbonyl reductase CnKRED mutant is selected from the following group: (a) carbonyl reductase mutants with E49G, E64K, V78G, A98T, I158V, A222T, D238N, T240A; (b) carbonyl reductase mutants having E49G, D62N, V67A, T72V, I73V, S75N, V78G, A178S, R182I, A222T; (c) carbonyl reductase mutants having S22A, S46K, E49G, D62N, V67A, T72V, I73V, S75N, V78G, N118R, S136V, A222T; Wherein, the amino acid numbering is based on the amino acid numbering of the wild-type carbonyl reductase shown in SEQ ID No.

2.

9. A reaction system, It is characterized in that The reaction system comprises: (i) an aqueous solvent; (ii) a substrate, wherein the substrate is 1-(3-bromopyridin-2-yl)ethanone represented by formula I; and (iii) carbonyl reductase CnKRED or a mutant thereof.

10. A method for preparing (S)-1-(3-bromopyridin-2-yl)ethanol, It is characterized in that The method comprises the steps of: using the reaction system as claimed in claim 9, carrying out an enzymatic reaction under enzyme catalysis conditions, thereby preparing (S)-1-(3-bromopyridin-2-yl)ethanol.

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