Application of halogenated alkane dehalogenase in selective preparation of S-configuration aryl ethanol

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to application of halogenated alkane dehalogenase in selective preparation of S-configuration aryl ethanol, the halogenated alkane dehalogenase is DbjA, and the amino acid sequence of the halogenated alkane dehalogenase is shown as SEQ ID NO.1. According to the invention, the halogenated alkane dehalogenase DbjA has stereoselectivity for the reaction of catalyzing phenyl chloride to prepare S-configuration aryl ethanol, the enantiomer selectivity for the catalytic reaction of different substrates can reach 500 or more, the ee value of the generated substrate can reach 99%, and meanwhile, the yield of 40% or more can be maintained; and a new solution is provided for the problems of complex operation and large workload in the preparation process of S-configuration aryl ethanol.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme engineering, and particularly relates to application of haloalkane dehalogenase in selective preparation of S-configuration aryl ethanol. BACKGROUND

[0002] Many optically active alcohols such as 1-phenyl ethanol are important intermediates for fine chemicals and drug synthesis, and have important applications in the fields of medicine, pesticide and chemical materials. For example, optically pure (S)-1-phenyl ethanol and its derivatives are synthetic precursors of many drugs, such as the immunopotentiator levamisole, the drug (S)-isopropyl adrenaline for treating asthma, and the antidepressant drug desipramine.

[0003] At present, a single enantiomer of optically active alcohol is mainly prepared by asymmetric synthesis and resolution, wherein the resolution is divided into enzyme catalytic resolution and chemical resolution, and enzyme catalytic kinetic resolution has the advantages of easy scale-up, environmental friendliness, product meeting food grade requirements of greenness and safety, etc., and therefore is widely valued. However, enzyme catalysis generally needs to be mutagenized, and there are technical problems of large amount of operation, complicated operation and high cost. SUMMARY

[0004] In view of the defects of insufficient product purity and complicated operation in the process of preparing optically active alcohol in the prior art, the application provides application of haloalkane dehalogenase in selective preparation of S-configuration aryl ethanol, and the specific technical solutions are as follows:

[0005] In a first aspect, the application provides application of haloalkane dehalogenase in selective preparation of S-configuration aryl ethanol, characterized in that the amino acid sequence of the haloalkane dehalogenase is shown in SEQ ID NO. 1; and the S-configuration aryl ethanol is any one of formula (I) to formula (II).

[0006]

[0007] wherein R1 is one of H, Cl and CH3; and R2 is one of H, F, Cl, Br and CH3.

[0008] Further, the formula (I) is one of the following:

[0009] (1) R1 is H, and R2 is H; (2) R1 is H, and R2 is F; (3) R1 is H, and R2 is Cl;

[0010] (4) R1 is H, and R2 is Br; (5) R1 is H, and R2 is CH3; (6) R1 is Cl, and R2 is H;

[0011] (7) R1 is CH3, and R2 is Cl.

[0012] In a second aspect, the present application provides a method for preparing S-configuration aryl ethanol from phenyl chloro compound by using enzyme catalysis, characterized in that the method comprises: using phenyl chloro compound as a substrate, using haloalkane dehalogenase as a catalyst, and forming an enzyme catalysis reaction system to obtain S-configuration aryl ethanol.

[0013] The amino acid sequence of the haloalkane dehalogenase is shown in SEQ ID NO. 1.

[0014] The S-configuration aryl ethanol is any one of formula (I) to formula (II).

[0015]

[0016] wherein R1 is one of H, Cl and CH3; and R2 is one of H, F, Cl, Br and CH3.

[0017] The present application uses haloalkane dehalogenase DbjA to catalyze different phenyl chloro compounds, has high enantioselectivity, can obtain S-configuration product with high ee, and can maintain a yield of more than 40%. This method does not need to mutate the enzyme, is simple to operate, and has good application prospect.

