A self-initiated one-pot enzymatic synthesis method for (1R, 2R)-p-methylsulfonylbenzylaminoethanol

Abstract

This invention discloses a self-initiated one-pot enzymatic synthesis method for (1R,2R)-p-methylsulfonylbenzylserine, specifically a D-serine-driven self-initiated one-pot enzymatic synthesis method for (1R,2R)-p-methylsulfonylbenzylserine, belonging to the fields of biocatalysis and biopharmaceutical technology. The method provided by this invention utilizes engineered transaminases and transketolases as catalysts, using D-serine as an amino donor to catalyze the reaction with p-methylsulfonylbenzaldehyde to produce hydroxypyruvate as an initiator, achieving efficient conversion of p-methylsulfonylbenzaldehyde to (1R,2R)-p-methylsulfonylbenzylserine. The method provided by this invention utilizes D-serine to generate hydroxypyruvate in situ and achieves the recycling of hydroxypyruvate, eliminating the need for the additional addition of expensive hydroxypyruvate (HPA) and the exogenous cofactor thiamine pyrophosphate (TPP), saving production costs and making it suitable for industrial production.

Description

Technical Field

[0001] This invention relates to an enzyme cascade catalytic preparation method for a chiral florfenicol aminodiol intermediate, specifically to a self-initiated one-pot enzymatic synthesis method for (1R, 2R)-p-methylsulfonylbenzylaminodiol, belonging to the fields of biocatalysis and biopharmaceutical technology. Background Technology

[0002] Florfenicol is a β-amino alcohol antibiotic developed through structural modification using chloramphenicol as a lead compound. Its chiral aminodiol intermediate is (1R, 2R)-p-methylsulfonylbenzylaminoethanol, with the following chemical structure:

[0003]

[0004] Florfenicol is a novel chloramphenicol-type veterinary antibiotic with broad-spectrum antibacterial activity, widely used for bacterial infections in animals such as cattle, pigs, and chickens. Thiamphenicol is clinically used primarily to treat respiratory infections, typhoid fever, and intestinal infections. (1R,2R)-p-methylsulfonylbenzimidol is the most direct chiral intermediate for synthesizing this type of drug; 2-amino group undergoes dichloroacetylation to obtain thiamphenicol, and 1-hydroxy group undergoes fluorination to obtain florfenicol.

[0005] In 1952, the Cutler group in the United States first synthesized racemic thiophene using p-methylthioacetophenone as the starting material in 7 and 9 steps respectively (Journal of the American Chemical Society 1952, 75 (17), 4330–4333). However, only florfenicol and thiophene possessing chiral cis-2-amino-1,3-diol subunits exhibit antibacterial activity. Optically pure florfenicol and thiophene contain two adjacent chiral centers, posing a significant challenge to large-scale synthesis. In 1969, the Akiyama group in Japan obtained an optically pure thiophene precursor by condensing p-methylsulfonylbenzaldehyde with glycine and then resolving it with tartaric acid. This precursor was then reduced to obtain optically pure thiophene. This synthetic route, using p-methylsulfonylbenzaldehyde as the starting material, involves only 5 steps and is currently the most widely used method for industrial production of optically pure thiophene both domestically and internationally. Its flowchart is as follows:

[0006] .

[0007] Although the chemical synthesis methods for thiamphenicol and florfenicol are constantly being improved, the theoretical yield of chemical resolution is only 50%, and environmental problems such as wastewater pollution exist. This has prompted the search for milder, more efficient, and environmentally friendly synthetic methods. Enzyme-catalyzed synthesis has the advantages of high regioselectivity, high chemoselectivity, and high stereoselectivity. Furthermore, enzymes, as green biocatalysts, are environmentally friendly and can serve as an effective alternative to the chemical synthesis of optically pure florfenicol and thiamphenicol. In recent years, combined strategies using ketone reductases, aldolases, transaldolases, transketases, and transaminases have been developed for the synthesis of chiral intermediates of florfenicol.

[0008] In 2018, a method for synthesizing florfenicol (2S,3R)-α-amino-β-hydroxy ester intermediates via enzymatic dynamic kinetic resolution using ketone reductase was first reported (European Journal of Organic Chemistry 2018, 2018 (36), 5044). This method has high reaction yield and good selectivity, but the substrate for the enzymatic reaction must be chemically synthesized, and the product still requires 6 chemical reactions to be converted into the final product florfenicol. In 2007, Steinreiber et al. used L-threonine aldolase to catalyze the production of (2S,3R)-p-methylsulfonylbenzaldehyde (Tetrahedron 2007, 63(4), 918.), but the diastereoselectivity of the reaction was relatively low, with a de value of only 53%. In 2021, Professor Wu Jianping's research group at Zhejiang University synthesized (2S,3R)-p-methylsulfonylphenylserine using a modified L-threonine aldolase mutant, achieving a yield of 75% and a de value > 99% (ACS Catalysis 2021, 11 (6), 3198). Currently, this method still suffers from drawbacks such as high glycine consumption and difficulty in product separation and purification. In 2019, they first reported the synthesis of (2S,3R)-p-methylsulfonylphenylserine using p-methylsulfonylbenzaldehyde and L-threonine as substrates, catalyzed by L-threonine aldolase (Catalysis Science & Technology 2019, 9 (21), 5943). The modified mutant showed a 7.2-fold increase in catalytic efficiency, with a yield of (2S,3R)-p-methylsulfonylphenylserine of 90% and a de value of 94.6% (Bioresour Technol 2020, 310, 123439). In 2021, Professor Lin Shuangjun's research group at Shanghai Jiao Tong University established an enzymatic synthesis route for (1R, 2R)-p-methylsulfonylbenzylserine (ACSCatalysis 2021, 11 (12), 7477). This method uses p-methylsulfonylbenzaldehyde as a substrate, coupled with transketolase and transaminase, and introduces lactate dehydrogenase and glucose dehydrogenase to drive the reaction and regenerate the cofactor NADH, achieving a final yield of 76% and a de value of 96%. The advantage of this method is that it can omit the esterification and reduction steps, directly synthesizing (1R, 2R)-p-methylsulfonylbenzylserine. However, this method uses hydroxypyruvate as a substrate for the transketolase, resulting in higher costs. Furthermore, this method requires two additional enzymes to catalyze the transamination reaction, making it unsuitable for industrial production. The reaction flow diagram is as follows:

[0009] .

