Aldehyde / ketone-initiated enzymatic synthesis method for (1r,2r)-p-methylsulfonylphenylserinol

Abstract

The present invention relates to the technical fields of biocatalysis and biopharmaceuticals. Disclosed is an aldehyde / ketone-initiated enzymatic synthesis method for (1R,2R)-p-methylsulfonylphenylserinol. In the method provided in the present invention, using transaminase and transketolase as catalysts, in the presence of an amino donor and an aldehyde compound or a ketone compound, a substrate p-methylsulfonylbenzaldehyde is catalyzed to produce (1R,2R)-p-methylsulfonylphenylserinol. The method provided in the present invention reduces operation steps and can realize the replacement and recycling of expensive non-industrial starting material hydroxypyruvic acid, thereby reducing production costs and thus being suitable for industrial production.

Description

An aldehyde-ketone-initiated enzymatic synthesis method for (1R,2R)-p-methylsulfonylbenzylamine. Technical Field

[0001] This invention relates to an enzymatic method for preparing a chiral florfenicol aminodiol intermediate, specifically to an aldehyde-ketone-initiated enzymatic synthesis method for (1R,2R)-p-methylsulfonylbenzylaminoethanol, 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] Florfenicol is a novel chloramphenicol antibiotic for veterinary use, possessing broad-spectrum antibacterial activity and widely used in treating 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-methylsulfonylbenzylamine is the most direct chiral intermediate for synthesizing this type of drug; 2-amino group undergoes dichloroacetylation to yield thiamphenicol, and 1-hydroxy group undergoes fluorination to obtain florfenicol.

[0004] In 1952, the Cutler group in the United States first synthesized racemic thiophene by using p-methylthioacetophenone as a starting material in 7 and 9 steps respectively (Journal of the American Chemical Society 1952, 75(17), 4330-4333). However, only florfenicol and thiophene with chiral cis-2-amino-1,3-diol subunits possess 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 optically pure thiophene from p-methylsulfonylbenzaldehyde by resolution with tartaric acid. This synthetic route uses p-methylsulfonylbenzaldehyde as a starting material and involves only 5 steps, which is currently the most commonly used method for industrial production of optically pure thiophene both domestically and internationally. The reaction process is as follows:

[0005] 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. Enzymatic synthesis has the advantages of high regioselectivity, high chemoselectivity, and high stereoselectivity, and green biocatalysts are environmentally friendly, making it 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.

[0006] 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 enzyme reaction substrate must be chemically synthesized, and the product still requires 6 chemical reactions to obtain 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 an enzyme-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, the first report described the synthesis of (2S,3R)-p-methylsulfonylphenylserine using p-methylsulfonylbenzaldehyde and L-threonine as substrates, catalyzed by an L-threonine aldolase (Catalysis Science & Technology 2019, 9(21), 5943). The enzyme-modified mutant showed a 7.2-fold increase in catalytic efficiency, achieving a yield of 90% and a de value of 94.6% for (2S,3R)-p-methylsulfonylphenylserine (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-methylsulfonylbenzylamine (ACS Catalysis 2021, 11(12), 7477). This method uses p-methylsulfonylbenzaldehyde as a substrate, couples transketolase and transaminase, 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%. This method utilizes hydroxypyruvate as a transketolase substrate, resulting in high costs. Furthermore, it requires two additional enzymes to catalyze the transamination reaction, making it unsuitable for industrial production. The reaction process is as follows:

[0007] Summary of the Invention

[0008] The problem the invention aims to solve

[0009] In view of the aforementioned drawbacks of existing technologies, such as large raw material consumption and high production costs, this invention provides a one-pot two-enzyme coupling reaction method initiated by aldehydes and ketones to synthesize optically pure florfenicol aminodiol intermediates. This method eliminates the need for expensive hydroxypyruvate and requires only two enzymes, significantly reducing production costs and making it suitable for industrial production.

