Application of alcohol dehydrogenase mutant in synthesis of axially chiral biaryl dimethyl carbinol

Abstract

The invention relates to the technical field of biological engineering and biological catalysis, in particular to an alcohol dehydrogenase mutant and application thereof. The invention discloses an alcohol dehydrogenase mutant CpAR2-M5 (F85A / T125S / F192L / E209A / I213F). The corresponding amino acid sequence of the alcohol dehydrogenase mutant CpAR2-M5 is shown as SEQ ID No. 3. The invention also discloses a preparation method of the alcohol dehydrogenase mutant CpAR2-M5. The S-configuration axial chiral biaryl dimethyl carbinol compound can be applied to dynamic kinetic resolution asymmetric reduction of the biaryl dimethyl carbinol compound so as to prepare the S-configuration axial chiral biaryl dimethyl carbinol compound, the highest yield can reach 95%, the highest stereoselectivity can reach 99% ee, and a green, efficient and high-stereoselectivity synthesis strategy is provided for subsequent synthesis of the 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 biaryldimethylethanols are an important class of chiral structural units, widely found in drug molecules, chiral catalysts, and functional materials, and have significant application value in fields such as medicinal chemistry, asymmetric catalysis, and materials science. Therefore, achieving the efficient and highly stereoselective synthesis of axially chiral biaryldimethylethanol compounds has important scientific significance and application value.

[0003] In recent years, the dynamic kinetic resolution (DKR) strategy has attracted widespread attention for its ability to rapidly racemize bridging intermediates with unstable configurations, enabling highly enantioselective synthesis of axially chiral biaryl compounds under chiral catalysts. However, current research mainly focuses on five- or six-membered bridging intermediate systems. Achieving this goal in transient bridging systems with larger rings, including seven-membered bridging intermediate systems, remains extremely challenging. This is because the racemic half-life of seven-membered structures is relatively long at relevant reaction timescales, typically ranging from minutes to hours, thus imposing extremely stringent requirements on the stereocontrol of chiral catalysis. Therefore, there is an urgent need for a structurally simple, widely applicable, and universally applicable intermediate, and for the establishment of an efficient catalytic system based on this intermediate, to achieve highly stereoselective synthesis of axially chiral biaryl compounds.

[0004] This invention reports the first biocatalysis-based method that achieves axially selective dynamic kinetic resolution (DKR) carbonyl reduction via a transient seven-membered bridged intermediate to construct a series of axially chiral biaryldiethanols. In this method, an engineered alcohol dehydrogenase catalyzes the axially selective reduction of biaryl aldehydes, achieving the stereoselective synthesis of axially chiral biaryldiethanol compounds, and exhibiting excellent catalytic activity and high stereoselectivity. Summary of the Invention

[0005] Objective of this invention: The technical problem to be solved by this invention is to fill the gap in the synthesis of axially chiral biaryldiethanol using existing catalytic systems, and to provide a specific biocatalytic method for the stereoselective synthesis of this compound. Another technical problem to be solved by this invention is to provide an alcohol dehydrogenase mutant to achieve highly stereoselective biocatalytic conversion of biaryl aldehyde substrates to chiral biaryldiethanol compounds with specific configurations.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: An alcohol dehydrogenase mutant CpAR2-M5, wherein the amino acid sequence of the alcohol dehydrogenase mutant CpAR2-M5 is obtained by mutating phenylalanine at position 85 to alanine, threonine at position 125 to serine, phenylalanine at position 192 to leucine, glutamic acid at position 209 to alanine, and isoleucine at position 213 to phenylalanine.

[0007] The wild-type alcohol dehydrogenase CpAR2 is derived from... Monilia parapsilosis (strain CDC317 / ATCC MYA-4646), whose amino acid sequence is shown in SEQ ID NO.2, and whose corresponding nucleotide sequence encoding wild-type alcohol dehydrogenase CpAR2 is shown in SEQ ID NO.1.

[0008] Specifically, the alcohol dehydrogenase mutant CpAR2-M5 is obtained by mutating phenylalanine at position 85 to alanine, threonine at position 125 to serine, phenylalanine at position 192 to leucine, glutamic acid at position 209 to alanine, and isoleucine at position 213 to phenylalanine in the wild-type alcohol dehydrogenase CpAR2. Its amino acid sequence is shown in SEQ ID NO. 3.

[0009] The nucleotide sequence encoding the above-mentioned alcohol dehydrogenase mutant is shown in SEQ ID NO.4.

[0010] In some embodiments of the present invention, the alcohol dehydrogenase mutant is the alcohol dehydrogenase mutant CpAR2-M5.

[0011] Specifically, the amino acid sequence of the alcohol dehydrogenase mutant CpAR2-M5 is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.4.

[0012] Specifically, the alcohol dehydrogenase mutant CpAR2-M5 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 CpAR2-M5 was finally obtained.