[0018] Further, the enzyme catalysis reaction for preparing S-configuration aryl ethanol from phenyl chloro compound is one of the following:

[0019] (1) catalyzing 1-chloro-1-phenylethane to synthesize (S)-1-phenylethanol;

[0020] (2) catalyzing 1-chloro-4-(1-chloroethyl)benzene to synthesize (S)-1-(4-chlorophenyl)-1-ethanol;

[0021] (3) catalyzing 2-(1-chloroethyl)naphthalene to synthesize (S)-2-naphthalene ethanol;

[0022] (4) catalyzing 1-(1-chloro-ethyl)-4-fluoro-benzene to synthesize (S)-1-(4-fluorophenyl)-1-ethanol;

[0023] (5) catalyzing 1-bromo-4-(1-chloroethyl)benzene to synthesize (S)-1-(4-bromophenyl)-1-ethanol;

[0024] (6) catalyzing 1-(1-chloroethyl)-4-methylbenzene to synthesize (S)-1-(4-methylphenyl)-1-ethanol;

[0025] (7) catalyzing 1-chloro-3-(1-chloroethyl)benzene to synthesize (S)-1-(3-chlorophenyl)-1-ethanol;

[0026] (8) catalyzing 1-chloro-4-(1-chloroethyl)-2-methylbenzene to synthesize (S)-1-(4-chloro-3-methylphenyl)ethanol.

[0027] Further, in the reaction system, the substrate and the catalyst are dispersed in water or an organic solvent.

[0028] Further, the organic solvent is methyl tert-butyl ether.

[0029] Further, the organic solvent further comprises 1-3 mM of water; wherein the 1-3 mM refers to the concentration of water dispersed in the methyl tert-butyl ether solvent.

[0030] Further, the water is ultrapure water.

[0031] In the present application, it is found that, compared with using water as the dispersion system, using methyl tert-butyl ether solvent with trace amount of ultrapure water as the dispersion system can greatly increase the enantioselectivity of the enzyme catalytic resolution reaction, and for the reaction of catalyzing 1-chloro-1-phenylethane to synthesize (S)-1-phenylethanol, the ee value can be increased from 36% to more than 99%.

[0032] Further, after the reaction is completed, the reaction liquid is subjected to post-treatment to obtain the S-configuration arylethanol product.

[0033] The post-treatment comprises adding water to the reaction liquid, and performing extraction, drying, dissolution, and filtration.

[0034] Further, the post-treatment specifically comprises:

[0035] Water is added to the reaction liquid, ethyl acetate is used for extraction, the extracted liquid is spin-dried, then isopropanol is used for dissolution, and then 0.22 μm organic membrane is used for filtration.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The present application finds that the halogenated alkane dehalogenase DbjA has stereoselectivity for catalyzing the reaction of preparing S-configuration arylethanol from phenyl chlorides, and the enantiomeric selectivity of the catalytic reaction of different substrates can reach more than 500, the ee value of the generated substrate can reach 99%, and meanwhile, the yield can be maintained at more than 40%, thereby providing a new solution to the problems of complex operation and large operation amount existing in the process of preparing S-configuration arylethanol. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The gas chromatogram of the racemic substrate 1-chloro-1-phenylethane.

[0039] Figure 2 The gas chromatogram of the halogenated alkane dehalogenase DbjA catalytic resolution of 1-chloro-1-phenylethane.

[0040] Figure 3 The image shows the gas chromatogram of the racemic product 1-phenylethanol.

[0041] Figure 4 The gas chromatogram of the reaction of 1-chloro-1-phenylethane to (S)-1-phenylethanol catalyzed by the haloalkane dehalogenase DbjA.

[0042] Figure 5 The gas chromatogram of the racemic product 1-(4-chlorophenyl)-1-ethanol is shown.

[0043] Figure 6 Gas chromatogram of the reaction of 1-chloro-4-(1-chloroethyl)benzene to (S)-1-(4-chlorophenyl)-1-ethanol catalyzed by the haloalkane dehalogenase DbjA.

[0044] Figure 7 This is the high-performance liquid chromatogram of the racemic product 2-naphthylethanol.

