A method for synthesizing an r configuration axially chiral biaryl glycol mediated by alcohol dehydrogenase

Abstract

The application belongs to the field of bioengineering and biocatalysis technology, and particularly relates to an alcohol dehydrogenase mutant and application thereof. The application discloses an alcohol dehydrogenase mutant CcPAR-M1 (V158L), and an amino acid sequence of the mutant is shown as SEQ ID No. 3. The mutant can be used for catalyzing asymmetric reduction reaction of a biaryl aldehyde substrate through dynamic kinetic resolution to generate R an axially chiral biaryl dimethanol compound. By using the biocatalytic system disclosed in the application, the highest yield of the target product can reach 96%, and the enantiomeric excess value can reach 99% ee at most. The method provides a green, efficient and excellent stereoselective biocatalytic strategy for construction of the axially chiral biaryl dimethanol compound.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and biocatalysis, specifically relating to an alcohol dehydrogenase mutant and its application as a biocatalyst in the highly stereoselective synthesis of axially chiral biaryl compounds. Background Technology

[0002] Axially chiral biaryl alcohols are an important class of multifunctional synthetic intermediates, widely used in the construction of valuable axially chiral molecules such as advanced materials, pharmaceuticals, natural products, and organocatalysts. Despite their wide applications, existing catalytic asymmetric synthesis methods for these compounds are still not mature enough, especially in the field of biocatalysis, where efficient catalytic systems with excellent stereoselectivity are still lacking.

[0003] Dynamic kinetic resolution (DKR) offers an attractive approach for the efficient asymmetric synthesis of axially chiral compounds. However, achieving DKR for axially chiral substrates typically requires a catalytic system that can adapt to dynamic configurational changes in the substrate and efficiently and precisely stereoinduce the target configuration during the reaction. This places high demands on the catalyst's activity, selectivity, and substrate adaptability. Therefore, for axially chiral biaromatic alcohols, especially... R Developing a green, efficient, and stereoselective biocatalytic system for axially chiral biaryldiethanol compounds remains of significant theoretical and practical value.

[0004] It has been achieved in the early stages. S Based on the highly stereoselective biocatalytic synthesis of configuration-axis chiral biaryldiethanol compounds, this invention further provides a method for preparing... R A biocatalytic method for the determination of biaryl dimethyl compounds with axial configuration. This method utilizes a transient seven-membered bridged intermediate-mediated axially selective dynamic kinetic resolution (DKR) carbonyl reduction reaction, catalyzed by an engineered alcohol dehydrogenase, to achieve the conversion of biaryl aldehyde substrates into carbonyl groups. R The configuration-axis chiral biaryldiethanol compounds exhibit efficient and highly stereoselective conversion, and demonstrate good catalytic activity and excellent stereoselectivity. Summary of the Invention

[0005] Objective of the Invention: This invention addresses the limitations of existing biocatalytic systems in the stereoselective synthesis of axially chiral biaryldiethanol compounds, particularly in... R There are shortcomings in the selective construction of configuration products. This paper proposes a biocatalytic method to achieve this. R Highly stereoselective synthesis of axially chiral biaryldimethyl compounds.

[0006] The technical problem to be solved by this invention is to provide an alcohol dehydrogenase mutant with excellent catalytic performance and stereoselectivity, so as to realize the asymmetric reduction reaction of biaryl aldehyde substrates through dynamic kinetic resolution. R Highly efficient and selective biocatalytic conversion of axially chiral biaryldiethanol compounds.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: An alcohol dehydrogenase mutant CcPAR-M1, wherein the amino acid sequence of the alcohol dehydrogenase mutant CcPAR-M1 is obtained by mutating valine at position 158 of the wild-type alcohol dehydrogenase CcPAR to leucine.

[0008] The wild-type alcohol dehydrogenase CcPAR mentioned above is derived from... Corynespora cassiicola Philippines, whose amino acid sequence is shown in SEQ ID NO.2, and whose corresponding nucleotide sequence encoding wild-type alcohol dehydrogenase CcPAR is shown in SEQ ID NO.1.

[0009] Specifically, the alcohol dehydrogenase mutant CcPAR-M1 is obtained by mutating valine at position 158 of the wild-type alcohol dehydrogenase CcPAR to leucine, and its amino acid sequence is shown in SEQ ID NO.3.