[0010] In addition, to reduce costs, researchers in this field have also attempted to use inexpensive aldehydes and ketones (such as acetone and pyruvic acid) as initiators to generate hydroxypyruvic acid in situ via transamination. While this strategy avoids the use of expensive raw materials to some extent, the introduction of exogenous chemicals increases the complexity of raw material control and may lead to problems such as solvent residue. As for the theoretically most economical "substrate self-initiation" strategy (i.e., utilizing the substrate p-methylsulfonylbenzaldehyde to generate hydroxypyruvic acid through its own transamination), existing research and experiments show that in enzyme catalytic systems without special adaptation, this self-initiation process often suffers from problems such as difficulty in initiation and sluggish reaction kinetics, resulting in a generally low final yield of the target product (e.g., less than 50%), which is difficult to meet the requirements of high yields for industrial applications.

[0011] Therefore, developing a one-pot enzyme catalysis process that does not require the addition of exogenous initiators and is driven by the substrate itself is a key technical challenge to achieve green and low-cost manufacturing of this drug intermediate. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] In view of the aforementioned shortcomings of existing technologies, such as large raw material consumption, high production costs, multiple reaction steps, relatively complex post-processing and quality control, and low yields, this invention provides a method for synthesizing optically pure florfenicol aminodiol intermediates via a self-initiated one-pot two-enzyme coupling reaction. This method eliminates the need for expensive hydroxypyruvate and requires only two enzymes, significantly reducing production costs and making it suitable for industrial production.

[0014] Solution for solving the problem

[0015] This invention uses p-methylsulfonylbenzaldehyde, a chemical raw material for the synthesis of florfenicol, as the starting material and D-serine as the amino donor. A modified transaminase catalyzes a transamination reaction I of p-methylsulfonylbenzaldehyde, producing an initial amount of hydroxypyruvate, which then initiates a transketal reaction to generate the intermediate shown in Formula 1. The transaminase then uses this intermediate as an amino acceptor to react with D-serine to generate the target product (1R, 2R)-p-methylsulfonylbenzylserine alcohol (this reaction is defined as transamination reaction II). Hydroxypyruvate is also generated, which then serves as a substrate for the transketal reaction, forming a cyclic reaction process. The hydroxypyruvate produced in transamination reaction II is consumed in the transketal reaction, which in turn shifts the chemical equilibrium of transamination reaction II towards the formation of the target product, improving the conversion efficiency of transamination reaction II. By optimizing the transaminase and transketase, as well as the reaction conditions, a one-pot, two-enzyme cascade catalytic reaction is achieved to synthesize high-optical-purity (1R, 2R)-p-methylsulfonylbenzylserine alcohol. The synthetic route is as follows: Figure 1 As shown.

[0016] The technical solution of the present invention is as follows:

[0017] [1]. A method for the self-initiated preparation of (1R, 2R)-p-methylsulfonylbenzylamine, wherein D-serine is used as an amino donor and an enzyme catalyst is used to catalyze the synthesis of (1R, 2R)-p-methylsulfonylbenzylamine from p-methylsulfonylbenzaldehyde;

[0018] The enzyme catalyst comprises transketolase and transaminase;

[0019] Optionally, the transketolase comprises the sequence shown in SEQ ID NO:4, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4;

[0020] The transaminase comprises the sequence shown in SEQ ID NO:6, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:6.

[0021] [2]. According to the method described in [1], hydroxypyruvate generated from p-methylsulfonylbenzaldehyde by transaminase is used as an initiator to initiate the transketal reaction.

[0022] [3]. According to the method described in [1] or [2], wherein the transketolization reaction is a condensation reaction of p-methylsulfonylbenzaldehyde and hydroxypyruvate under the action of the transketase to produce the compound shown in Formula 1.

[0023] Formula 1;

[0024] In the presence of D-serine, the compound shown in Formula 1 is converted to (1R, 2R)-p-methylsulfonylbenzylserine alcohol and hydroxypyruvate by the transaminase catalysis, and the resulting hydroxypyruvate then participates in the next round of ketone reaction.

[0025] [4]. The method according to any one of [1] to [3], wherein the transketolase and transaminase are in the form of pure enzyme, crude enzyme or cell lysate;

[0026] Optionally, the pure enzyme, crude enzyme, or cell lysate may be in free or immobilized form.

[0027] [5]. The method according to any one of [1] to [4], wherein the enzyme-catalyzed system also contains a cofactor;

[0028] Optionally, the cofactor includes Mg 2+ Thiamine pyrophosphate and pyridoxal phosphate.

[0029] [6]. The method according to any one of [1] to [5], wherein the enzyme catalysis is carried out under conditions of pH 7.5 ± 0.5;

[0030] Optionally, a buffer solution, preferably Tris-HCl or phosphate buffer, is used to maintain the pH at 7.5 ± 0.5.

[0031] [7]. The method according to any one of [1] to [6], wherein the enzyme-catalyzed system further contains (A) and / or (B):

[0032] (A) Hydrogen peroxide and catalase;

[0033] (B) Cation exchange resin;

[0034] Optionally, the cation exchange resin is sodium-type cation exchange resin IR120 or Amberlite 732.

[0035] [8]. A transaminase, wherein the transaminase is used in any one of the methods described in [1] to [7], the transaminase comprising the sequence shown in SEQ ID NO:6, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:6.

[0036] [9]. A transketolase, wherein the transketolase is used in any one of the methods described in [1] to [7], the transketolase comprising the sequence shown in SEQ ID NO:4, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:4.

[0037]

[10] . A product for the self-initiated preparation of (1R, 2R)-p-methylsulfonylbenzylamine, wherein it comprises a transaminase as described in [8] and a transketase as described in [9].

[0038]

[11] . The product according to

[10] , wherein the product further comprises at least one of (a) to (d):

[0039] (a) A cofactor, said cofactor including Mg 2+ Pyridoxal phosphate; optionally, the cofactor further includes thiamine pyrophosphate;

[0040] (b) Buffer solution, preferably Tris-HCl or phosphate buffer, to maintain the pH of the reaction system at 7.5 ± 0.5;

[0041] (c) Hydrogen peroxide and catalase;

[0042] (d) A cation exchange resin, preferably a sodium-type cation exchange resin; more preferably, the cation exchange resin is a sodium-type cation exchange resin IR120 or Amberlite 732.

