Preparation method of sulfo-amino acid compound
The α-amino acid dehydrogenase mutant obtained through directed evolution by genetic engineering catalyzes the asymmetric reductive amination reaction of thiomethyl α-butanone, solving the problems of low yield and difficult purification in chemical synthesis methods, and realizing the production of thioamino acid compounds with high efficiency and low cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-03-13
AI Technical Summary
Existing chemical synthesis methods for aminothiomethylbutyric acid suffer from low yields, difficult purification, and challenges in constructing chiral centers, making efficient and low-cost industrial production impossible.
Highly active and selective α-amino acid dehydrogenase mutants were obtained through directed evolution via genetic engineering. These mutants, combined with buffer, ammonia source, and coenzyme, catalyze the asymmetric reductive amination reaction of thiomethyl α-butanone to generate high-purity thioamino acid compounds.
A high yield (98%) and high stereoselectivity (ee value 99%) of thioamino acid compounds were achieved, providing a green and efficient biocatalytic synthesis method suitable for industrial production.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, and specifically relates to a method for preparing a thioamino acid compound. Background Technology
[0002] Aminothiomethylbutyric acid (MTBA) is an important intermediate and active pharmaceutical ingredient in clinical drugs, suitable for designing HIV protease inhibitors or tumor-targeting peptides. It can also be used as a heavy metal detoxifier, linking peptide chains targeting the liver and kidneys (such as glutathione analogs) to enhance the detoxification efficiency of organs rich in heavy metals.
[0003] Currently, the chemical synthesis of aminothiomethylbutyric acid faces numerous challenges, with theoretically low yields and extremely low actual yields, coupled with high purification costs. Furthermore, the construction of chiral carbon atoms is difficult to achieve in the field of chemical synthesis. Therefore, there is an urgent need for a stereoselective α-amino acid dehydrogenase and its mutants to obtain a high-purity product with a single conformation through enzymatic action. Summary of the Invention
[0004] To address the aforementioned shortcomings of the existing technology, the present invention provides the following technical solution: A method for preparing the thioamino acid compound of Formula I, characterized in that the method comprises the following steps: in the presence of a buffer, an ammonia source, nicotinamide adenine dinucleotide and formate dehydrogenase, using an α-amino acid dehydrogenase containing the amino acid sequence shown in SEQ ID NO:1 or a mutant thereof, catalyzing the thiomethyl α-butanone compound of Formula II to undergo an asymmetric reductive amination reaction to generate the compound of Formula I. Wherein, R is a C1-C6 alkyl group or is formed by one or more R groups. a Substituted C1-C6 alkyl groups; R a C6-C10 aryl, with one or more R a-1 Substituted C6-C10 aryl or ; R a-1 It is a halogen, a C1-C6 alkyl, a C1-C6 alkoxy, or a C1-C6 alkyl substituted with one or more halogens; R a-2 It is a C1-C6 alkyl group or a C1-C6 alkyl group substituted with one or more halogens; the compound of formula I is an R-configuration isomer.
[0005] In some embodiments, the mutant is obtained by mutating one or more of the following positions in the sequence shown in SEQ ID NO:1: position 37, position 105, position 138, position 140, position 282, position 284, and position 285.
[0006] In some implementations, the mutants are selected from Table 1: Table 1. Mutation Type Table In some embodiments, the mass ratio of the α-amino acid dehydrogenase or its mutant to the compound of formula II is 0.02:1 to 5:1, preferably 0.02:1 to 0.2:1; the mass ratio of the formate dehydrogenase to the compound of formula II is 0.01:1 to 0.1:1; and the mass ratio of the nicotinamide adenine dinucleotide (NAD) to the compound of formula II is 0.001:1 to 0.5:1.
[0007] In some embodiments, the C1-C6 alkyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, preferably methyl or tert-butyl; the C6-C10 aryl groups are independently phenyl or naphthyl, preferably phenyl; the C1-C6 alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy, preferably methoxy; and the halogen is F, Cl, Br, or I.
[0008] In some embodiments, R is preferably methyl, ethyl, , , , , or More preferably .
[0009] In some embodiments, the compound represented by Formula II is preferably... The compound represented by Formula I is preferably .