[0010] Solution for solving the problem

[0011] This invention uses p-methylsulfonylbenzaldehyde, a chemically synthesized raw material for florfenicol, as the starting material and D-serine as the amino donor. The transketal reaction is initiated by the conversion of aldehydes and ketones to hydroxypyruvate. Simultaneously, the consumption of hydroxypyruvate pulls the transamination reaction equilibrium, improving the conversion efficiency. By optimizing the reaction conditions, a one-pot two-enzyme cascade reaction catalyzes the synthesis of high-optical-purity (1R,2R)-p-methylsulfonylbenzylserine alcohol. The synthetic route is shown below:

[0012] The technical solution of the present invention is as follows:

[0013] [1]. A method for preparing (1R,2R)-p-methylsulfonylbenzylamine, wherein (1R,2R)-p-methylsulfonylbenzylamine is synthesized by enzyme catalyst from p-methylsulfonylbenzaldehyde in a system containing aldehydes and / or ketones.

[0014] Preferably, the enzyme catalyst comprises transketase and transaminase;

[0015] Preferably, the system further comprises an amino donor;

[0016] 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;

[0017] Optionally, 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.

[0018] [2]. The method according to [1], wherein the method comprises the following steps:

[0019] Step 1: Under the action of transaminase, amino donors and ketones and / or aldehydes are converted into hydroxypyruvate;

[0020] Step 2: Under the action of transketolase, p-methylsulfonylbenzaldehyde and the hydroxypyruvate obtained in Step 1 are catalyzed to form the compound shown in Formula 1.

[0021] Step 3: Under the action of transaminase, the compound shown in Formula 1 is catalyzed to (1R,2R)-p-methylsulfonylbenzylamine and hydroxypyruvate;

[0022] Optionally, the amino donor includes serine, isopropylamine, and α-phenylethylamine;

[0023] The preferred form is serine;

[0024] D-serine is preferred;

[0025] Preferably, the hydroxypyruvate is β-hydroxypyruvate.

[0026] [3]. The method according to [1] or [2], wherein the aldehyde and / or ketone compounds are compounds containing a carbonyl group;

[0027] The aldehyde compounds include aliphatic aldehydes and aromatic aldehydes; optionally, the aldehyde compounds include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and benzaldehyde;

[0028] The ketone compounds include aliphatic ketones and aromatic ketones; optionally, the ketone compounds include acetone, pyruvate, cyclohexanone and phenylmethyl ketone.

[0029] [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;

[0030] Optionally, the pure enzyme, crude enzyme, or cell lysate may be in free or immobilized form.

[0031] [5]. The method according to any one of [1] to [4], wherein the enzyme-catalyzed system also contains a cofactor;

[0032] Optionally, the cofactor includes Mg 2+ Thiamine pyrophosphate and pyridoxal phosphate.

[0033] [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;

[0034] Alternatively, the pH can be maintained at 7.5 ± 0.5 using Tris-HCl or phosphate buffer.

[0035] [7]. A transaminase, wherein the transaminase is used in any one of the methods described in [1] to [6], the transaminase comprising 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.

[0036] [8]. A transketolase, wherein the transketolase is used in any one of the methods described in [1] to [6], 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] [9]. A product for preparing (1R,2R)-p-methylsulfonylbenzylamine, wherein it comprises a transaminase as described in [7] and a transketase as described in [8].

[0038]

[0010] . The product according to [9], wherein the product further comprises a cofactor;

[0039] Optionally, the cofactor includes Mg 2+ Thiamine pyrophosphate and pyridoxal phosphate.

[0040] The effects of the invention

[0041] 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.

[0042] In addition, the hydroxypyruvate generated during the preparation process is recycled, eliminating the need for the addition of exogenous hydroxypyruvate. The reaction process does not require the addition of the transketolase substrate β-hydroxypyruvate, and the intermediate products do not need to be separated, thus realizing the reuse of intermediate products and significantly saving costs. Attached Figure Description

[0043] Figure 1 is a high-performance liquid chromatogram of the reaction solution in Example 3. Detailed Implementation

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M. 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 NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0054] 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 the 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 the 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% "sequence identity" or "percentage of identity" of nucleotides 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% "sequence identity" or "percentage of identity" of nucleotides 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).

[0055] In some embodiments, the "mutation" of this invention may be selected from "conservative mutations." In this invention, the term "conservative mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conserved substitution.