[0045] Figure 8 High-performance liquid chromatography (HPLC) chromatogram of the reaction of 2-(1-chloroethyl)naphthalene to (S)-2-naphthylethanol catalyzed by the haloalkane dehalogenase DbjA.

[0046] Figure 9 10 represents the substrate 1-chloro-1-phenylethane. 1 H and 13 C-spectrum.

[0047] Figure 11 12 are the product 1-phenylethanol, respectively. 1 H and 13 C-spectrum.

[0048] Figure 13 14, respectively, are the substrates 1-chloro-4-(1-chloroethyl)benzene. 1 H and 13 C-spectrum.

[0049] Figure 15 16 are the product 1-(4-chlorophenyl)-1-ethanol, respectively. 1 H and 13 C-spectrum.

[0050] Figure 17 18 are the substrates 2-(1-chloroethyl)naphthalene, respectively. 1 H and 13 C-spectrum.

[0051] Figure 19 20 represents the product 2-naphthylethanol. 1 H and 13 C-spectrum. Detailed Implementation

[0052] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0053] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0054] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0055] The chemicals used in the following examples:

[0056] (1) 1-Chloro-1-phenylethane, CAS No.: 672-65-1;

[0057] (2) 1-Chloro-4-(1-Chloroethyl)benzene, CAS No.: 20001-65-4;

[0058] (3) 2-(1-chloroethyl)naphthalene, CAS No.: 58464-06-5;

[0059] (4) 1-(1-chloro-ethyl)-4-fluoro-benzene, CAS No.: 456-16-6;

[0060] (5) 1-Bromo-4-(1-Chloroethyl)benzene, CAS No.: 20488-10-2;

[0061] (6) 1-(1-Chloroethyl)-4-toluene, CAS No.: 2362-36-9;

[0062] (7) 1-Chloro-3-(1-Chloroethyl)benzene, CAS No.: 34887-78-0;

[0063] (8) 1-Chloro-1-phenylpropane, CAS No.: 934-11-2.

[0064] The culture medium used in the following examples is:

[0065] The LB liquid medium comprises: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L NaCl; the LB solid medium comprises: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, and 15.0 g / L agar. Both the LB liquid and solid media are autoclaved at 121°C for 20 min.

[0066] In the following examples, the amino acid sequence of the haloalkane dehalogenase DbjA is shown in SEQ ID NO.1.

[0067] SEQ ID NO.1:

[0068] MSKPIEIEIRRAPVLGSSMAYRETGAQDAPVVLFLHGNPTSSHIWRNILPLVSPVAHCIAPDLIGFGQSGKPDIAYRFFDHVRYLDAFIEQRGVTSAYLVAQDWGTALAFHLAARRPDFVRGLAFMEFIRPMPTWQDFHHTEVAEEQDHAEAARAVFR KFRTPGEGEAMILEANAFVERVLPGGIVRKLGDEEMAPYRTPFPTPESRRPVLAFPRELPIAGEPADVYEALQSAHAALAASSYPKLLFTGEPGALVSPEFAERFAASLTRCALIRLGAGLHYLQEDHADAIGRSVAGWIAGIEAVRPQLAAHHHHHH.

[0069] Example 1: Construction of genetically engineered bacteria expressing haloalkane dehalogenase DbjA

[0070] Using *E. coli* as a host, the specific steps for expressing the target strain of the corresponding plasmid are as follows:

[0071] The obtained DbjA gene was synthesized by Jiutian Gene Technology (Tianjin) Co., Ltd. after codon optimization. The target gene was inserted into the Nde I and BamH I sites of the E. coli expression vector pET-11b to obtain the pET11b-DbjA ligation product.

[0072] The plasmid pET11b-DbjA was transformed into the recipient strain E. coli BL21(DE3)pLYsS and plated on LB agar plates containing ampicillin (100 mg / mL). The plates were then incubated at 37°C for 12 h to allow single colonies to grow. Single colonies were randomly selected and inoculated into LB liquid medium and incubated for 12 h to obtain the target strain.