[0010] In some embodiments, the buffer is selected from one or more of phosphate buffer (PBS), tris(hydroxymethyl)amine hydrochloride buffer (Tris-HCl), 3-morpholine propanesulfonic acid buffer or triethanolamine hydrochloride buffer (TEOA), preferably triethanolamine hydrochloride buffer.
[0011] In some embodiments, the ammonia source is formate, and its concentration in the reaction system is from 0.1 M to 4 M, preferably from 1 M to 3 M, and most preferably 2 M.
[0012] In some embodiments, the pH of the reaction system is preferably 7.0 to 11.0, more preferably 8.0 to 9.0, and most preferably 9.0; the reductive amination reaction is carried out at a temperature of 4 to 45°C, preferably 25 to 37°C; and the reaction time is 0.5 to 42 h, preferably 16 to 24 h.
[0013] In some embodiments, the post-treatment of the reductive amination reaction includes the following steps: quenching and concentration, washing with a reconstituted salt, washing with an organic acid, filtration, extraction with an organic solvent, adjusting the pH of the filtrate to 6.5-7.5 with an organic acid and a high-concentration potassium hydroxide solution, and drying to obtain the compound shown in Formula I; the organic solvent is an ether solvent, dichloromethane, 2-methyltetrahydrofuran, n-heptane, ethyl acetate, preferably ethyl acetate. The organic acid is oxalic acid, benzoic acid, acetic acid, propionic acid, butyric acid, preferably acetic acid.
[0014] The reagents and raw materials used in this invention are all commercially available.
[0015] Given that aminothiomethylbutyric acid (AMB) is a key intermediate and active pharmaceutical ingredient in the synthesis of important drugs such as HIV protease inhibitors, tumor-targeting peptides, and heavy metal detoxifiers, and that existing chemical synthesis methods suffer from low yields, difficult purification, and challenges in constructing chiral centers, developing an efficient, highly selective, and industrially viable biosynthetic process is of significant practical and economic value for ensuring the research and production of such drugs and controlling costs. This invention, through systematic protein-directed evolution and reaction condition screening, ultimately obtained a series of highly active and selective α-amino acid dehydrogenase mutants and corresponding reaction systems capable of efficiently catalyzing the asymmetric reductive amination of aminothiomethyl α-butanone. Literature review has revealed no reports of using α-amino acid dehydrogenases to catalyze the direct and efficient construction of chiral centers from such thiosubstrate, nor of a complete methodology combining mutants obtained through directed evolution of this substrate with complete NAD and formate dehydrogenases to achieve gram-scale high-efficiency preparation.
[0016] The significant advancements of this invention are as follows: By using genetic engineering to direct the evolution of an α-amino acid dehydrogenase, several mutants with higher activity and stereospecificity are obtained. Using compound II as a substrate, compound I is generated under the action of the α-amino acid dehydrogenase with a yield of up to 98%, stereoselectivity (ee value) of 99%, and purity of 99%. This invention achieves a production technology for the biocatalytic synthesis of compound I.
[0017] The above description is merely an overview of the technical solutions of the embodiments of the present invention. In order to better understand the technical methods of the embodiments of the present invention and to implement them in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the embodiments of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Detailed Implementation
[0018] To make the technical means, inventive features, achieved objectives, and effects of the invention readily understandable, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0019] It should be noted that the structures, proportions, sizes, etc. illustrated in this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modification to the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed in the present invention, provided that it does not affect the effects and objectives that the present invention can produce.
[0020] The inventors have discovered that the synthesis of aminothiomethylbutyric acid mainly relies on traditional chemical synthesis methods. However, these methods suffer from low yields, difficult purification, and challenges in efficiently constructing the required chiral centers. In known technologies, asymmetric reductive amination using enzyme catalysis is theoretically a green and highly selective route. However, this route is impractical due to the extremely low catalytic activity and stereoselectivity of natural α-amino acid dehydrogenases for thiomethyl α-butanone substrates. Therefore, this invention provides an α-amino acid dehydrogenase mutant and its application method. Through directed evolution technology, a highly active and selective mutant was obtained, thereby achieving the efficient and precise synthesis of this type of thioamino acid.
[0021] The present invention will be further described in detail below with reference to specific embodiments, but this should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The α-amino acid dehydrogenase of the present invention can be expressed and purified by recombinant expression technology, or it can be prepared by fully artificial synthesis. These methods are conventional means well known in the art for obtaining enzyme proteins.