[0043] The effects of the invention

[0044] This invention utilizes specially modified, highly active and selective transketolase and transaminase mutants to successfully overcome the problem of kinetic lag in substrate self-initiation reactions. This allows the substrate to be converted into the initiator hydroxypyruvate during the reaction, thus enabling the recycling of the hydroxypyruvate produced during preparation and eliminating the need for the addition of exogenous hydroxypyruvate. Furthermore, the intermediate products do not require separation, achieving their reuse. Moreover, the endogenous cofactor thiamine pyrophosphate in the enzyme preparation is sufficient to maintain the reaction, eliminating the need for additional addition and further reducing costs.

[0045] Furthermore, the method for synthesizing p-methylsulfonylphenylserine alcohol intermediate provided by this invention is a novel one-pot enzymatic method for preparing p-methylsulfonylphenylserine alcohol intermediate. By adding two enzyme catalysts, it eliminates the two-step chemical operation from acid to alcohol. Based on this, the yield of the product is further improved through modifications to the reaction conditions. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the self-initiated cyclic cascade enzyme-catalyzed synthesis of (1R, 2R)-p-methylsulfonylbenzylserine alcohol according to the present invention. First, a transaminase uses D-serine as a donor to catalyze a small amount of substrate aldehyde transamination reaction I, producing a small amount of hydroxypyruvate; then, a transketase uses the hydroxypyruvate produced in transamination reaction I as a donor to catalyze the condensation with the substrate aldehyde to obtain dihydroxyketone (R)-2; finally, a transaminase uses D-serine as an amino donor to catalyze the transamination reaction of (R)-2 to generate (1R, 2R)-p-methylsulfonylbenzylserine alcohol 3a.

[0047] Figure 2 The results are shown in the graphs for Examples 3-9 and Comparative Example 1. The results demonstrate the conversion yield and chiral purity of the target product under different examples. Specifically, TK:TA 1:4 is Example 3; TK:TA 2:3 is Example 4; TK:TA 3:2 is Example 5; 10 eq. D-ser, 15 eq. D-ser, and 20 eq. D-ser are Examples 6; TPP 0 mM, TPP 0.1 mM, and TPP 0.2 mM are Examples 7; TA24_M3 is Comparative Example 1; +H2O2 & Catalase is Example 8; and +Na-form IR120 is Example 9.

[0048] Figure 3The results are HPLC results for the reaction in Example 9. The retention times of aminodiols 3a and 3b after elution with ammonia were 20.2 min and 17.2 min, respectively. Detailed Implementation

[0049] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0050] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0051] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0052] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0053] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0054] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0055] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0056] The terms “preferred” and “ideal” as used herein are not intended to limit the scope of the claimed invention or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to emphasize alternative or additional features that may or may not be used in particular embodiments of the invention.

[0057] In this invention, unless otherwise specified, the term "comprising" as used herein can be open-ended or closed-ended. For example, "comprising" may mean that it may also include other components not listed, or it may only include the listed components.

[0058] In this specification, the terms "sequence identity" or "percentage of identity" in comparisons of two nucleic acids or peptides refer to the percentage of identical sequences or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed. The methods disclosed herein for determining “sequence identity” or “percentage of identity” include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0059] In some embodiments, the transketolase mutant of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or percentage of identity of amino acid residues compared to a transketolase containing the sequence shown in SEQ ID NO:1. The transaminase mutant of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity or percentage of identity of amino acid residues compared to a transaminase containing the sequence shown in SEQ ID NO:2. In other embodiments, the polynucleotide encoding the transketase mutant of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide "sequence identity" or "percentage of identity" compared to the polynucleotide encoding the sequence shown in SEQ ID NO:1 (the sequence of said polynucleotide is the nucleotide sequence shown in SEQ ID NO:3). In other embodiments, the polynucleotide encoding the transaminase mutant of the present invention has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide "sequence identity" or "percentage of identity" compared to the polynucleotide encoding the sequence shown in SEQ ID NO:2 (the sequence of said polynucleotide is the nucleotide sequence shown in SEQ ID NO:5). The determination / calculation of "sequence identity" or "percentage of identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues, a region of at least about 100 residues, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In some embodiments, the sequence is substantially identical along the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides).

[0060] EC designations, or EC numbers, are a numbering and classification system for enzymes developed by the Enzyme Commission. The classification is based on the chemical reactions catalyzed by each enzyme. This system also provides a suggested name for each enzyme, hence it is also known as the Enzyme Commission nomenclature system.

[0061] The technical solution of the present invention will be described in detail below:

[0062] <Method for preparing (1R, 2R)-p-methylsulfonylbenzylamine>

[0063] This invention provides a one-pot, self-initiated two-enzyme cascade catalytic method for synthesizing (1R,2R)-p-methylsulfonylbenzylaminoethanol, the target product having the following structural formula:

[0064] .

[0065] In some embodiments, the method involves the enzymatic catalytic reaction of p-methylsulfonylbenzaldehyde with transketolase (TK) and transaminase (TA) in a system containing an amino donor (e.g., D-serine) to achieve the synthesis of the florfenicol aminodiol intermediate (1R, 2R)-p-methylsulfonylbenzylamine.

[0066] (Transaminase)

[0067] In some embodiments of the present invention, "aminotransferase" and "transaminase" are used interchangeably. A transaminase is a transferase that catalyzes a transamination reaction, transferring the α-amino group of an amino acid to an α-keto acid. More specifically, it refers to a protein with the enzymatic ability to transfer an amino (NH2) and hydrogen atom from a primary amine to an acceptor carbonyl (ketone) compound, converting the amino donor to its corresponding carbonyl (ketone) compound and the acceptor to its corresponding primary amine.

[0068] Based on the role of transaminase in this invention, the reaction can be "self-initiated". The "self-initiation" means that without the addition of exogenous aldehyde or ketone initiators (such as acetone or pyruvic acid), the chemical reaction is shifted towards the generation of the target product by using hydroxypyruvic acid produced by catalyzing p-methylsulfonylbenzaldehyde as the initiator.

[0069] In some embodiments, the present invention is not limited to the specifically disclosed transaminases, but can be extended to their functional equivalents. Preferably, the transaminase may be selected from transaminases with EC number EC2.6, and further, the transaminase may be selected from transaminases with EC number EC2.6.1.