[0056] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0057] In this specification, "conservative substitution" generally refers to the exchange of one amino acid at one or more sites in a protein. This substitution can be conserved. Examples of substitutions considered conserved include, for instance, the substitution of Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Asp to Asn, Glu, or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp, or Arg, Glu to Gly, Asn, Gln, Lys, or Asp, Gly to Pro, and His to Asn, Lys, Gln, Arg, or Tyr. Substitutions include: Ile to Leu, Met, Val, or Phe; Leu to Ile, Met, Val, or Phe; Lys to Asn, Glu, Gln, His, or Arg; Met to Ile, Leu, Val, or Phe; Phe to Trp, Tyr, Met, Ile, or Leu; Ser to Thr or Ala; Thr to Ser or Ala; Trp to Phe or Tyr; Tyr to His, Phe, or Trp; and Val to Met, Ile, or Leu. In addition, conserved mutations also include naturally occurring mutations arising from individual differences, strain differences, or species differences in gene origin.

[0058] 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.

[0059] The technical solution of the present invention will be described in detail below:

[0060] <Method for preparing (1R,2R)-p-methylsulfonylbenzirtine>

[0061] This invention provides a one-pot biocatalytic method using a two-enzyme cascade reaction to synthesize chiral (1R,2R)-p-methylsulfonylbenzylamine, the target product having the following structural formula:

[0062] In some embodiments, the method involves using transketolase (TK) and transaminase (TA) to enzymatically catalyze the reaction of p-methylsulfonylbenzaldehyde in a reaction system containing aldehydes and / or ketones, thereby achieving the synthesis of the florfenicol aminodiol intermediate (1R,2R)-p-methylsulfonylbenzylaminoethanol.

[0063] In some embodiments, the reaction system also contains an amino donor.

[0064] (transaminase)

[0065] 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.

[0066] In some embodiments, the present invention is not limited to the specifically disclosed transaminase, but can be extended to its 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.

[0067] In some exemplary embodiments, the transaminase is based on the transaminase sequence shown in SEQ ID NO:2, with four mutation sites (F113I, V148F, Q143N, and I146R) introduced to obtain the transaminase mutant TA24_M4. 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_M4 sequence (as shown in SEQ ID NO:6), for example, by introducing two mutation sites (F113I and V148F) based on the transaminase sequence shown in SEQ ID NO:2.

[0068] (Transketonease)

[0069] 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).

[0070] 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.

[0071] In some exemplary embodiments, the transketase is based on the transketase sequence shown in SEQ ID NO:1, and six mutation sites, namely I189G, G67H, E468W, T469P, F437W and I247A, are introduced to obtain the transketase mutant EcTK_M12.

[0072] 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 EcTK_M12 (as shown in SEQ ID NO:4).

[0073] [Enzyme form]

[0074] 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.

[0075] 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.

[0076] 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.

[0077] In some embodiments of the present invention, the transaminase and transketase described above can be jointly immobilized in 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.

[0078] Steps for aldehyde-ketone transamination

[0079] In some preferred embodiments, the aldehydes and / or ketones act as initiators for enzyme-catalyzed reactions. Specifically, they generate hydroxypyruvate via an aldehyde-ketone transamination reaction, thereby initiating a subsequent transketal reaction. Specifically, the aldehydes and / or ketones react with the amino donor under the action of a transaminase to generate hydroxypyruvate. The resulting hydroxypyruvate can then be used as a donor substrate in the transketal step of p-methylsulfonylbenzaldehyde, reacting with p-methylsulfonylbenzaldehyde. This enables the recycling of the expensive non-industrial raw material hydroxypyruvate (e.g., β-hydroxypyruvate).

[0080] (Aldehydes and / or ketones)

[0081] In some embodiments, the aldehydes and / or ketones are compounds containing a carbonyl group.

[0082] In some alternative embodiments, the aldehyde compounds include aliphatic aldehydes and aromatic aldehydes; optionally, the aldehyde compounds include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and benzaldehyde; the ketone compounds include aliphatic ketones and aromatic ketones; optionally, the ketone compounds include acetone, pyruvate, cyclohexanone, and phenylmethyl ketone.

[0083] In some exemplary embodiments, acetone or pyruvate is selected as the reaction initiator.

[0084] (Amino donor)

[0085] In this invention, the "amino donor" refers to an amino compound that can supply an amino group to an acceptor carbonyl compound (i.e., an amino acceptor), thereby becoming a carbonyl byproduct.

[0086] In some alternative embodiments, any suitable amino donor can be used for the transamination reaction. The amino donor may include serine, isopropylamine, and α-phenylethylamine. Preferably, the amino donor includes serine; exemplarily, the amino donor is D-serine.