[0022] The chirality and purity testing methods in the following examples are as follows: The test method used was supercritical fluid chromatography (SFC): CHIRALPAK OX-3 column, 150×4.6 mm, 3 µm column, PN: 63524; mobile phases were CO2 and 0.2% isopropylamine MeOH solution, flow rate 3.0 mL / min, detection wavelength 210 nm, peak elution time of isomer II was 0.89 min, peak elution time of isomer I (R) was 1.2 min, and peak elution time of isomer I (S) was 1.95 min. Product yield: I / (I+II); product stereoselectivity (ee value): (I(R)-I(S)) / (I(R)+I(S)).
[0023] Confirm the mass ratio of α-amino acid dehydrogenase (0.02~5):1 to 20-500 mL / g of buffer solution, add 2 M formate, formate dehydrogenase (0.01~0.1):1, NAD (0.001~0.5):1, and 0.01-1 g of substrate. Stir continuously at 5~45℃ for 4 h-36 h.
[0024] In the following examples, compound 2 is Compound 1 is (R configuration).
[0025] The α-amino acid dehydrogenases and their mutant sequences involved in the following examples are shown in Table 1. They can be obtained through fully artificial synthesis or through computer-aided design and high-throughput directed evolution screening.
[0026] Example 1: In PBS buffer, α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0027] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 98%, ee value is 99%, and product purity is >98%.
[0028] Example 2: In TEOA buffer, α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0029] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of TEOA (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 80%, ee value is 98%, and product purity is >98%.
[0030] Example 3: In Tris-HCl buffer, α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0031] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of Tris-HCl (pH=7.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 82%, ee value is 99%, and product purity is >98%.
[0032] Example 4: Under pH=8 conditions, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0033] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=8.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 85%, ee value is 98%, and product purity is >98%.
[0034] Example 5: Under pH=10 conditions, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0035] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=10.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 88%, ee value is 98%, and product purity is >98%.
[0036] Example 6: Under the condition that the volume ratio of the cosolvent DMSO to compound 1 is 10, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0037] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 10 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 90%, ee value is 95%, and product purity is >98%.
[0038] Example 7: Under the condition that the volume ratio of the cosolvent DMSO to compound 1 is 20, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0039] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 20 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 85%, ee value is 92%, and product purity is >98%.
[0040] Example 8: Under the condition that the mass ratio of α-amino acid dehydrogenase to compound 2 is 0.02:1, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0041] To a 100 mL jacketed flask, add 2 M formate, 0.02 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield was 76%, ee value was 92%, and product purity was >98%.
[0042] Example 9: Under the condition that the mass ratio of α-amino acid dehydrogenase to compound 2 is 0.5:1, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0043] To a 100 mL jacketed flask, add 2 M formate, 0.5 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 98%, ee value is 99%, and product purity is >98%.
[0044] Example 10: Under the condition that the mass ratio of formate dehydrogenase to compound 2 is 0.05:1, α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0045] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.05 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 96%, ee value is 99%, and product purity is >98%.
[0046] Example 11: Under the condition that the mass ratio of formate dehydrogenase to compound 2 is 0.02:1, α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0047] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.02 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 95%, ee value is 99%, and product purity is >98%.
[0048] Example 12: Under the condition that the mass ratio of NAD to compound 2 is 0.03:1, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0049] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.03 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 90%, ee value is 99%, and product purity is >98%.
[0050] Example 13: Under the condition that the mass ratio of NAD to compound 2 is 0.1:1, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0051] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.1 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 94%, ee value is 99%, and product purity is >98%.
[0052] Example 14: Under the condition that the mass-to-volume ratio of compound 2 to buffer is 1:100 g / mL, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0053] To a 100 mL jacketed flask, add 2 M formate, 0.1 g of α-amino acid dehydrogenase mutant 075, 0.015 g formate dehydrogenase, 0.025 g NAD, and 47.5 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 0.5 g of compound 2 (dissolved in 2.5 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 94%, ee value is 98%, and product purity is >98%.