[0070] In a further embodiment, the transaminase in this invention can catalyze the reaction of an amino donor and p-methylsulfonylbenzaldehyde to generate hydroxypyruvate.

[0071] In some exemplary embodiments, the transaminase is based on the transaminase sequence shown in SEQ ID NO:2, with 12 mutation sites (F113V, V148F, Q143N, I146R, F216Y, Y145W, V112A, V114G, K123V, M190Q, S214A, and A199Q) introduced to obtain a transaminase mutant, which is named TA24_M7 in this invention. In some optional embodiments, the transaminase may further include a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the TA24_M7 sequence.

[0072] (Transketonease)

[0073] In some embodiments of the present invention, the transketase is an enzyme that catalyzes the transfer of a 2-hydroxyacetaldehyde group (CH2OHCO-) ​​from a 2-ketoose (such as sedoheptose 7-phosphate, ribulose 5-phosphate) to the first carbon atom of an aldose (such as ribose 5-phosphate, erythrose 4-phosphate, glyceraldehyde 3-phosphate).

[0074] In some embodiments, the present invention is not limited to the specifically disclosed transketase, but can be extended to its functional equivalents. Preferably, the transketase may be selected from the transketase with EC number EC2.2, and further, the transketase may be selected from the transketase with EC number EC2.2.1.

[0075] In some exemplary embodiments, the transketase is based on the transketase sequence shown in SEQ ID NO:1, with eight mutation sites (I189G, G67H, E468W, T469P, F437W, I247A, A29D, and P30W) introduced to obtain the transketase mutant Ec6_M6. These mutations can improve the transketase activity; for example, the transketase activity is increased by nearly 2.1 times compared to the mutant with the six mutation sites (I189G, G67H, E468W, T469P, F437W, and I247A).

[0076] In some alternative embodiments, the transketolase may further comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of Ec6_M6.

[0077] [Enzyme form]

[0078] The transaminases and transketolases used in this invention may be present in extracts (crude enzyme solutions) from cells that naturally or recombinantly produce the enzymes, in purified extracts (pure enzymes), or in substantially pure or completely pure forms. This invention also relates to fermentation products or cell lysates containing the enzymes of this invention.

[0079] The crude enzyme solution, purified enzyme, or cell lysate containing the enzyme used in this invention are water-soluble. Immobilized enzymes are generally preferred as they are typically more stable and durable. Immobilized enzymes are also easier to recover and reuse. Many methods of enzyme immobilization are known in the art. Enzymes can also be cross-linked to form cross-linked enzyme aggregates (CLEAs), which are generally more stable and easier to recover and reuse.

[0080] In some implementations, crude enzyme solutions, pure enzymes, or cell lysates can be immobilized, for example, by using immobilization carriers such as activated carbon, silica gel, quartz sand, sodium alginate, or ion exchange resins.

[0081] In some embodiments of the present invention, the transaminase and transketase described above can be jointly immobilized on activated carbon, silica gel, quartz sand, sodium alginate, or ion exchange resin. In the present invention, the activated carbon can be selected from wood-based activated carbon, coal-based activated carbon, synthetic resin-based activated carbon, etc. In the present invention, the activated carbon is used after pretreatment, and the pretreatment methods include, but are not limited to, physical activation, chemical activation, acid washing, and alkali washing.

[0082] The formation of the initiator hydroxypyruvate

[0083] In the method for preparing (1R,2R)-p-methylsulfonylbenzylserine provided by this invention, no additional exogenous aldehyde or ketone initiators (e.g., acetone, pyruvate, etc.) are added. Hydroxypyruvate, generated by the transaminase using D-serine as an amino donor, is used as an initiator to initiate subsequent transketal and transamination reactions, thereby achieving the synthesis of (1R,2R)-p-methylsulfonylbenzylserine. This invention reduces the use of exogenous initiators, generates the desired hydroxypyruvate during the reaction process, maintains the equilibrium of various reactions in the reaction system, and achieves the recycling of expensive non-industrial raw material hydroxypyruvate (e.g., β-hydroxypyruvate).

[0084] Steps for converting p-methylsulfonylbenzaldehyde to ketone

[0085] In some embodiments, p-methylsulfonylbenzaldehyde and the hydroxypyruvate, preferably β-hydroxypyruvate, prepared above are converted into the compound shown in Formula 1 by a transketolase:

[0086] Formula 1.

[0087] The steps of transamination of the compound described in Formula 1

[0088] In some embodiments, the compound shown in Formula 1 is converted with D-serine to (1R, 2R)-p-methylsulfonylbenzylserine and β-hydroxypyruvate under the action of the same transaminase.

[0089] In some implementations, the β-hydroxypyruvate obtained from this step further initiates the next round of ketogenic reaction, promoting the ketogenic reaction of p-methylsulfonylbenzaldehyde and pulling the transamination reaction toward the target product.

[0090] Reaction system and reaction conditions

[0091] In some embodiments, the method for preparing (1R, 2R)-p-methylsulfonylbenzylamine of the present invention is a one-pot method. The product hydroxypyruvate in the reaction process can be used as a raw material to continue the transketal reaction with p-methylsulfonylbenzaldehyde under the action of transketase. Therefore, the substrate, enzyme catalyst and other materials required for the reaction are added to the reaction system, and then (1R, 2R)-p-methylsulfonylbenzylamine can be obtained by reacting under suitable conditions.

[0092] In some embodiments, the reaction system further includes a cofactor; optionally, the cofactor includes Mg. 2+ Thiamine pyrophosphate and pyridoxal phosphate. Optionally, the Mg... 2+ Provided by divalent magnesium salts, wherein the divalent magnesium salts are soluble magnesium salts, such as magnesium chloride, magnesium sulfate, etc.

[0093] In some exemplary embodiments, when the reaction system contains 50-150 mM p-methylsulfonylbenzaldehyde, the reaction system also contains 0.5-2.0 M D-serine, 0-0.5 mM thiamine pyrophosphate, and 0.2-1.0 mM Mg. 2+ 0.05~0.5mM pyridoxal phosphate.

[0094] In some preferred embodiments, the reaction system contains 100-150 mM p-methylsulfonylbenzaldehyde, 1.0-2.0 M D-serine, 0-0.2 mM thiamine pyrophosphate, and 0.3-0.8 mM Mg. 2+ 0.05~0.3 mM pyridoxal phosphate.