[0073] The target strain was inoculated into 5 mL of LB medium containing ampicillin (100 mg / mL) and initially cultured at 37°C and 200 rpm for 6 h. The entire inoculum from the initial culture was then inoculated into 200 mL of LB medium containing ampicillin (100 mg / mL) and cultured at 37°C and 200 rpm until the optical density (OD600) reached 0.6-0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) inducer was then added to a final concentration of 0.2 mM and induced for 12 h at 20°C and 180 rpm. Finally, the cells were collected by centrifugation at 8000 × g for 10 min, resuspended and washed with PBS buffer (pH 8.0), centrifuged at 10000 rpm for 10 min, and the wet cells were collected and stored at -20°C for later use.

[0074] The bacterial cells were suspended in a PBS buffer system at pH 8.0. The whole cells were then disrupted by sonication and centrifuged to obtain a crude enzyme solution. The crude enzyme solution was then freeze-dried overnight to obtain dehydrated crude enzyme powder, which was stored for later use.

[0075] Example 2: Dehalogenase DbjA catalyzes the synthesis of (S)-1-phenylethanol from 1-chloro-1-phenylethane.

[0076] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-phenylethanol from 1-chloro-1-phenylethane was catalyzed, and the specific steps are as follows:

[0077] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0078] (2) After adding 0.005 mM 1-chloro-1-phenylethane to the resolution reaction system, the reaction was shaken for 12 h at 30 °C and 800 r / min. After 12 h, water was added and the mixture was extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0079] The reaction is shown in the following equation:

[0080]

[0081] The substrate 1-chloro-1-phenylethane was analyzed by gas chromatography, specifically as follows:

[0082] The SHIMADZU GC-2014C system was used, with a column type of CP7502-0.25mm×0.25μm×25m; the chromatographic conditions were: column temperature 40℃.

[0083] The chromatographic parameters were: column temperature 40℃, heating rate 1℃ / min, vaporization chamber temperature 180℃, and detector temperature 200℃.

[0084] in, Figure 1 The gas chromatogram of the racemic substrate 1-chloro-1-phenylethane is shown.

[0085] according to Figure 1 It can be seen that the retention time of (R)-1-chloro-1-phenylethane in the standard sample 1-chloro-1-phenylethane is about 18.792 min, and the retention time of (S)-1-chloro-1-phenylethane is about 19.261 min.

[0086] The product 1-phenylethanol was analyzed by gas chromatography, specifically as follows:

[0087] The SHIMADZU GC-2014C system was used, with a column type of BGB-174-0.25mm×0.25μm×30m; the chromatographic conditions were: column temperature 70℃.

[0088] The chromatographic parameters were: column temperature 70℃, heating rate 1℃ / min, vaporization chamber temperature 180℃, and detector temperature 200℃. Figure 3 The gas chromatogram of the racemic product 1-phenylethanol is shown.

[0089] according to Figure 3 It can be seen that the retention time of (R)-1-phenylethanol in the standard 1-phenylethanol is about 23.725 min, and the retention time of (S)-1-phenylethanol is about 24.350 min.

[0090] The substrate 1-chloro-1-phenylethane was resolved using a haloalkane dehalogenase catalytic process, and the results are as follows: Figure 2 As shown in Figure 4. Among them, Figure 2 The gas chromatograms of the substrate resolved by the dehalogenase of haloalkanes are shown. Figure 4 The gas chromatogram of the product 1-phenylethanol is shown.

[0091] according to Figure 2 It can be seen that (R)-1-chloro-1-phenylethane elutes at 18.399 min, and (S)-1-chloro-1-phenylethane elutes at 19.174 min.

[0092] according to Figure 4 It can be seen that (S)-1-phenylethanol eluted at 24.331 min and (R)-1-phenylethanol eluted at 23.831 min.

[0093] The substrate 1-chloro-1-phenylethane 1 H and 13 C spectrum as shownFigure 9 , Figure 10 As shown. Product 1-phenylethanol 1 H and 13 C spectrum as shown Figure 11 , Figure 12 .