[0054] Example 15: Under the condition that the mass-to-volume ratio of compound 2 to buffer is 1:200 g / mL, α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0055] To a 100 mL jacketed flask, add 2 M formate, 0.05 g of α-amino acid dehydrogenase mutant 075, 0.0075 g of formate dehydrogenase, 0.0125 g of NAD, and 48.75 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 0.25 g of compound 2 (dissolved in 1.25 mL DMSO) at 30 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 96%, ee value is 98%, and product purity is >98%.
[0056] Example 16: Under the condition that the reaction temperature can be 25°C, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0057] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 25 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 85%, ee value is 95%, and product purity is >98%.
[0058] Example 17: Under the condition that the reaction temperature can be 37°C, the α-amino acid dehydrogenase mutant 075 catalyzes compound 2 to compound 1.
[0059] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 37 °C and react for 20 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield was 78%, ee value was 94%, and product purity was >98%.
[0060] Example 18: Under a reaction time of 4 h, the α-amino acid dehydrogenase mutant 075 catalyzed compound 2 to compound 1.
[0061] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 4 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 65%, ee value is 99%, and product purity is >98%.
[0062] Example 19: Under a reaction time of 36 h, α-amino acid dehydrogenase mutant 075 catalyzed compound 2 to compound 1.
[0063] To a 100 mL jacketed flask, add 2 M formate, 0.2 g of α-amino acid dehydrogenase mutant 075, 0.03 g formate dehydrogenase, 0.05 g NAD, and 45 mL of PBS (pH=9.0) buffer. Stir magnetically until the enzyme is completely dissolved to obtain a homogeneous solution. Add 1 g of compound 2 (dissolved in 5 mL DMSO) at 30 °C and react for 36 h. Quench and concentrate the reaction solution, add acetic acid and 50.0 mL of ethyl acetate, mix well, filter, adjust the pH of the filtrate to obtain crystals, filter again, and dry. The final product yield is 99%, ee value is 99%, and product purity is >98%.
[0064] Example 20 The reaction conditions were as shown in Example 1, except that the α-amino acid dehydrogenase mutant 075 was replaced with other mutants and wild-type α-amino acid dehydrogenases listed in Table 2, and the results are shown in Table 2.
[0065] Table 2. Catalytic performance data of wild-type and mutant α-amino acid dehydrogenases Example 21 The reaction conditions were as shown in Example 1, except that the R group of the above compound 2 was replaced with methyl, ethyl, benzyl, diphenylmethyl, triphenylmethyl, N-methylacetamide, or tert-butyl, and the results are shown in Table 3.
[0066] Table 3. Catalytic performance data of 75 α-amino acid dehydrogenase mutants against different compounds. SEQ ID NO: 1 (WP_038532428.1) is as follows: MEHEEVKVRRGPRSGLPVVVAIHSRALGPAAGGIRLRRYPDWRDGLVDALRLSEAMTAKNAAAGLDFGGGKTVIALGPGTELTPSLREAALLDVGELIESFGGTYFGGPDVGTGPADMVVVRRATSRVFCLPPEHGGTGSSSIPTALGVFAALRAGARHVFGSAELT GRTVVVSGLGSVGALVAQHVAAAGARVVVSDVDPAKRASGYEWVDPEKALRTPADIVVPAAVGGVLSAELVPELSAPLVVGPANNQLTSEAVADELAARGIVWVPDFIASAGGAVYTLLREVEGVPHEEANARVEAIGATVSAVLDAARDKGTTPLREARARVAARLS In summary, the α-amino acid dehydrogenase, its mutant, and the catalytic method provided by this invention can efficiently and selectively synthesize aminothiomethylbutyric acid and its derivatives. The enzymes used in this method can be prepared through genetic engineering, and the required raw materials, such as buffer solutions, ammonia sources, and coenzymes, are commercially available, making the cost controllable. The entire reaction system operates under mild conditions, with simple steps and easy scale-up. In the synthesis of pharmaceutical intermediates, this method can stably and environmentally supply high-optical-purity thioamino acid products, which has significant practical value for the research and development and production of related drugs.
[0067] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the detailed process of the specific operation steps in the above embodiments has been specifically described in the method description, and will not be repeated here.
