CN110777125B - Efficient preparation method of heterocyclic drug intermediate - Google Patents

Abstract

The invention discloses a high-efficiency preparation method of a heterocyclic drug intermediate. The invention uses an alcohol dehydrogenase mutant and glucose dehydrogenase coupling to catalyze a heterocyclic substrate to generate a heterocyclic drug intermediate, wherein the alcohol dehydrogenase mutant mutates tyrosine at 127 bit of an alcohol dehydrogenase female parent with an amino acid sequence shown as SEQ ID NO.2 into tryptophan. The mutant Y127W of the invention can reduce the product inhibition effect in a single aqueous phase system without adding any cosolvent, so that the conversion rate can reach more than 99% within 12 h. The mutant Y127W is coupled with glucose dehydrogenase (BmGDH), and the single-aqueous phase system without any exogenous coenzyme and organic cosolvent realizes the scale of 50mL and the substrate concentration as high as 600 g.L^‑1The catalyst loading was 3.3%. The e.e. value of the final product (S) -NBHP is as high as 99.4%, and the space-time yield is about 1400 g.L^‑1·d^‑1The purity of the product is 99.58%.

Description

Efficient preparation method of heterocyclic drug intermediate

Technical Field

The invention relates to a high-efficiency preparation method of a heterocyclic drug intermediate, belonging to the technical field of biocatalysis.

Background

(S)-N-B_OC-3-hydroxypiperidine [ (S) -NBHP]Is a key chiral intermediate for synthesizing a medicament for treating lymphoma, namely IBRUTINIB (IBRUTINIB). The method for biosynthesis of (S) -NBHP is more environmentally friendly and is receiving increasing attention. However, the conventional research has been conductedThe results show that the biological method for synthesizing (S) -NBHP requires the addition of organic cosolvent and expensive coenzyme, and the high substrate concentration can cause the inhibition of substrate or product, thus increasing the synthesis cost of (S) -NBHP.

(1)2009 a_CHERETZAnd the like, a biocatalytic synthesis method is adopted for the first time, reductase in carrot blocks is used for catalysis, and the catalyst is cheap and environment-friendly, so that a new idea is provided for catalytically synthesizing optically active cyclic 3-hydroxypiperidine. However, this reaction is not suitable for industrial scale-up due to the low substrate concentration (3MM), high concentration of added catalyst (23%, M/V) and low yield (73%). (ORGANIC LETTERS,2009,11(6):1245-

(2) 2014X_IN J_UAnd the (S) -NBHP is synthesized by screening commercial ketoreductase KRED and utilizing the capability of ketoreductase for oxidizing isopropanol to construct a method for regenerating substrate coupling coenzyme, and the substrate is added in batches in the preparation process to reduce the substrate inhibition effect and finally realize the substrate concentration of 100 G.L^-1The biotransformation of (1). (O)_RGANIC P_ROCESS R_ESEARCH&D_EVELOPMENT,2014,18(6):827-830.)

(3)2016 (Z)_HONG-L_IU W_UFrom C_{HRYSEOBACTERIUM SP}The 27 ketoreductases were called from the CA49 genome and screened to obtain C_HKRED03, reaction of C_HThe KRED03 is coupled with GDH to realize the biological synthesis method of the cofactor recycling system, and finally the substrate concentration of 200 G.L is realized in the reaction system added with methanol for assisting in dissolving^-1The biotransformation of (1). (P)_ROCESS B_IOCHEMISTRY,2016,51(7):881-885.)

(4) 2017J_INGJING C_HENBy using Source C_{ANDIDA PARAPSILOSIS}Nostol dehydrogenase C_PRCR and glucose dehydrogenase B derived from Bacillus megaterium_MGDH in E.coli R_OSETTA(DE3) Co-expression, the substrate concentration of 100 G.L was achieved in an organic-aqueous two-phase system using recombinant co-expressed whole cells^-1The biotransformation of (1). (W)_ORLD J_{OURNAL OF}M_{ICROBIOLOGY AND} B_IOTECHNOLOGY,2017,33(61):2-12.)