[0094] The results showed that the haloalkane dehalogenase DbjA preferentially hydrolyzed (S)-1-chloro-1-phenylethane, and the enantioselectivity of the catalytically generated (S)-1-phenylethanol reached 536, the ee value was greater than 99%, and the yield was 46%.

[0095] Example 3: Dehalogenase DbjA catalyzes the synthesis of (S)-1-(4-chlorophenyl)-1-ethanol from 1-chloro-4-(1-chloroethyl)benzene.

[0096] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(4-chlorophenyl)-1-ethanol from 1-chloro-4-(1-chloroethyl)benzene was catalyzed, and the specific steps are as follows:

[0097] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0098] (2) After adding 0.005 mM 1-chloro-4-(1-chloroethyl)benzene to the resolution reaction system, the reaction was shaken for 12 h at 30 °C and 800 r / min. After 12 h, water was added and the mixture was extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0099] The reaction is shown in the following equation:

[0100]

[0101] The product 1-(4-chlorophenyl)-1-ethanol was analyzed by gas chromatography, specifically as follows:

[0102] The SHIMADZU GC-2014C system was used, with a column type of BGB-174-0.25mm×0.25μm×30m; the chromatographic conditions were: column temperature 80℃.

[0103] The chromatographic parameters were: column temperature 80℃, heating rate 1℃ / min, vaporization chamber temperature 180℃, and detector temperature 200℃. Figure 5 The gas chromatogram of the racemic product 1-(4-chlorophenyl)-1-ethanol is shown.

[0104] according to Figure 5It can be seen that the retention time of (R)-1-(4-chlorophenyl)-1-ethanol in the standard sample 1-(4-chlorophenyl)-1-ethanol is about 41.025 min, and the retention time of (S)-1-(4-chlorophenyl)-1-ethanol is about 41.092 min.

[0105] Meanwhile, in the embodiments of this application, the substrate 1-chloro-4-(1-chloroethyl)benzene is catalytically resolved using a haloalkane dehalogenase, and the results are as follows: Figure 6 As shown.

[0106] The substrate 1-chloro-4-(1-chloroethyl)benzene 1 H and 13 C spectrum as shown Figure 13 , 14 As shown. The product 1-(4-chlorophenyl)-1-ethanol 1 H and 13 C spectrum as shown Figure 15 , 16 As shown.

[0107] according to Figure 6 It can be seen that (S)-1-(4-chlorophenyl)-1-ethanol eluted at 41.056 min, and (R)-1-(4-chlorophenyl)-1-ethanol eluted at 41.026 min, with a yield of 44%.

[0108] The results showed that the haloalkane dehalogenase DbjA preferentially hydrolyzed (S)-1-chloro-4-(1-chloroethyl)benzene, and the enantioselectivity of the catalytically generated (S)-1-(4-chlorophenyl)-1-ethanol reached 473, the ee value was greater than 99%, and the yield was 44%.

[0109] Example 4: Dehalogenase DbjA catalyzes the synthesis of (S)-2-naphthylethanol from 2-(1-chloroethyl)naphthalene.

[0110] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-2-naphthylethanol from 2-(1-chloroethyl)naphthalene was catalyzed, and the specific steps are as follows:

[0111] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0112] (2) After adding 0.005 mM 2-(1-chloroethyl)naphthalene to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0113] The reaction is shown in the following equation:

[0114]

[0115] The product 2-naphthylethanol was analyzed by high performance liquid chromatography, specifically as follows:

[0116] The system used was Hitachi Primaide, and the column type was CHIRALPAK AS-H (4.6 mm I.D. × 250 mm L, 5 μm).

[0117] The chromatographic parameters were as follows: mobile phase: n-hexane:isopropanol = 90:10 (v / v); flow rate: 1 mL / min; UV wavelength: 220 nm. Figure 7 The liquid chromatogram of the racemic product 2-naphthylethanol is shown.