[0095] In some specific implementation schemes, the reaction system also includes transaminase and transketase, the amounts of which are determined based on the amounts of substances participating in the reaction, such as p-methylsulfonylbenzaldehyde and D-serine. The amounts can be either exactly to complete the reaction or in excess. In the above exemplary reaction system, the concentration of transketolase is at least 0.1 U / mL, at least 0.2 U / mL, at least 0.3 U / mL, at least 0.4 U / mL, or at least 0.5 U / mL, and can be 0.1 U / mL, 0.2 U / mL, 0.3 U / mL, 0.4 U / mL, 0.5 U / mL, 0.6 U / mL, 0.7 U / mL, 0.8 U / mL, 0.9 U / mL, 1 U / mL, or 1.5 U / mL, etc.; the concentration of transaminase is at least 0.1 U / mL, at least 0.2 U / mL, at least 0.3 U / mL, at least 0.4 U / mL, at least 0.5 U / mL, at least 0.6 U / mL, at least 0.7 U / mL, at least 0.8 U / mL, at least 0.9 U / mL, at least 1 U / mL, or at least 1.5 U / mL, and can be 0.1 U / mL, 0.2 U / mL, 0.3 U / mL, 0.4 U / mL, etc. U / mL, 0.5 U / mL, 0.6 U / mL, 0.8 U / mL, 1 U / mL, 1.5 U / mL, 2.5 U / mL, etc. In some embodiments, the ratio of the transketolase to the transaminase is not specifically limited and can be (1~5):(2~6), for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 2:1, 2:2, 2:3, 2:4, 2:5, 2:6, 3:1, 3:2, 3:3, 3:4, 3:5, 3:6, 4:1, 4:2, 4:3, 4:4, 4:5, 4:6, 5:1, 5:2, 5:3, 5:4, 5:5, 5:6.

[0096] In some implementations, hydrogen peroxide and catalase can be added to the reaction system to inhibit the reducing power of NADH or NADPH. Specifically, the action of hydrogen peroxide and catalase is through direct oxidation (e.g., NADH → NADPH). + ) and indirectly activated antioxidant systems (such as NADPH→NADP) + The dual mechanism reduces the reducing power of NADH and NADPH.

[0097] In other embodiments, a cation exchange resin may be added to the reaction system to drive the equilibrium reaction. Specifically, the cation exchange resin drives the transamination / ketone reaction by selectively adsorbing products and breaking the reaction equilibrium limitation. In some preferred embodiments, the cation exchange resin is a sodium-type cation exchange resin, such as sodium-type cation exchange resin IR120, Amberlite 732, etc.

[0098] In some specific implementation schemes, the reaction is carried out under the conditions of pH 7.5±0.5 and 30±5℃. The reaction can be terminated after the raw materials (substrate) are completely converted, or the reaction can be carried out for at least 0.1 h, 0.2 h, 0.3 h, 0.4 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, or 8 h.

[0099] In some exemplary embodiments, a pH buffer solution is used to maintain the pH of the reaction system; optional pH buffer solutions include phosphate buffer, Tris-HCl buffer, etc. Further, in exemplary embodiments of the present invention, the reaction system also contains 50-100 mM phosphate buffer.

[0100] Stereoselectivity of the target product

[0101] Furthermore, the stereoselectivity of the target product (1R, 2R)-p-methylsulfonylbenzylserine obtained by the method described above was detected and calculated. The stereoselectivity ee and de values ​​can be calculated using the following formulas:

[0102]

[0103]

[0104] <Preparation of (1R, 2R)-p-methylsulfonylbenzylamine>

[0105] According to some embodiments of the present invention, a product (or enzyme composition) for preparing (1R, 2R)-p-methylsulfonylbenzirin is provided, said product comprising transaminase and transketase.

[0106] In some embodiments, the present invention is not limited to the specifically disclosed transaminases, but can be extended to their functional equivalents. Preferably, the transaminase may be selected from transaminases with EC number EC2.6, and further, the transaminase may be selected from transaminases with EC number EC2.6.1.

[0107] In some exemplary embodiments, the transaminase is based on the transaminase sequence shown in SEQ ID NO:2, with 12 mutation sites (F113V, V148F, Q143N, I146R, F216Y, Y145W, V112A, V114G, K123V, M190Q, S214A, and A199Q) introduced to obtain the transaminase mutant TA24_M7. In some optional embodiments, the transaminase may further include a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the TA24_M7 sequence.

[0108] In some embodiments, the present invention is not limited to the specifically disclosed transketase, but can be extended to its functional equivalents. Preferably, the transketase may be selected from the transketase with EC number EC2.2, and further, the transketase may be selected from the transketase with EC number EC2.2.1.

[0109] In some exemplary embodiments, the transketase is based on the transaminase sequence shown in SEQ ID NO:1, and eight mutation sites, namely I189G, G67H, E468W / T469P, F437W, I247A, and A29D / P30W, are introduced to obtain the transketase mutant Ec6_M6.

[0110] In some alternative embodiments, the transaminase may further comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of Ec6_M6.

[0111] In some embodiments, the product further comprises cofactors, including magnesium chloride, thiamine pyrophosphate, and pyridoxal phosphate. Optionally, it may also contain a buffer solution to maintain pH.

[0112] In some embodiments, the product may also contain hydrogen peroxide and catalase to inhibit the reducing power of NADH or NADPH. Specifically, the action of hydrogen peroxide and catalase is through direct oxidation (e.g., NADH → NADPH). + ) and indirectly activated antioxidant systems (such as NADPH→NADP) + The dual mechanism reduces the reducing power of NADH and NADPH.

[0113] In other embodiments, the reaction system may also include a cation exchange resin to drive the equilibrium reaction. Specifically, the cation exchange resin drives the transamination / ketone reaction by selectively adsorbing products and breaking the reaction equilibrium limitation. In some preferred embodiments, the cation exchange resin is a sodium-type cation exchange resin, such as sodium-type cation exchange resin IR120, Amberlite 732, etc.

[0114] Example

[0115] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0116] Example 1: Preparation of transketolase mutant

[0117] (1) Preparation of transketolase mutants

[0118] Mutations were introduced at eight sites—I189G, G67H, E468W, T469P, F437W, I247A, A29D, and P30W—into the transketolase Ec6, resulting in the final mutant Ec6_M6.