[0010] The nucleotide sequence encoding the above-mentioned alcohol dehydrogenase mutant is shown in SEQ ID NO.4.

[0011] In some embodiments of the present invention, the alcohol dehydrogenase mutant is the alcohol dehydrogenase mutant CcPAR-M1.

[0012] Specifically, the amino acid sequence of the alcohol dehydrogenase mutant CcPAR-M1 is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.4.

[0013] Specifically, the alcohol dehydrogenase mutant CcPAR-M1 is derived from... Monilia parapsilosis Using the gene expression plasmid (strain CDC 317 / ATCC MYA-4646) as a template, a mutant library was constructed based on structure-guided protein engineering. Combined with screening methods, superior mutants with enhanced or reversed stereoselectivity were obtained. After screening and iteration at advantageous mutation sites, the alcohol dehydrogenase mutant CcPAR-M1 was finally obtained.

[0014] The alcohol dehydrogenase mutant CcPAR-M1 described in this invention can be used in whole cell, lysed crude enzyme solution, or pure enzyme form. Alternatively, the alcohol dehydrogenase mutant CcPAR-M1 enzyme involved in this invention can be made into an immobilized enzyme or an immobilized cell form using immobilization techniques known in the art.

[0015] Recombinant expression vectors containing the above-mentioned alcohol dehydrogenase mutant nucleotide sequence are also within the scope of protection of this invention.

[0016] Recombinant strains containing the above-mentioned recombinant expression vector are also within the scope of protection of this invention.

[0017] The application of the above-mentioned alcohol dehydrogenase mutant in the catalytic synthesis of axially chiral biaryldiethanol compounds is also within the scope of protection of this invention.

[0018] The catalytic synthesis described herein uses aldehydes as substrates; Specifically, the structure of the biaryl aldehyde is as follows:

[0019] Specifically, the catalytic synthesis described herein uses a reaction system comprising: an initial aldehyde concentration of 10 mM, an initial addition of 0.2 g / mL of crude alcohol dehydrogenase lysis solution, 2% dimethyl sulfoxide (DMSO) (based on total reaction volume), a 50 mM Tris-HCl buffer at pH 7.5, 3% isopropanol (based on total reaction volume), and NADP. + 0.16 mM; the reaction conditions were: temperature 30 ℃, rotation speed 1000 rpm, reaction time 3 h.

[0020] After the catalytic reaction was complete, the product was extracted three times using ethyl acetate as the extractant. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and then removed under vacuum. The yield and stereoselectivity of the product were analyzed by high-performance liquid chromatography (HPLC) with a chiral column. The results showed that the corresponding product was obtained by catalysis with the alcohol dehydrogenase mutant CcPAR-M1. R The axially chiral arylbenzyl alcohol II configuration can be obtained in yields up to 96% and with optical purity up to 99% ee. Beneficial effects:

[0021] The alcohol dehydrogenase mutant provided by this invention exhibits good catalytic activity and excellent stereoselectivity, enabling the conversion of biaryl aldehyde substrates to alcohols. R Efficient conversion of axially chiral biaryldiethanol compounds. The enantioselective biocatalytic synthesis method described in this invention has advantages such as high reaction efficiency, mild reaction conditions, excellent stereoselectivity, and readily available and easily prepared catalysts. This invention builds upon existing... SBased on the research on configuration-selective synthesis, further progress has been made. R The controllable construction of configurations provides a new technical means for the directed biocatalytic synthesis of different configurations of axially chiral biaryl compounds. Attached Figure Description

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer. Figure 1 : A technical route for synthesizing axially chiral aryldimethyl compounds II using the alcohol dehydrogenase mutant CcPAR-M1 as a catalyst; Figure 2 : The 1H NMR spectrum of the axially chiral aryldimethylamine compound II prepared by the reaction; Figure 3 : Carbon NMR spectrum of axially chiral aryldimethylamine compound II prepared by reaction; Figure 4 High-performance liquid chromatography (HPLC) chromatogram of the optical purity of axially chiral aryldimethylamine compound II prepared by the reaction. Detailed Implementation