[0118] according to Figure 7 It can be seen that the retention time of (R)-2-naphthylethanol in the standard sample 2-naphthylethanol is about 9.053 min, and the retention time of (S)-2-naphthylethanol is about 10.173 min.

[0119] The substrate 2-(1-chloroethyl)naphthalene was resolved by catalytic catalysis using a haloalkane dehalogenase. Figure 8 The high-performance liquid chromatogram of the dehalogenase-catalyzed reaction of haloalkanes is shown.

[0120] substrate 2-(1-chloroethyl)naphthalene 1 H and 13 C spectrum as shown Figure 17 , 18 As shown. The product 2-naphthylethanol 1 H and 13 C spectrum as shown Figure 19 , 20 As shown.

[0121] according to Figure 8 It can be seen that (S)-2-naphthylethanol eluted at 10.227 min and (R)-2-naphthylethanol eluted at 9.090 min, with a yield of 44%.

[0122] The haloalkane dehalogenase DbjA preferentially hydrolyzes (S)-2-(1-chloroethyl)naphthalene, and the enantioselectivity of the catalytically generated (S)-2-naphthylethanol reaches 261, with an ee value of 98%.

[0123] Example 5: Dehalogenase DbjA catalyzes the synthesis of (S)-1-(4-fluorophenyl)-1-ethanol from 1-(1-chloro-ethyl)-4-fluorobenzene.

[0124] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(4-fluorophenyl)-1-ethanol from 1-(1-chloro-ethyl)-4-fluoro-benzene was catalyzed, and the specific steps are as follows:

[0125] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0126] (2) After adding 0.005 mM of 1-(1-chloro-ethyl)-4-fluoro-benzene to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0127] The reaction is shown in the following equation:

[0128]

[0129] The results showed that the enantioselectivity of (S)-1-(4-fluorophenyl)-1-ethanol catalyzed by the haloalkane dehalogenase DbjA reached 245, the ee value was 98%, and the yield was 45%.

[0130] Example 6: Dehalogenase DbjA catalyzes the synthesis of (S)-1-(4-bromophenyl)-1-ethanol from 1-bromo-4-(1-chloroethyl)benzene.

[0131] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(4-bromophenyl)-1-ethanol from 1-bromo-4-(1-chloroethyl)benzene was catalyzed, and the specific steps are as follows:

[0132] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0133] (2) After adding 0.005 mM 1-bromo-4-(1-chloroethyl)benzene to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0134] The reaction is shown in the following equation:

[0135]

[0136] The results showed that the enantioselectivity of (S)-1-(4-bromophenyl)-1-ethanol catalyzed by the haloalkane dehalogenase DbjA reached 536, the ee value was 99%, and the yield was 46%.

[0137] Example 7: Dehalogenase DbjA catalyzes the synthesis of (S)-1-(4-methylphenyl)-1-ethanol from 1-(1-chloroethyl)-4-toluene.

[0138] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(4-methylphenyl)-1-ethanol from 1-(1-chloroethyl)-4-toluene was catalyzed, and the specific steps are as follows:

[0139] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0140] (2) After adding 0.005 mM of 1-(1-chloroethyl)-4-toluene to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and the mixture was extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0141] The reaction is shown in the following equation:

[0142]

[0143] The results showed that the enantioselectivity of (S)-1-(4-methylphenyl)-1-ethanol produced from 1-(1-chloroethyl)-4-toluene by the haloalkane dehalogenase DbjA reached 18, the ee value was 80%, and the yield was 45%.

[0144] Example 8: Dehalogenase DbjA catalyzes 1-chloro-2-(1-chloroethyl)benzene

[0145] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(2-chlorophenyl)-1-ethanol from 1-chloro-2-(1-chloroethyl)benzene was catalyzed, and the specific steps are as follows:

[0146] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0147] (2) After adding 0.005 mM 1-chloro-2-(1-chloroethyl)benzene to the resolution reaction system, the mixture was shaken for 12 h at 30 °C and 800 r / min. After 12 h, water was added, and the mixture was extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane.