[0119] The starting sequence of transketolase Ec6 is:

[0120] MSSRKELANAIRALSMDAVQKAKSGRPGAPMGMADIAEVLWRDFLKHNPQNPSWADRDRFVLSNGHGSMLIYSLLHLTGYDLPMEELKNFRQLHSKTPGHPEVGYTAGVETTTGPLGQGIANAVGMAIAEKTLAAQFNRPGHDIVDHYTYAFMGDGCMMEGISHEVCSLAGTLKLGKLIAFYDDNGISIDGHVEGWFTDDTAMRFEAYGWHVIRDIDGHDAASIKRAVEEARAVTDKPSLLMCKTIIGFGSPNKAGTHDSHGAPLGDAEIALTREQLGWKYAPFEIPSEIYAQWDAKEAGQAKESAWNEKFAAYAKAYPQEAAEFTRRMKGEMPSDFDAKAKEFIAKLQANPAKIASRKASQNAIEAFGPLLPEFLGGSADLAPYNLTLWSGSKAINEDAAGNYIHYGVREFGMTAIANGISLHGGFLPYTSTYLMFVEYARNAVRMAALMKQRQVMVYTHDSIGFGETGPTHQPVEQVASLRVTPNMSTWRPCDQVESAVAWKYGVERQDGPTALILSQQNLAQQERTEEQLANIARGGYVLKDCAGQPELIFIATGSEVELAVAAYEKLTAEGVKARVVSMPSTDAFDKQDAAYRESVLPKAVTARVAVEAGIADYWYKYVGLNGAIVGMTTFGESAPAELLFEEFGFTVDNVVAKAKELL (SEQ ID NO:1) [[]]

[0121] [[]]

[0121] Nucleotide sequence of Ec6: [[]]

[0122] [[]]

[0122]

[0123] The specific modification process is as follows: The nucleotide sequence encoding transketolase Ec6 is ligated into the pET28a vector to construct a recombinant plasmid expressing Ec6. By designing primer pairs targeting the I189G mutation of Ec6, Overlap PCR is performed using the recombinant plasmid expressing Ec6 as a template to mutate the 189th amino acid from isoleucine to glycine.

[0124] The PCR system (50 μL) is as follows: KOD ONE Mix 25 μL, plasmid template final concentration 1 ng / μL, primer final concentration 0.4 μM, ddH2O to 50 μL.

[0125] The PCR amplification program is as follows: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 25 sec; (3) 55℃ annealing for 30 sec; (4) 68℃ extension for 2 min. Repeat steps (2) to (4) for 25-30 cycles, and finally extend for 5 min. Store the PCR amplification product at 4℃.

[0126] After PCR, 1 μL of restriction endonuclease Dpn I was added to the PCR product and incubated at 37°C for 15 min to eliminate the plasmid template. The principle is that plasmids extracted from organisms have methylation modifications, while PCR products do not. Dpn I enzyme can specifically cleave methylated DNA, thereby reducing the probability of false positives.

[0127] The treated PCR product was added to 100 μL of E. coli BL21(DE3) competent cells at a ratio of 1:10 (v / v). After standing on ice for 30 min, the cells were heat-shocked at 42°C for 90 sec. After cooling on ice, 500 μL of LB liquid medium was added and the cells were incubated on a shaker at 37°C for 30 min. The cells were then evenly plated onto LB agar plates containing 50 μg / mL kanamycin sulfate and incubated upside down at 37°C for 12 hours. The resulting clones, after being verified by sequencing, were identified as single clones containing the target mutation.

[0128] Construction of the Ec6_M2 mutant: Using the successfully sequenced Ec6_M1 plasmid as a template, PCR amplification was performed using primers targeting the G67H mutation in Ec6_M1. Following the same procedure, glycine at position 67 was mutated to histidine, yielding Ec6_M2. This iterative mutagenesis was repeated until a single clone of Ec6_M6 containing eight mutation sites (I189G, G67H, E468W, T469P, F437W, I247A, A29D, and P30W) was obtained.

[0129] Single clones of Ec6_M6 were selected and cultured overnight in LB seed medium containing kanamycin (50 mg / L). The next day, recombinant Escherichia coli strains were inoculated into fresh LB medium containing kanamycin (50 mg / L) at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm in a shaker until OD500. 600 = 0.6; 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was added for induction at 20℃. After induction for 16 h, the cells were centrifuged at 8,000 rpm for 10 min to obtain cells that could efficiently express Ec6_M6. The collected cells were suspended in phosphate buffer (50 mM, pH 7.5) and sonicated (280 W, 2 sec on, 2 sec off, 10 min) until the cells were clear, or the cells were disrupted by pressure using a high-pressure cell disruptor. The cells were centrifuged at 10,000 rpm for 30 min, and the supernatant was the crude Ec6_M6 enzyme solution.

[0130] Affinity chromatography was performed using the histidine tag on the recombinant protein, employing a Ni-NTA Beads6FF nickel affinity column. After equilibrating the nickel column with Buffer A (50 mM phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 7.5), the crude enzyme solution was loaded. Following equilibration, a gradient elution was performed using Buffer B (50 mM phosphate, 300 mM sodium chloride, 50 mM imidazole, pH 7.5). Finally, the recombinant protein bound to the nickel column was eluted using the elution buffer (50 mM phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 7.5) to obtain the purified Ec6_M6 enzyme.

[0131] The amino acid sequence of Ec6_M6:

[0132] (SEQ ID NO:4)

[0133] Example 2: Preparation of transaminase mutants

[0134] Based on the wild-type transaminase TA24, mutations were introduced at 12 sites: F113V, V148F, Q143N, I146R, F216Y, Y145W, V112A, V114G, K123V, M190Q, S214A, and A199Q. The resulting mutant was named TA24_M7.