(5) 2017M_ENGYANH et al obtained Thermotoga maritima (T) by screening ketoreductase libraries_{HERMOTOGAMARITIMA}) The catalytic process of the high-temperature-resistant ketoreductase AKR-43 also utilizes GDH in the biological synthesis method 2 to regenerate and recycle coenzyme, and realizes the substrate concentration of 200 G.L in an aqueous phase system added with isopropanol cosolvent^-1The biotransformation of (1). (A)_PPLIED B_{IOCHEMISTRY AND} B_IOTECHNOLOGY,2017,181(4):1304-1313.)

(6) L of 2017_I-F_ENG C_HENIsolated from Kluyveromyces marxianus ATCC 748 (K)_{LUYVEROMYCESMARXIANUS}ATCC 748) NADPH-dependent reductase (YGL039W) to produce (R) -N-B_OC-3-hydroxypiperidine shows excellent catalytic activity, and cyclic coenzyme regeneration is carried out using GDH, and substrate concentration 400 G.L is achieved by adding isopropanol as a cosolvent to the reaction system^-1The biotransformation of (1). (C)_ATALYSIS C_{OMMUNICATIONS},2017,97:5-9.)

(7) L of 2017_I-F_ENG C_HENIsolated by et al from Saccharomyces cerevisiae (S)_{ACCHAROMYCESCEREVISIAE}) The NADPH-dependent reductase (YDR541C) was found to have excellent catalytic activity in production. Meanwhile, GDH is also adopted to construct coenzyme regeneration cycle, but serious product inhibition phenomenon is found in a single-aqueous phase reaction system, and finally a two-phase system of 1:1(V/V) ethyl caprylate and water is introduced to reduce product inhibition and realize substrate concentration of 240 G.L^-1The biotransformation of (1). (T)_ETRAHEDRONL_ETTERS, 2017,58(16):1644-1650.)

(8) 2017G_AO-W_EI Z_HENGProtein engineered ketoreductase C of et al_GKR1-F92C/F94W preparation of chiral alcohols with diverse structures, coenzyme circulation by coupling GDH, and substrate concentration of 100 G.L in a system with ethanol cosolvent^-1The biotransformation of (1). (ACS C)_ATALYSIS,2017,7(10):7174-7181.)

(9)X of 2018_IANGXIAN Y_INGEt al, by genome mining, obtain reductase R capable of catalyzing chiral ketone_ECR, derived from Rhodococcus erythropolis WZ010 (R)_{HODOCOCCUSERYTHROPOLIS}WZ010) and the authors explored R_EUse of CR in the synthesis of chiral alcohols. Finally, the authors make use of R_ECR mutant Y54F, using isooctanol as co-substrate to construct coenzyme cycle, realizing substrate concentration 300 G.L in isooctanol two-phase system^-1The biotransformation of (1). (M)_OLECULES,2018,23(3117):2-13.)

(10) Y in 2019_ITONG C_HENEt al T from Thermoanaerobacter_BADH (T_{HERMOANAEROBACTERBROCKII}) Co-expressing with glucose dehydrogenase of Bacillus subtilis in Escherichia coli BL21(DE3), optimizing cell culture system, and realizing substrate concentration of 100 G.L in system added with methanol cosolvent^-1The biotransformation of (1). (RSC A)_DVANCES,2019,9(4):2325-2331.)