[0148] The results showed that the haloalkane dehalogenase DbjA did not have the ability to catalyze the resolution of 1-chloro-2-(1-chloroethyl)benzene.

[0149] Example 9: Dehalogenase DbjA catalyzes the synthesis of (S)-1-(3-chlorophenyl)-1-ethanol from 1-chloro-3-(1-chloroethyl)benzene.

[0150] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(3-chlorophenyl)-1-ethanol from 1-chloro-3-(1-chloroethyl)benzene was catalyzed, and the specific steps are as follows:

[0151] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0152] (2) After adding 0.005 mM 1-chloro-3-(1-chloroethyl)benzene to the resolution reaction system, the reaction was shaken for 12 h at 30 °C and 800 r / min. After 12 h, water was added and extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0153] The reaction is shown in the following equation:

[0154]

[0155] The results showed that the enantioselectivity of (S)-1-(3-chlorophenyl)-1-ethanol catalyzed by the haloalkane dehalogenase DbjA reached 383, the ee value was 99%, and the yield was 39%.

[0156] Example 10: Dehalogenase DbjA catalyzes the synthesis of (S)-1-(4-chloro-3-methylphenyl)ethanol from 1-chloro-4-(1-chloroethyl)-2-methylbenzene.

[0157] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-(4-chloro-3-methylphenyl)ethanol from 1-chloro-4-(1-chloroethyl)-2-methylbenzene was catalyzed, and the specific steps are as follows:

[0158] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0159] (2) After adding 0.005 mM 1-chloro-4-(1-chloroethyl)-2-methylbenzene to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0160] The reaction is shown in the following equation:

[0161]

[0162] The results showed that the enantioselectivity of (S)-1-(4-chloro-3-methylphenyl)ethanol, catalyzed by the haloalkane dehalogenase DbjA to 1-chloro-4-(1-chloroethyl)-2-methylbenzene reached 536, the ee value was 99%, and the yield was 46%.

[0163] Example 11: Dehalogenase DbjA catalyzes the synthesis of (S)-1-phenylpropanol from 1-chloro-1-phenylpropane.

[0164] Using the crude enzyme powder prepared in Example 1 as a catalyst, the synthesis of (S)-1-phenylpropanol from 1-chloro-1-phenylpropane was catalyzed, and the specific steps are as follows:

[0165] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0166] (2) After adding 0.005 mM (1-chloropropyl)benzene to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0167] The results showed that the haloalkane dehalogenase DbjA did not have the ability to resolve 1-chloro-1-phenylpropane.

[0168] Example 12: Dehalogenase DbjA catalyzes the synthesis of (S)-2-(1-hydroxyethyl)pyridine from 2-(1-chloroethyl)pyridine.

[0169] Using the crude enzyme powder prepared in Example 1 as a catalyst, 2-(1-chloroethyl)pyridine was synthesized into 2-(1-hydroxyethyl)pyridine. The specific steps are as follows:

[0170] (1) Take 1g of crude enzyme powder and suspend it in 1mL of methyl tert-butyl ether solvent containing 2mM ultrapure water to obtain the resolution reaction system;

[0171] (2) After adding 0.005 mM 2-(1-chloroethyl)pyridine to the resolution reaction system, the reaction was carried out with shaking at 30 °C and 800 r / min for 12 h. After 12 h, water was added and the mixture was extracted with 3 mL of ethyl acetate. The extract was evaporated to dryness, dissolved in 3 mL of isopropanol, and filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate.

[0172] The reaction is shown in the following equation:

[0173]

[0174] The results showed that the enantioselectivity of (S)-2-(1-hydroxyethyl)pyridine produced by the haloalkane dehalogenase DbjA from 2-(1-chloroethyl)pyridine reached 205, the ee value was 99%, and the yield was 3%.

[0175] Example 13: Synthesis of (S)-1-phenylethanol from 1-chloro-1-phenylethane using water as the dispersion medium and catalyzed by the haloalkane dehalogenase DbjA.