[0023] The following detailed description illustrates the specific implementation method: The three-letter or single-letter expressions for amino acids used in this application are based on the amino acid codes specified by IUPAC (Eur. J. Biochem., 138:9-37, 1984). Example 1: Obtaining the CcPAR-M1 mutant of alcohol dehydrogenase

[0024] CcPAR-M1: PCR was performed using pET-28a-CpAR2 plasmid (the pET-28a vector contains the CcPAR WT gene sequence, as shown in SEQ ID NO.1) as template DNA to obtain the corresponding plasmid containing the alcohol dehydrogenase mutant gene fragment. The forward primer was (GCAAgtccagttgat). GCc gaagttagtg / gtcaatcc G act CTG gtatttggccctcaag / gagaagctaaat CTg tcagcagagatcaTC) and the default reverse primer (GCTAGTTATTGCTCAGCGG).

[0025] The PCR reaction system (final volume 50 μL) consisted of: ddH2O (20 μL), 2×Phanta Max Master Mix (Dye Plus) (25 μL), forward primers (2 μL, 10 μM each), default reverse primers (2 μL, 10 μM each), and template plasmid (1.0 μL, 50 ng / μL each). The PCR program was as follows: Step 1, 95℃ / 3 min; Step 2, (95℃ / 15 s; 62℃ / 0.5 min; 72℃ / 7 min), 30 cycles; Step 3, 72℃ / 5 min.

[0026] After PCR amplification, the resulting system was digested. Specifically, 50 μL of the PCR reaction mixture was mixed with 1 μL of DpnI and 4.5 μL of buffer, and incubated at 37°C for 1 h. Next, the DpnI digestion product was purified using an Omega PCR spin column. 8 μL of the digested PCR product was then electroporated into 80 μL of *E. coli* (BL21-DE3) competent cells. After incubation at 37°C with shaking for 1 h, the cells were plated onto LB agar containing kanamycin (34 μg / mL) and incubated at 37°C for 12 h. Single colonies were picked from the plates, transferred to LB agar, and incubated at 37°C for 12 h before plasmid extraction. Gene sequencing revealed the corresponding alcohol dehydrogenase mutant strain CcPAR-M1. Subsequently, the CcPAR-M1 strain was cultured and its protein expression was induced to obtain a crude enzyme solution, which was then used as a biocatalyst. Example 2: Preparation of crude enzyme solution of alcohol dehydrogenase mutant CcPAR-M1

[0027] First, 50 μL of the stored alcohol dehydrogenase mutant CcPAR-M1 strain was inoculated into 5 mL LB medium (containing 34 μg / mL kanamycin) and cultured at 37℃ and 220 rpm for 12 h with shaking. The culture was then inoculated into TB medium (250 mL TB containing 34 μg / mL kanamycin, 1 L shake flask) at a 1% volume concentration and cultured at 37℃ and 220 rpm for approximately 4 h with shaking, until the bacterial OD(600) reached 0.6–0.8. After cooling the bacterial culture at 4℃ for 1 h, 0.3 mM isopropyl alcohol was added. β-Induced culture with thiogalactoside (IPTG) at 16°C and 220 rpm for 16 h. Then, the induced bacterial culture was centrifuged at 4°C and 8500 rpm for 5 min, and the supernatant was removed to obtain wet cells. The wet cells were resuspended in buffer (1 g of wet cells in 5 mL of different buffers) and frozen at -80°C. The cells were subjected to three freeze-thaw cycles, and then the target protein was released by sonication. The cell lysate was centrifuged at 4°C and 12,000 rpm for 30 min, and the supernatant was stored at -20°C for later use. Example 3: Selective catalytic synthesis of axially chiral biaryldiethanol compound II via the alcohol dehydrogenase mutant CcPAR-M1

[0028] The technical route for synthesizing axially chiral biarylbenzyl alcohol compound II using the alcohol dehydrogenase mutant CcPAR-M1 as a catalyst is as follows: Figure 1 As shown.