[0135] The wild-type sequence of transaminase TA24 is:

[0136] MASMDKLFAGYKSRLAALKASDNPLSQGVAWVEGEIVPLSQARIPLLDQGFLHSDLTYDVPAVWDGRFFRLDDHLSRLEKSCSKLRLKLPFPREEVKRLLIDMVTKSGIRDVFVELIVTRGLKGVREAKNPEDLVNTLYMFIQPYIWVMEPEVQRVGGSAVIARTVRRTPPGSMDPTVKNLQWGDLVRAMLEASDRGAAYPFLTDGDANITEGSGFNIVLVKDGVIYTPDRGVLEGVTRKSVFDVAKANGIEVRLEVVPVELAYQADEIFMCTTAGGIMPITSLDGQPVKDGKIGPVTQKIWDDYWALHYDPAYSFEIDYEGVGKNGSNGMNGIH (SEQ ID NO:2)

[0137] The nucleotide sequence of transaminase TA24 is as follows:

[0138]

[0139] The specific modification process is as shown in Example 1 above. The nucleotide sequence encoding transaminase TA24 is ligated into the pET28a vector to construct a recombinant plasmid expressing TA24. Using a primer pair involving the F113V mutation against TA24, Overlap PCR is performed with the recombinant plasmid expressing TA24 as a template to mutate the 113th amino acid from phenylalanine to valine.

[0140] The PCR product was treated with Dpn I enzyme and transformed into E. coli BL21(DE3) competent cells. After culture and sequencing verification, a single clone containing the target mutation was obtained. Based on this, plasmids were extracted and mutated iteratively until a single clone of TA24_M7 containing 12 mutation sites, namely F113V, V148F, Q143N, I146R, F216Y, Y145W, V112A, V114G, K123V, M190Q, S214A, and A199Q, was obtained.

[0141] Select a single colony containing TA24_M7 and inoculate it into 5 mL of LB liquid medium (containing 50 µg / mL kanamycin sulfate) and incubate overnight at 37°C and 220 rpm; then inoculate 5 mL of the bacterial culture into a 2 L shake flask containing 500 mL of LB medium and incubate at 37°C and 220 rpm until OD200. 600 When the bacterial count reaches 0.6-0.8, 0.2 mM IPTG is added for induction, and the cells are cultured at 20℃ and 220 rpm for 16 h. After incubation, the cells are collected by centrifugation at 6000 rpm for 10 min. The collected cells are resuspended in 25 mL of pre-cooled buffer A (containing 50 mM Tris-HCl at pH 7.5 and 300 mM NaCl) and sonicated on an ice-water mixture for 2 s followed by a 2 s pause, for a total of 10 min. The lysed mixture is then centrifuged at 12000 rpm for 40 min at 4℃, and the resulting supernatant is the TA24_M7 cell lysis buffer.

[0142] The supernatant after centrifugation was filtered through a 0.22 µm filter. The filtrate was loaded onto a 2 mL nickel column pre-equilibrated with buffer A. Impurities were washed with 20 mL of buffer A containing 50 mM imidazole, followed by elution of the target protein with 4 mL of buffer A containing 250 mM imidazole. The concentrate was then concentrated to 2.5 mL. The protein concentrate was loaded onto a desalting column pre-equilibrated with buffer B (containing 50 mM pH 7.5 Tris-HCl, 150 mM NaCl, and 5% glycerol). After drying, 3.5 mL of buffer B was added to elute the protein, yielding the purified TA24_M7 enzyme.

[0143] The amino acid sequence of TA24_M7:

[0144] MASMDKLFAGYKSRLAALKASDNPLSQGVAWVEGEIVPLSQARIPLLDQGFLHSDLTYDVPAVWDGRFFRLDDHLSRLEKSCSKLRLKLPFPREEVKRLLIDMVTKSGIRDAVGELIVTRGLVGVREAKNPEDLVNTLYMFINPWRWFMEPEVQRVGGSAVIARTVRRT PPGSMDPTVKNLQWGDLVRAQLEASDRGAQYPFLTDGDANITEGAGYNIVLVKDGVIYTPDRGVLEGVTRKSVFDVAKANGIEVRLEVVPVELAYQADEIFMCTTAGGIMPITSLDGQPVKDGKIGPVTQKIWDDYWALHYDPAYSFEIDYEGVGKNGSNGMNGIH (SEQ ID NO:6)

[0145] Example 3: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using engineered transketolase-transaminase (1:4)

[0146] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.2 U / mL Ec6_M6 transketase (crude enzyme solution), 0.8 U / mL TA24_M7 transaminase (crude enzyme solution), 0.1 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, the reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0147] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 67.2%, and the stereoselectivity of the product was 94.3% de, >99% ee.

[0148] Example 4: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using engineered transketolase-transaminase (2:3)

[0149] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.4 U / mL transketolase Ec6_M6 (crude enzyme solution), 0.6 U / mL transaminase TA24_M7 (crude enzyme solution), 0.1 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, the reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0150] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 62.4%, and the stereoselectivity of the product was 94.5% de, >99% ee.

[0151] Example 5: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using engineered transketolase-transaminase (3:2)

[0152] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.6 U / mL transketolase Ec6_M6 (crude enzyme solution), 0.4 U / mL transaminase TA24_M7 (crude enzyme solution), 0.1 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, the reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0153] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 59.4%, and the stereoselectivity of the product was 94.6% de, >99% ee.

[0154] Example 6: Synthesis of (1R,2R)-p-methylsulfonylbenzylserine using different equivalents of D-serine

[0155] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, D-serine at concentrations of 1.0 M (10 eq), 1.5 M (15 eq), or 2.0 M (20 eq), transketolase Ec6_M6 at 0.2 U / mL (crude enzyme solution), transaminase TA24_M7 at 0.8 U / mL (crude enzyme solution), thiamine pyrophosphate at 0.1 mM, magnesium chloride at 0.5 mM, and pyridoxal phosphate at 0.1 mM, the reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzylserine from p-methylsulfonylbenzaldehyde.

[0156] After the reaction was completed, the reaction solution was collected and analyzed. The yields of the target products (1R,2R)-p-methylsulfonylbenzylamine were 55.5%, 67.2% and 67.0%, respectively, and the stereoselectivity of the products was 94.6% de and >99% ee.

[0157] Example 7: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using different concentrations of thiamine pyrophosphate

[0158] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.2 U / mL transketolase Ec6_M6 (crude enzyme solution), 0.8 U / mL transaminase TA24_M7 (crude enzyme solution), 0 mM, 0.1 mM, or 0.2 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, the reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0159] After the reaction was completed, the reaction solution was collected and analyzed. The yields of the target products (1R,2R)-p-methylsulfonylbenzylamine were 66.7%, 67.2% and 67.5%, respectively, and the stereoselectivity of the products was 94.6% de and >99% ee.