[0176] The test method is the same as the specific operation and parameter settings in Example 2, except that the solvent of the haloalkane dehalogenase is changed from methyl tert-butyl ether containing trace amounts of ultrapure water to ultrapure water.

[0177] Test results show that in the aqueous phase, the haloalkane dehalogenase DbjA can completely convert 1-chloro-1-phenylethane to 1-phenylethanol within 2 hours, but its ee value drops to 36%. Therefore, the ee value of the product catalyzed by the haloalkane dehalogenase in the organic phase is 2.78 times that of the haloalkane dehalogenase in the aqueous phase.

[0178] In summary, this haloalkane dehalogenase exhibits excellent performance with high stereoselectivity in the organic solvent methyl tert-butyl ether.

Claims

1. The application of haloalkane dehalogenases in the selective preparation of S-configuration arylethanol, characterized in that, The amino acid sequence of the haloalkane dehalogenase is shown in SEQ ID NO.1; the S-configuration arylethanol is one of formula (I) or formula (II); Among them, R1 is one of H, Cl, and CH3; R2 is one of H, F, Cl, Br, and CH3.

2. The application according to claim 1, characterized in that, Formula (Ⅰ) is one of the following: (1) R1 is H, R2 is H; (2) R1 is H, R2 is F; (3) R1 is H, R2 is Cl; (4) R1 is H, R2 is Br; (5) R1 is H, R2 is CH3; (6) R1 is Cl, R2 is H; (7) R1 is CH3, and R2 is Cl.

3. A method for preparing S-configuration arylethanol from phenyl chlorides by enzyme catalysis, characterized in that, include: Using phenyl chlorides as substrates and haloalkane dehalogenases as catalysts, an enzyme-catalyzed reaction system was formed to obtain S-configuration arylethanol. The amino acid sequence of the haloalkane dehalogenase is shown in SEQ ID NO.1; The S-configuration arylethanol is any one of formula (I) to formula (II); Among them, R1 is one of H, Cl, and CH3; R2 is one of H, F, Cl, Br, and CH3.

4. The method according to claim 3, characterized in that, The enzyme-catalyzed reaction for the preparation of S-configuration arylethanol from phenyl chloride is one of the following: (1) Catalytic synthesis of (S)-1-phenylethanol from 1-chloro-1-phenylethane; (2) Catalytic synthesis of (S)-1-(4-chlorophenyl)-1-ethanol from 1-chloro-4-(1-chloroethyl)benzene; (3) Catalytic synthesis of (S)-2-naphthylethanol from 2-(1-chloroethyl)naphthalene; (4) Catalytic synthesis of (S)-1-(4-fluorophenyl)-1-ethanol from 1-(1-chloro-ethyl)-4-fluoro-benzene; (5) Catalytic synthesis of (S)-1-(4-bromophenyl)-1-ethanol from 1-bromo-4-(1-chloroethyl)benzene; (6) Catalytic synthesis of (S)-1-(4-methylphenyl)-1-ethanol from 1-(1-chloroethyl)-4-toluene; (7) Catalytic synthesis of (S)-1-(3-chlorophenyl)-1-ethanol from 1-chloro-3-(1-chloroethyl)benzene; (8) Catalytic synthesis of (S)-1-(4-chloro-3-methylphenyl)ethanol from 1-chloro-4-(1-chloroethyl)-2-methylbenzene.

5. The method according to claim 3, characterized in that, In the reaction system, the substrate and catalyst are dispersed in water or an organic solvent.

6. The method according to claim 5, characterized in that, The organic solvent is methyl tert-butyl ether.

7. The method according to claim 5, characterized in that, The organic solvent also includes 1-3 mM of water.

8. The method according to any one of claims 3 or 4, characterized in that, After the reaction was completed, the reaction solution was post-treated to obtain the S-configuration arylethanol product; The post-processing includes: adding water to the reaction solution, and performing extraction, drying, dissolution, and filtration.

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