(11) Patent CN201310173088.2 discloses an asymmetric reduction of N-B by recombinant Ketoreductase (KRED) enzyme powder_OC-3-piperidone, but no gene sequence or amino acid sequence of Ketoreductase (KRED) is disclosed. Patent CN201310054684.9 discloses asymmetric synthesis of (S) -1-tert-butoxycarbonyl-3-hydroxypiperidine by using alcohol dehydrogenase PAR, but the coenzyme circulation is performed by using an organic reagent isopropanol, and the organic reagent has great damage to the enzyme activity and obvious inhibition effect. Patent CN201610132936.9 discloses the use of carbonyl reductase R_ECR enzymes asymmetric reductone reductases (KRED), but the enzyme requires N-passage_INTA purification and the two-phase reaction of sec-octanol and water are not favorable for scale-up production or the production cost is higher. Pichia pastoris P reported in patent CN108220358A_ICHIASIT2014 can be used as a biocatalyst for preparing (S) -NBHP, but the excessive addition of the catalyst increases the production cost. CN10822061A reports that ketoreductase MT-KRED is used for preparing (S) -NBHP, but expensive coenzyme is required to be added in the reaction process.

Although the ketoreductase reported above can be used for preparing (S) -NBHP, expensive coenzyme, large amount of enzyme and organic solvent are required in the reaction process, which is not favorable for the amplification application in the actual industry.

Disclosure of Invention

In order to solve the technical problem, the alcohol dehydrogenase KPADH carries out molecular modification, and the method for efficiently preparing the drug intermediate (S) -N-Boc-3-hydroxypiperidine is realized. The invention utilizes site-directed saturation mutagenesis to obtain the optimal mutant, and improves the stereoselectivity, the conversion rate and the substrate tolerance of NBPO.

The first purpose of the invention is to provide a high-efficiency preparation method of a heterocyclic drug intermediate, the method is to couple and catalyze a heterocyclic substrate to generate the heterocyclic drug intermediate by alcohol dehydrogenase mutant and glucose dehydrogenase, and the alcohol dehydrogenase mutant is to mutate tyrosine at 127 bit of an alcohol dehydrogenase female parent with an amino acid sequence shown as SEQ ID NO.2 into tryptophan.

Furthermore, the heterocyclic drug intermediate is (S) -N-Boc-3-hydroxypyrrolidine, (S) -N-Boc-3-hydroxypiperidine or N-Boc-4-hydroxypiperidine.

Further, the heterocyclic substrate is N-Boc-3-pyrrolidone, N-Boc-3-piperidone (NBPO) or N-Boc-4-piperidone.

Further, in the coupling catalysis process, glucose is added, and the molar mass ratio of the glucose to the NBPO is 1-2: 1.

Further, the mass ratio of the alcohol dehydrogenase mutant to the glucose dehydrogenase is 1-2: 1.

Furthermore, the addition amount of the alcohol dehydrogenase mutant and the glucose dehydrogenase is 3-5% of the mass of the NBPO.

Further, the loading capacity of the N-Boc-3-piperidone is 100-g.L^-1。

Further, the nucleotide sequence of the female parent of the alcohol dehydrogenase is shown in SEQ ID NO. 1.

Furthermore, the temperature of the coupling catalytic reaction is 25-35 ℃, and the pH value is 6.5-7.5.

The invention has the beneficial effects that:

(1) mutations of the inventionBody Y127W k compared to wild type_cat/K_mThe improvement is 21.5 times, the half-life period at 30 ℃ is up to 147 hours, and the improvement is 67 hours compared with the wild type.

(2) The mutant Y127W of the invention has improved catalytic efficiency and stereoselectivity for substrate with Boc group and carbonyl at C-3 position, and has improved catalytic efficiency and stereoselectivity for N-3-Boc-pyrrolidone compared with k_cat/K_mThe wild type is increased by 10.5 times, and the e.e. value is increased to 99.9% (S).

(3) The mutant Y127W of the invention can reduce the product inhibition effect in a single aqueous phase system without adding any cosolvent, so that the conversion rate can reach more than 99% within 12 h. The mutant Y127W is coupled with glucose dehydrogenase (BmGDH), under the optimal condition, a single aqueous phase system without any exogenous coenzyme and organic cosolvent is realized, the scale of 50mL is realized, and the substrate concentration is as high as 600 g.L⁰¹The catalyst loading was 3.3%. The e.e. value of the final product (S) -NBHP is as high as 99.4%, and the space-time yield is about 1400 g.L^-1·d ^-1The purity of the product is 99.58%.