[0013] The alcohol dehydrogenase mutant CpAR2-M5 described in this invention can be used in whole cell, crude enzyme lysate, or pure enzyme form. Alternatively, the alcohol dehydrogenase mutant CpAR2-M5 enzyme involved in this invention can be made into an immobilized enzyme or an immobilized cell form using immobilization techniques known in the art.

[0014] Recombinant expression vectors containing the above-mentioned alcohol dehydrogenase mutant nucleotide sequence are also within the scope of protection of this invention.

[0015] Recombinant strains containing the above-mentioned recombinant expression vector are also within the scope of protection of this invention.

[0016] 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.

[0017] The catalytic synthesis described herein uses aldehydes as substrates; Specifically, the structure of the biaryl aldehyde is as follows:

[0018] 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 7.0, 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.

[0019] 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 S-configuration axially chiral arylbenzyl alcohol II could be obtained by catalysis with the alcohol dehydrogenase mutant CpAR2-M5 in a yield of up to 95% and an optical purity of up to 99% ee. Beneficial effects

[0020] This invention provides, for the first time, a biocatalytic-based axially selective dynamic kinetic resolution method for carbonyl reduction, enabling the efficient construction of a series of axially chiral biaryldiethanol compounds via transient seven-membered bridging intermediates, filling a gap in existing biocatalytic synthesis methods. Compared with existing technologies, the engineered alcohol dehydrogenase provided in this invention can catalyze the selective reduction of biaryl aldehyde substrates, thereby achieving highly stereoselective synthesis of specific configurations of the target product, while exhibiting both good catalytic activity and excellent stereoselectivity. The method of this invention also has advantages such as mild reaction conditions, environmental friendliness, high substrate utilization, and broad application prospects, providing a new technical means for the green and efficient synthesis of axially chiral biaryldiethanol compounds. Attached Figure Description

[0021] 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.

[0022] Figure 1 A technical route for synthesizing axially chiral aryldimethyl compounds II using the alcohol dehydrogenase mutant CpAR2-M5 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 mutant CpAR2-M5 of alcohol dehydrogenase

[0024] CpAR2-M5: PCR was performed using pET-28a-CpAR2 plasmid (the pET-28a vector contains the CpAR2 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 primer (2 μL), default reverse primer (2 μL), 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 CpAR2-M5. Subsequently, the CpAR2-M5 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 CpAR2-M5

[0027] First, 50 μL of the stored alcohol dehydrogenase mutant CpAR2-M5 strain was inoculated into 5 mL LB medium (containing 34 μg / mL kanamycin) and cultured at 37℃ with shaking at 220 rpm for 12 h. The culture was then inoculated into TB medium (250 mL TB containing 34 μg / mL kanamycin, 1 L shake flask) at a volume concentration of 1% and cultured at 37℃ with shaking at 220 rpm for approximately 4 h, 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 CpAR2-M5

[0028] The technical route for synthesizing axially chiral biarylbenzyl alcohol compound II using the alcohol dehydrogenase mutant CpAR2-M5 as a catalyst is as follows: Figure 1 As shown.

[0029] Add 0.475 mL of Tris-HCl buffer (50 mM, pH 7.0) containing the alcohol dehydrogenase mutant CpAR2-M5 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 CpAR2 catalyzed reaction yielded (… S The product was obtained with a configuration of 50% yield and 15% optical purity; the alcohol dehydrogenase mutant CpAR2-M5 further improved the reaction performance, catalyzing compound I to obtain ( S 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% ( S ).

[0031] Table 1 Comparison of responses between wild type and mutant alcohol dehydrogenase name Yield % Optical purity %ee Configuration of chiral compound II Wild-type CpAR2 50 15 mutant CpAR2-M5 95 99

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant CpAR2-M5 is obtained by mutating phenylalanine at position 85 to alanine, threonine at position 125 to serine, phenylalanine at position 192 to leucine, glutamic acid at position 209 to alanine, and isoleucine at position 213 to phenylalanine in the wild-type alcohol dehydrogenase CpAR2.

2. The alcohol dehydrogenase mutant according to claim 1, characterized in that, The wild-type alcohol dehydrogenase CpAR2 is derived from Monilia parapsilosis (strain CDC 317 / ATCC MYA-4646), whose amino acid sequence is shown in SEQ ID NO.2, and whose corresponding nucleotide sequence encoding wild-type alcohol dehydrogenase CpAR2 is shown in SEQ ID NO.

1.

3. The alcohol dehydrogenase mutant CpAR2-M5 according to claim 1, characterized in that, The amino acid sequence of the alcohol dehydrogenase mutant is obtained by mutating phenylalanine at position 85 to alanine, threonine at position 125 to serine, phenylalanine at position 192 to leucine, glutamic acid at position 209 to alanine, and isoleucine at position 213 to phenylalanine in the wild-type alcohol dehydrogenase CpAR2. 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 S 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 7.0, 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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