[0029] Add 0.475 mL of Tris-HCl buffer (50 mM, pH 7.5) containing the alcohol dehydrogenase mutant CcPAR-M1 to each 5 mL centrifuge tube, along with 5 μmol of substrate, 10 μL of dimethyl sulfoxide, 15 μL of isopropanol, and NADP. + (0.16 mM). The reaction mixture was shaken at 30 °C and 1000 rpm for 3 h, then extracted with ethyl acetate (3 × 1 mL), dried over anhydrous sodium sulfate, concentrated under vacuum, and the yield and ee value were determined by chiral high-performance liquid chromatography. Specific analytical conditions were as follows: CHIRACEL® IC column, column temperature 25 °C, mobile phase n-hexane:isopropanol = 85:15, flow rate 1 mL / min, retention time 15 min.

[0030] The proton NMR spectrum of product II 1 H NMR (400MHz, CDCl3) such as Figure 2 As shown, 13 C NMR (100MHz, CDCl3) such as Figure 3 As shown in Table 1, the retention time of S configuration II was 7.6 min, and the retention time of R configuration II was 8.8 min. The stereoselective transformation results of wild-type alcohol dehydrogenase and mutant-catalyzed compound I to axially chiral biarylbenzyl alcohol II are shown in Table 1 below. The wild-type CcPAR-catalyzed reaction yielded (… R The product was obtained with a configuration of ) and a yield of 65% and an optical purity of 90%. The alcohol dehydrogenase mutant CcPAR-M1 further improved the reaction performance, catalyzing compound I to obtain ( R The product configuration was achieved with a yield of 90% and an optical purity of 99%. Specific liquid phase results are as follows: Figure 4As shown, the results indicate that the ee value of the isolated compound II is 99% ( R ). Table 1 Comparison of responses between wild type and mutant alcohol dehydrogenase name Yield % Optical purity %ee Configuration of chiral compound II Wild-type CcPAR 65 90 mutant CcPAR-M1 96 99

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant CcPAR-M1 is obtained by mutating valine at position 158 of the wild-type alcohol dehydrogenase CcPAR to leucine.

2. The alcohol dehydrogenase mutant according to claim 1, characterized in that, The wild-type alcohol dehydrogenase CcPAR is derived from Corynespora cassiicola Philippines, and its amino acid sequence is shown in SEQ ID NO.

2. The corresponding nucleotide sequence encoding the wild-type alcohol dehydrogenase CcPAR is shown in SEQ ID NO.

1.

3. The amino acid sequence of the alcohol dehydrogenase mutant CcPAR-M1 according to claim 1 is obtained by mutating valine at position 158 of the wild-type alcohol dehydrogenase CcPAR to leucine, and its amino acid sequence is shown in SEQ ID NO.

3.

4. The alcohol dehydrogenase mutant of claim 3, characterized in that, The nucleotide sequence corresponding to the alcohol dehydrogenase mutant is shown in SEQ ID NO.

4.

5. A recombinant expression vector, characterized in that, The nucleotide sequence containing the alcohol dehydrogenase mutant of claim 4.

6. A recombinant bacterial strain, characterized in that, The nucleotide sequence contains the recombinant expression vector of claim 5 or the alcohol dehydrogenase mutant of claim 5.

7. The application of the alcohol dehydrogenase mutant according to claim 1 in the catalytic synthesis of axially chiral biaryldiethanol compounds.

8. The application according to claim 7, characterized in that, The catalytic synthesis uses biaryl aldehydes as substrates; wherein, the biaryl aldehyde has the following structural formula: The axially chiral biaryldiethanol is R The configuration of the biaryl compound is as follows:

9. The application according to claim 7, characterized in that, The catalytic synthesis described herein uses biaryl aldehydes as substrates, dimethyl sulfoxide as a co-solvent, and isopropanol and NADP as catalysts. + As a reduction cycle system, an alcohol dehydrogenase mutant was used to catalyze the dynamic kinetic resolution of carbonyl groups to synthesize axially chiral biaryldiethanol compounds.

10. The application according to claim 9, characterized in that, The catalytic synthesis described herein uses the following reaction system: an initial aldehyde concentration of 10 mM, an initial addition of crude alcohol dehydrogenase solution of 0.2 g / mL, a dimethyl sulfoxide volume fraction of 2%, an isopropanol volume fraction of 3%, a pH of 7.5, a 50 mM Tris-HCl buffer, and NADP. + 0.16 mM; the reaction conditions were: temperature 30 ℃, rotation speed 1000 rpm, reaction time 3 h.

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