Drawings

FIG. 1 is an SDS-PAGE electrophoretic analysis M: protein marker; lanes 1& 2: pure and crude enzyme for KpADH; 3& 4: pure enzyme & crude enzyme of Y127A; 5& 6: pure enzyme & crude enzyme of Y127C; 7& 8: pure enzyme & crude enzyme of Y127F; 9& 10: pure enzyme & crude enzyme of Y127I; 11& 12: pure enzyme & crude enzyme of Y127M; 13& 14: pure enzyme & crude enzyme of Y127Q; 15& 16: crude & pure enzyme of Y127V; 17& 18: crude & pure enzyme of Y127W;

FIG. 2 shows the enzyme coupling construction of coenzyme cyclic regeneration synthesis of (S) -NBHP;

FIG. 3 is a diagram of the purity of the product.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

The enzyme activity determination method comprises the following steps: depending on the characteristic absorption peak of NAD (P) H at 340nm, ketoreductases will produce or consume NAD (P) H during the oxidation or reduction reaction. Thus, enzyme activity can be calculated indirectly using changes in NAD (P) H at 340 nm. One unit of enzyme activity (U) is defined as the amount of enzyme required to oxidize 1. mu. mol of NAD (P) H per minute under the above-mentioned conditions of activity measurement.

Reduction activity assay system:

measurement System for Oxidation Activity:

and (3) a liveness measuring process: the assay temperature was set at 30 ℃ and all buffers were preheated to 30 ℃. Substrate, coenzyme and buffer were added separately to clean 96 wells.

The concentration of the crude enzyme solution protein was measured by Bradford method, and the color of the protein-pigment conjugate was measured at 595nm based on the change of color of Coomassie brilliant blue G-250 after binding to the protein, and the absorbance was proportional to the concentration of the protein. With a concentration of 5 mg.L^-1The BSA bovine serum standard protein is mother liquor, and the concentration interval of the prepared solution by gradient dilution is 0.01-0.12 mg.L^-1Protein concentration of (a). Diluting the protein to be detected to the protein concentration standard range, sucking 20 μ L protein liquid, adding 180 μ L Coomassie brilliant blue, standing at 30 deg.C for 5min, and detecting at 595 nm. In order to reduce errors, the samples measured each time are measured together with the protein standard curve, a standard protein concentration curve is drawn, and the protein concentration of the sample to be measured is calculated according to the curve. 3 replicates were measured for each sample.

The concentration of the pure enzyme protein is determined according to the fact that most proteins have the maximum absorption peak at 280nm, and therefore concentration data can be directly obtained through a Nanodrop instrument. After the purified protein is concentrated and desalted, the molar extinction coefficient and the protein molecular weight of the protein are found by a website https:// web.expasy.org/protparam/the 5 microliter of pure enzyme solution is titrated on an instrument, and the protein concentration is read according to the molar extinction coefficient and the protein molecular weight. The protein is sequentially diluted by different times, and the determination results have good linear relation under different dilution times. The protein concentration of the pure enzyme can be obtained.

The conversion was analyzed by High Performance Liquid Chromatography (HPLC). The sample to be tested is extracted by ethyl acetate, dried by anhydrous sodium sulfate, evaporated by a vacuum concentrator and finally dissolved in the mobile phase. The analytical column was a C18 column (4.6X 250mm, Diamonsil, Shanghai DIKMA Co. Ltd.) and the mobile phase was 55% volume fraction acetonitrile and 45% volume fraction water. The detector wavelength was 210nm and the column temperature was 30 ℃. The stereoselective analytical column was a Superchiral S-AY column (4.6X 150mm, Shanghai Chiralway Biotech Co. Ltd.) with a mobile phase of 95% volume fraction n-hexane and 5% volume fraction ethanol. The detector wavelength was 210nm and the column temperature was 30 ℃.