[0160] Example 8: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine with the additional addition of hydrogen peroxide and catalase become

[0161] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.2 U / mL Ec6_M6 transketase (crude enzyme solution), 0.8 U / mL TA24_M7 transaminase (crude enzyme solution), 0 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, 0.2% (v / v) hydrogen peroxide and 50 U / mL catalase (Maclean, catalog number: C6319) were added before the reaction to reduce the reducing power of NADH or NADPH in the system. The reaction was then carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0162] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 73.9%, and the stereoselectivity of the product was 94.2% de, >99% ee.

[0163] Example 9: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine by adding an additional cation exchange resin

[0164] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.2 U / mL Ec6_M6 transketase (crude enzyme solution), 0.8 U / mL TA24_M7 transaminase (crude enzyme solution), 0 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, 0.2% (v / v) hydrogen peroxide and 50 U / mL catalase were added before the reaction. The reaction was then carried out at 30 °C. After 5 h of reaction, 300 g / L sodium cation exchange resin IR120 was added to pull the reaction to equilibrium. The total reaction time was 18 h. The reaction of p-methylsulfonylbenzaldehyde was used to synthesize (1R,2R)-p-methylsulfonylbenzylserine.

[0165] After the reaction was completed, the cation exchange resin was eluted with ammonia and the product was tested. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 88.4%, and the stereoselectivity of the product was 96.4% de, >99% ee.

[0166] Comparative Example 1: Preparation of the transaminase mutant TA24_M3 and its use in (1R,2R)-p-methylsulfonyl... Synthesis of benzethrin

[0167] For specific implementation details, please refer to Example 2. Based on the wild-type transaminase TA24, mutations F113V, V148F, Q143N, and I146R were introduced, and the resulting mutant was named TA24_M3. The amino acid sequence of TA24_M3 is as follows:

[0168] MASMDKLFAGYKSRLAALKASDNPLSQGVAWVEGEIVPLSQARIPLLDQGFLHSDLTYDVPAVWDGRFFRLDDHLSRLEKSCSKLRLKLPFPREEVKRLLIDMVTKSGIRDVIVELIVTRGLKGVREAKNPEDLVNTLYMFINPYRWFMEPEVQRVGGSAVIARTVRRT PPGSMDPTVKNLQWGDLVRAMLEASDRGAAYPFLTDGDANITEGSGFNIVLVKDGVIYTPDRGVLEGVTRKSVFDVAKANGIEVRLEVVPVELAYQADEIFMCTTAGGIMPITSLDGQPVKDGKIGPVTQKIWDDYWALHYDPAYSFEIDYEGVGKNGSNGMNGIH (SEQ ID NO:7)

[0169] Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using TA24_M3:

[0170] In 50 mL of phosphate buffer (pH 7.5) containing 100 mM p-methylsulfonylbenzaldehyde, 1.5 M D-serine, 0.2 U / mL transketolase Ec6_M6 (crude enzyme solution), 0.8 U / mL transaminase TA24_M3 (crude enzyme solution), 0.1 mM thiamine pyrophosphate, 0.5 mM magnesium chloride, and 0.1 mM pyridoxal phosphate, the reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0171] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 42.2%, and the stereoselectivity of the product was 97.3% de, >99% ee.

Claims

1. A method for the self-initiated preparation of (1R, 2R)-p-methylsulfonylbenzylaminoethanol, characterized in that, Using D-serine as the amino donor, (1R, 2R)-p-methylsulfonylbenzaldehyde was synthesized by enzyme catalysis. The enzyme catalyst comprises transketolase and transaminase; Optionally, the transketolase comprises the sequence shown in SEQ ID NO:4, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:4; The transaminase comprises the sequence shown in SEQ ID NO:6, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

6.

2. The method according to claim 1, characterized in that, Hydroxypyruvate, generated from the transaminase-catalyzed transamination reaction of p-methylsulfonylbenzaldehyde and D-serine, is used as an initiator to initiate a transketal reaction.

3. The method according to claim 1 or 2, characterized in that, The transketal reaction is a catalytic reaction between p-methylsulfonylbenzaldehyde and hydroxypyruvate, under the action of the transketase, to produce the compound shown in Formula 1. Formula 1; In the presence of D-serine, the transaminase is used to catalyze the compound shown in Formula 1 to generate (1R, 2R)-p-methylsulfonylbenzylserine alcohol and hydroxypyruvate, and the resulting hydroxypyruvate then participates in the next round of transketal reaction.

4. The method according to any one of claims 1 to 3, characterized in that, The transketolase and transaminase are in the form of pure enzymes, crude enzymes, or cell lysates; Optionally, the pure enzyme, crude enzyme, or cell lysate may be in free or immobilized form.

5. The method according to any one of claims 1 to 4, characterized in that, The enzyme-catalyzed system also contains cofactors; Optionally, the co-factors include Mg 2+ , thiamine pyrophosphate and pyridoxal phosphate.

6. The method according to any one of claims 1 to 5, characterized in that, Enzyme catalysis was carried out at pH 7.5 ± 0.5; Optionally, the pH is maintained at 7.5 ± 0.5 using a buffer solution, preferably Tris-HCl or phosphate buffer.

7. The method according to any one of claims 1-6, characterized in that, The enzyme-catalyzed system also contains (A) and / or (B): (A) Hydrogen peroxide and catalase; (B) Cation exchange resin; Optionally, the cation exchange resin is sodium-type cation exchange resin IR120 or Amberlite 732.

8. A transaminase, characterized in that, The transaminase is used in the method according to any one of claims 1 to 7, wherein the transaminase comprises the sequence shown in SEQ ID NO:6, and the sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

6.

9. A transketolase, characterized in that, The transketolase is used in the method according to any one of claims 1 to 7, wherein the transketolase comprises the sequence shown in SEQ ID NO:4, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

4.

10. A product for the self-initiated preparation of (1R, 2R)-p-methylsulfonylbenzylaminoethanol, characterized in that, It contains the transaminase as described in claim 8 and the transketase as described in claim 9.

11. The product according to claim 10, characterized in that, The product also contains at least one of (a) to (d): (a) A cofactor, said cofactor including Mg 2+ Pyridoxal phosphate; optionally, the cofactor further includes thiamine pyrophosphate; (b) Buffer solution, preferably Tris-HCl or phosphate buffer, to maintain the pH of the reaction system at 7.5 ± 0.5; (c) Hydrogen peroxide and catalase; (d) Cation exchange resin, preferably sodium-type cation exchange resin.

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