[0068] In the embodiments provided by this invention, it should be understood that the disclosed methods and parameters can be implemented in other appropriate ways. For example, the process parameters such as reaction temperature, pH value, reaction time, enzyme-to-substrate mass ratio, and coenzyme dosage given in the embodiments are illustrative and can be reasonably adjusted according to the solubility of the specific substrate, the reaction scale, and the purity requirements of the target product in actual applications. For example, the PBS can be replaced with other suitable buffer systems commonly used in the art, the DMSO cosolvent can be replaced with other polar solvents that can solubilize the substrate, and the formic acid amine can be replaced with other equivalent reagents that provide amino groups. Furthermore, other amino acid substitution combinations that can enhance catalytic activity and stereoselectivity within the range of positions 37, 105, 138, 140, 282, 284, and 285 of the sequence shown in SEQ ID NO:1 are also reasonable variations based on the core concept of this invention. All such equivalent substitutions or adaptive changes based on the principles of this invention should be covered within the substance and scope of protection of this invention.
[0069] The operational steps involved in the above embodiments can be implemented individually, or they can be combined or separated according to experimental needs. Some or all of the steps can be selected to achieve the technical effects of the present invention based on actual sample processing requirements.
[0070] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions such as parameter adjustments and equivalent equipment replacements that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A method for preparing a thioamino acid compound, characterized in that, The thioamino acid compound is the compound shown in Formula I; the method includes the following steps: in the presence of buffer, ammonia source, nicotinamide adenine dinucleotide and formate dehydrogenase, using an α-amino acid dehydrogenase containing the amino acid sequence shown in SEQ ID NO:1 or its mutant, the thiomethyl α-butanone compound shown in Formula II is catalyzed for an asymmetric reductive amination reaction to generate the compound of Formula I. Wherein, R is a C1-C6 alkyl group or is formed by one or more R groups. a Substituted C1-C6 alkyl groups; R a C6-C10 aryl, with one or more R a-1 Substituted C6-C10 aryl or ; R a-1 It is a halogen, a C1-C6 alkyl, a C1-C6 alkoxy, or a C1-C6 alkyl substituted with one or more halogens; R a-2 It is a C1-C6 alkyl group or a C1-C6 alkyl group substituted with one or more halogens; The compound of formula I is an R-configuration isomer.
2. The method according to claim 1, characterized in that, The amino acid sequence of the mutant is the sequence corresponding to one or more of the following positions in SEQ ID NO:1: position 37, position 105, position 138, position 140, position 282, position 284, and position 285.
3. The method according to claim 2, characterized in that, The mutants are selected from Table 1: Table 1. Mutation Type Table 4. The method according to any one of claims 1-3, characterized in that, The mass ratio of the α-amino acid dehydrogenase or its mutant to the compound of formula II is 0.02:1 to 5:1, preferably 0.02:1 to 0.2:1; The mass ratio of the formate dehydrogenase to the compound of formula II is from 0.01:1 to 0.1:1; The mass ratio of the nicotinamide adenine dinucleotide to the compound of formula II is from 0.001:1 to 0.5:
1.
5. The method according to claim 1, characterized in that, The C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl, preferably methyl or tert-butyl; The C6-C10 aryl group is phenyl or naphthyl, preferably phenyl; The C1-C6 alkoxy group is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy, preferably methoxy; The halogen is F, Cl, Br or I.
6. The method according to claim 1, characterized in that, R represents methyl, ethyl, or... , , , , or Preferred .
7. The method according to claim 1, characterized in that, The compound represented by Formula II is The compound represented by Formula I is preferably .
8. The method according to claim 1, characterized in that, The buffer solution is selected from one or more of phosphate buffer, trimethylolpropanediol hydrochloride buffer, 3-morpholine propanesulfonic acid buffer and triethanolamine hydrochloride buffer, preferably triethanolamine hydrochloride buffer.
9. The method according to claim 1, characterized in that, The ammonia source is formate, and its concentration in the reaction system is 0.1 M to 4 M, preferably 1 M to 3 M, and most preferably 2 M.
10. The method according to claim 1, characterized in that, The pH of the reaction system is 7.0 to 11.0, preferably 8.0 to 9.0, and most preferably 9.0; The reductive amination reaction is carried out at a temperature of 4 to 45°C, preferably 25 to 37°C, and for a reaction time of 0.5 to 42 h, preferably 16 to 24 h.