Example 1:

the method for rationally mutating and constructing the key amino acid of the alcohol dehydrogenase KPADH comprises the following steps:

first, find out the key amino acid for controlling stereoselectivity and catalytic activity

Finding out the amino acid interacting with the substrate NBPO through the crystal structure (PDB:5Z2X) derived from Kluyveromyces alcohol dehydrogenase KPADH; and screening out a key amino acid residue 127 site through conservative analysis of amino acid alignment with more than 30% of homology.

Second, construction of mutants

Site-directed saturation mutagenesis was carried out on the 127 th amino acid of the alcohol dehydrogenase KPADH having an amino acid sequence shown in SEQ ID No.1 by a whole-plasmid PCR method using a pET28a-KPADH recombinant plasmid deposited in the laboratory as a template (described in the patent application publication No. CN 105936909A). And mutants Y127A, Y127C, Y127F, Y127I, Y127M, Y127Q, Y127V and Y127W with catalytic activity improved compared with that of WT are obtained through reduction activity determination and screening.

Example 2:

the mutant with improved catalytic activity was purified by inducible expression and subjected to kinetic assay according to example 1, which comprises the following steps:

first, inducible expression

The mutant of example 1 was inoculated into 50. mu.L/mL LB medium and cultured with shaking at 37 ℃ and 180 rpm. When OD600 reached 0.8, isopropyl-. beta. -D-thiogalactopyranoside (IPTG, 0.2M) was added to a final concentration of 0.2mM, and the temperature was lowered to 25 ℃ to induce protein expression. At the end of the incubation, the cells were harvested by centrifugation and sonicated in PBS buffer (pH 7.4). The cell lysate was centrifuged at 8000rpm for 30 min.

Secondly, protein purification

The method adopts a nickel column affinity chromatography, and according to the fact that the N end of KPADH is provided with a His-Tag label, the His-Tag label can be combined with nickel competitively, and two elution modes, namely gradient elution and linear elution, can be adopted. KPADH and its mutant could be eluted completely when the imidazole concentration reached 200mM during the gradient elution. After the purification, samples were collected and analyzed for the band of interest by SDS-PAGE, and the eluate was concentrated to give clear bands without contaminating proteins. The purification results are shown in FIG. 1.

Third, kinetic determination

The activity of the KpADH mutant was measured at different substrate concentrations and fitted to nonlinear regression of the michaelis equation using Origin software based on the activity and substrate concentration data, respectively. The results are shown in Table 2 for kinetic parameters of alcohol dehydrogenase KPADH mutant on NBPO. As is clear from Table 1, the catalytic efficiencies of mutants Y127C, Y127W and Y127Q (k)_cat/K_m) Compared with wild type, the wild type gene has the advantages of 7.9, 8.7 and 21.5 times higher activity.

Kinetic parameters of mutants at site 1127 in Table

Fourth, determination of mutant stability

Purified KPADH and its mutant enzyme solution (protein concentration maintained at 1 mg. mL)^-1Left and right) 1 mL of the solution is put into a 30 ℃ constant-temperature water bath kettle, and sampling is carried out at regular time to determine the activity. The activity of 0h is 100%, the specific activity is sequentially measured to obtain a curve changing along with time, and the time for the activity to be reduced to 50% is the curveHalf-life of the protein at 30 ℃. The half-life of WT, Y127C, Y127Q and Y127W were 80h, 24h, 26h and 147h, respectively. The mutant with the longest half-life at 30 ℃ was shown to be Y127W. Binding kinetic parameters finally Y127W was chosen as the target mutant for gram-scale production.

Example 3:

in order to explore the substrate spectrum of the heterocyclic ketone of the KpADH and the mutant, the substrate kinetics of the heterocyclic ketone of the KpADH and the mutant are respectively determined by selecting the heterocyclic ketone dihydro-3 (2H) -furanone, tetrahydrothiophene-3-ketone, cyclohexanone, 4-ethylcyclohexanone, N-Boc-3-pyrrolidone, N-Boc-2-piperidone, N-Boc-3-piperidone and N-Boc-4-piperidone. As shown in Table 2, catalytic activity was exhibited on all substrates except for the ortho-position N-Boc-2-piperidone substrate. Mutant Y127W in substrate spectra, k to substrate N-Boc-3-pyrrolidone_cat/K_mFrom 0.2s^-1·mM^-1Increased to 2.1s^-1·mM^-1And k to substrate 7a_cat/K_mFrom 3.6s^-1·mM^-1Increased to 31.0s^-1·mM^-1. Meanwhile, the e.e. value of the two substrates is also improved to more than 99 percent. This indicates that mutant Y127W has improved catalytic efficiency specificity for substrates bearing a Boc group and a carbonyl group at the C-3 position. It also shows that 127 site on the substrate Boc group has a fine-tuning effect on stereoselectivity. Therefore, it is advantageous to select residues around the Boc group for mutation.

TABLE 2 kinetic analysis of the substrate spectra of KPADH and mutant Y127W for different heterocyclic ketones

Example 4:

the mutants with improved catalytic activity were optimized systematically according to example 2, with the following steps:

optimization of one, different additives

We selected 3 systems (single aqueous phase system without solubilizing aid, aqueous phase system with 5% ethanol, two-phase system with 50% butyl acetate) to explore the optimal reaction system for mutant Y127W, using lyophilized enzyme powder of 35mg Y127W and 20mg BmGDH added to 10mL reaction system containing 1g substrate and 1.2g glucose. In the reaction process, freeze-dried enzyme powder and PBS 7.0 buffer solution are added firstly, the mixture is mechanically stirred evenly, and substrate and glucose are added at one time. It can be seen from Table 3 that the optimum reaction system for Y127W is a single aqueous phase system without any co-solvent.

TABLE 3 optimization of the catalytic System

Second, optimization of different enzyme adding amount

Under the condition of no cosolvent and no additional coenzyme, the biotransformation concentration of NBPO is increased to 800 g.L^-1To test the feasibility of the reaction at high concentrations of substrate. As shown in Table 4, as the concentration of the substrate increases, the ratio of the substrate to the catalyst also increases synchronously, and a large amount of the catalyst carries a large amount of the cofactor, which contributes to the improvement of the catalytic efficiency. Through the optimization of enzyme addition, the final selection of catalyst is 3.3% of the substrate addition, and the substrate concentration is 600 g.L^-1Carrying out amplification reaction.

TABLE 4 optimization of enzyme addition

Preparation of three, 50mL gram grade

The optimized 10mL reaction system was scaled up to 50mL using lyophilized enzyme powder of Y127W and BmGDH, containing 30g of substrate and 36g of glucose. In the reaction process, freeze-dried enzyme powder and PBS 7.0 buffer solution are added firstly, the mixture is mechanically stirred evenly, and substrate and glucose are added at one time. The process curves are shown in Table 5, and the 50mL reaction system is in agreement with the progress of the 10mL reaction system, without encountering the difficulties of the scale-up process and the product and substrate inhibition. The reaction was carried out at a conversion rate of 30% per hour in the early phase and already at 6h, the conversion rate was as high as 83.3%. The reaction rate gradually decreases with decreasing substrate concentration. After 10h, the conversion rate reaches 99.9%, which indicates that the mutant has the potential of continuously catalyzing NBPO. And the e.e. value is constant over 99% during the reaction, indicating that the e.e. value of the mutant is not affected by the reaction time or the concentration of the substrate product. After the reaction was completed for 12 hours, the reaction solution was collected.

TABLE 550 mL reaction progress

Reaction time (h)	Conversion (%)	e.e.(％)
			2	38.1	99.4
4	63.9	99.4
			6	83.3	99.4
8	96.9	99.4
			10	99.9	99.4
12	100.0	99.4

Fourthly, extracting and nuclear magnetic identification of products

According to the distribution coefficient of the product in different organic phases, the dichloromethane with the highest distribution coefficient of the product is used for extraction. The collected reaction solution was left at 70 ℃ for 2h to denature part of the protein and reduce the emulsification during extraction. The reaction solution was extracted 3 times with 3 volumes of dichloromethane, and no serious emulsification was observed in the process. Collecting extract, volatilizing dichloromethane in a vacuum rotary evaporator in water bath at 30 ℃, increasing the temperature of the water bath to 50 ℃ after most of dichloromethane is volatilized, and volatilizing residual dichloromethane as far as possible. After the concentration, the product (S) -NBHP was obtained as a pale yellow solid after standing in a refrigerator at 4 ℃. Subjecting the product (S) -NBHP to nuclear magnetic NMR substance identification, gas chromatography purity identification, liquid chromatography stereoselectivity identification and polarimeter optical rotation identification. Nuclear magnetic results: 1H NMR (300MHz, CDCl3) δ 3.83-3.65 (m,2H),3.53(dd, J ═ 13.3,6.0Hz,1H), 3.17-2.98 (m,2H),2.03(s, 1H),1.86(dt, J ═ 12.4,8.5Hz,1H), 1.80-1.68 (m,1H), 1.57-1.36 (m, 11H). Optical rotation: [ α ]25D ═ 21.52(c 0.1 EtOH).

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Sequence listing

<110> university of south of the Yangtze river

<120> high-efficiency preparation method of heterocyclic drug intermediate

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1029

<212> DNA

<213> (Artificial sequence)

<400> 1

atgagcgtat taattagtgg tgcttctgga tacattgcca aacatatcgt cagagttctt 60

ttggaacaaa attacaaagt aattggtact gttagaagtc aagacaaagc tgataagtta 120

ttgaaacaat ataataatcc taatttgtct tatgaaattg tacctgaaat agcaaactta 180

gatgcttttg atgacatttt taagaaacat ggtaaggaaa taaaatatgt cattcatgca 240

gcttcaccag tgaacttcgg cgcaaaagat ttggaaaaag atttagttat tcctgccatt 300

aatggtacca agaatatgtt cgaagctatt aaaaagtatg ccccagatac tgtcgaacgt 360

gttgtaatga ctgcttctta tgcttcaatt atgaccccac atagacaaaa tgatccaact 420

ttaactttag atgaagaaac ttggaatcca gtaactgaag aaaatgctta tgaaaatgtc 480

ttcactgctt attgtgcttc aaaaactttt gctgaaaagg aagcttggaa gttcgttaaa 540

gaaaatagtg atgctgttaa gtttaaacta accactatcc acccatcctt cgttttcgga 600

cctcagaact ttgatgaaga cgtcactaag aaactaaatg aaacttgtga aattatcaat 660

ggtttattac atgctccatt tgacaccaaa gtcgaaaaga ctcacttcag tcaattcatt 720

gatgttcgtg atgtcgccaa aactcacgtt ttgggtttcc aaaaagatga attaatcaac 780

caaagattgt tattatgtaa cggcgccttc tctcaacaag atattgttaa tgtatttaat 840

gaagatttcc cagagttaaa aggccaattc ccaccagaag ataaggacac cgatttaaac 900

aaaggtgtaa caggttgtaa aattgataat gaaaagacta aaaaattatt agcatttgaa 960

tttactcctt tccataaaac aattcatgac actgtctatc aaattttaca taaagaaggt 1020

agagtttaa 1029

<210> 2

<211> 342

<212> PRT

<213> (Artificial sequence)

<400> 2

Met Ser Val Leu Ile Ser Gly Ala Ser Gly Tyr Ile Ala Lys His Ile

1 5 10 15

Val Arg Val Leu Leu Glu Gln Asn Tyr Lys Val Ile Gly Thr Val Arg

20 25 30

Ser Gln Asp Lys Ala Asp Lys Leu Leu Lys Gln Tyr Asn Asn Pro Asn

35 40 45

Leu Ser Tyr Glu Ile Val Pro Glu Ile Ala Asn Leu Asp Ala Phe Asp

50 55 60

Asp Ile Phe Lys Lys His Gly Lys Glu Ile Lys Tyr Val Ile His Ala

65 70 75 80

Ala Ser Pro Val Asn Phe Gly Ala Lys Asp Leu Glu Lys Asp Leu Val

85 90 95

Ile Pro Ala Ile Asn Gly Thr Lys Asn Met Phe Glu Ala Ile Lys Lys

100 105 110

Tyr Ala Pro Asp Thr Val Glu Arg Val Val Met Thr Ala Ser Tyr Ala

115 120 125

Ser Ile Met Thr Pro His Arg Gln Asn Asp Pro Thr Leu Thr Leu Asp

130 135 140

Glu Glu Thr Trp Asn Pro Val Thr Glu Glu Asn Ala Tyr Glu Asn Val

145 150 155 160

Phe Thr Ala Tyr Cys Ala Ser Lys Thr Phe Ala Glu Lys Glu Ala Trp

165 170 175

Lys Phe Val Lys Glu Asn Ser Asp Ala Val Lys Phe Lys Leu Thr Thr

180 185 190

Ile His Pro Ser Phe Val Phe Gly Pro Gln Asn Phe Asp Glu Asp Val

195 200 205

Thr Lys Lys Leu Asn Glu Thr Cys Glu Ile Ile Asn Gly Leu Leu His

210 215 220

Ala Pro Phe Asp Thr Lys Val Glu Lys Thr His Phe Ser Gln Phe Ile

225 230 235 240

Asp Val Arg Asp Val Ala Lys Thr His Val Leu Gly Phe Gln Lys Asp

245 250 255

Glu Leu Ile Asn Gln Arg Leu Leu Leu Cys Asn Gly Ala Phe Ser Gln

260 265 270

Gln Asp Ile Val Asn Val Phe Asn Glu Asp Phe Pro Glu Leu Lys Gly

275 280 285

Gln Phe Pro Pro Glu Asp Lys Asp Thr Asp Leu Asn Lys Gly Val Thr

290 295 300

Gly Cys Lys Ile Asp Asn Glu Lys Thr Lys Lys Leu Leu Ala Phe Glu

305 310 315 320

Phe Thr Pro Phe His Lys Thr Ile His Asp Thr Val Tyr Gln Ile Leu

325 330 335

His Lys Glu Gly Arg Val

340

Claims (8)

1. An efficient preparation method of a heterocyclic drug intermediate is characterized in that an alcohol dehydrogenase mutant and glucose dehydrogenase are coupled to catalyze a heterocyclic substrate to generate the heterocyclic drug intermediate, wherein the alcohol dehydrogenase mutant is obtained by mutating tyrosine at 127 th position of an alcohol dehydrogenase wild type shown in SEQ ID NO.2 in an amino acid sequence to tryptophan;

the heterocyclic substrate is N-Boc-3-pyrrolidone,N-Boc-3-piperidone orN-Boc-4-piperidone.

2. The method of claim 1, wherein said heterocyclic pharmaceutical intermediate is (S) -N-Boc-3-hydroxypyrrolidine, (S) -N-Boc-3-hydroxypiperidine orN-Boc-4-hydroxypiperidine.

3. The method of claim 1, wherein during the coupling catalysis, glucose is also added, said glucose being in contact withNThe molar mass ratio of the Boc-3-piperidone is 1-2: 1.

4. The method according to claim 1, wherein the mass ratio of the alcohol dehydrogenase mutant to the glucose dehydrogenase is 1-2: 1.

5. The method of claim 1, wherein the alcohol dehydrogenase mutant and the glucose dehydrogenase are added in amounts ofN3% -5% of the mass of the-Boc-3-piperidone.

6. The method of claim 1, wherein said step of removing is performed by a laserNThe concentration of (E) -Boc-3-piperidone was 100 g.L^-1-600 g·L^-1。

7. The method of claim 1, wherein the nucleotide sequence of the alcohol dehydrogenase wild-type is shown in SEQ ID No. 1.

8. The method of claim 1, wherein the temperature of the coupling catalysis reaction is25^oC -35^oC, the pH value is 6.5-7.5.

Images