[0087] Furthermore, acetone or pyruvate reacts with D-serine under the action of transaminase to catalyze the formation of β-hydroxypyruvate.

[0088] Steps for converting p-methylsulfonylbenzaldehyde to ketone

[0089] 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:

[0090] The steps of transamination of the compound described in Formula 1

[0091] In some embodiments, the compound shown in Formula 1 is converted into (1R,2R)-p-methylsulfonylbenzylamine and β-hydroxypyruvate under the action of the same transaminase.

[0092] In some embodiments, the β-hydroxypyruvate obtained from this step further initiates a ketogenic reaction, promoting the ketogenic reaction of p-methylsulfonylbenzaldehyde.

[0093] Reaction system and reaction conditions

[0094] 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 reacting with p-methylsulfonylbenzaldehyde under the action of transketolase. Therefore, the substrate, enzyme catalyst and other materials required for the reaction are added to the reaction system, and then reacted under suitable conditions to obtain (1R,2R)-p-methylsulfonylbenzylamine.

[0095] 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.

[0096] In some exemplary embodiments, the reaction system contains 30–100 mM p-methylsulfonylbenzaldehyde, 0.5 mM–2 M acetone and / or pyruvate, 0.2–1 M D-serine, 0.05–0.5 mM thiamine pyrophosphate, and 0.2–1.0 mM Mg. 2+ 0.05–0.5 mM pyridoxal phosphate.

[0097] In some preferred embodiments, the reaction system contains 40–80 mM p-methylsulfonylbenzaldehyde, 1 mM–2 mM acetone and / or pyruvate, 0.5–1 M D-serine, 0.1–0.2 mM thiamine pyrophosphate, and 0.3–0.8 mM Mg. 2+ 0.05–0.3 mM pyridoxal phosphate.

[0098] In some specific implementations, the reaction system further includes transaminase and transketase, the amounts of which are determined based on the amounts of reactants such as p-methylsulfonylbenzaldehyde, acetone and / or pyruvate, and D-serine. The amounts can be either exactly to complete the reaction or in excess. In some exemplary reaction systems, the concentration of transketase 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, 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, 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, at least 1.5 U / mL, or 0.1 U / mL, 0.2 U / mL, 0.3 U / mL, 0.4 U / mL, 0.5 U / mL, 0.6 U / mL, 0.8 U / mL, 1 U / mL, 1.5 U / mL, 2.5 U / mL, etc.

[0099] In some specific implementation schemes, the reaction is carried out under conditions of pH 7.5±0.5 and 30±5℃. The reaction can be terminated after the raw material (substrate) is completely converted, or it can be carried out for at least 0.1h, 0.2h, 0.3h, 0.4h, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, or 8h.

[0100] 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.

[0101] Stereoselectivity of the target product

[0102] Furthermore, the stereoselectivity of the target product (1R,2R)-p-methylsulfonylbenzylserine prepared by the method described above was detected and calculated. The stereoselectivity ee and de values ​​can be calculated using the following formulas:

[0103] <Preparation of (1R,2R)-p-methylsulfonylbenzylserine alcohol products>

[0104] According to some embodiments of the present invention, a product for preparing (1R,2R)-p-methylsulfonylbenzylamine is provided, the product comprising transaminase and transketase.

[0105] In some embodiments, the present invention is not limited to the specifically disclosed transaminase, but can be extended to its 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.

[0106] In some exemplary embodiments, the transaminase is based on the transaminase sequence shown in SEQ ID NO:2, with four mutation sites (F113I, V148F, Q143N, and I146R) introduced to obtain the transaminase mutant TA24_M4. 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_M4 sequence, for example, by introducing two mutation sites (F113I and V148F) based on the transaminase sequence shown in SEQ ID NO:2, i.e., the transaminase mutant TA24_M2 shown in SEQ ID NO:7.

[0107] 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.

[0108] In some exemplary embodiments, the transketase is based on the transaminase sequence shown in SEQ ID NO:1, and six mutation sites, namely I189G, G67H, E468W, T469P, F437W, and I247A, are introduced to obtain the transketase mutant EcTK_M12.

[0109] 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 EcTK_M12.

[0110] 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.

[0111] Example

[0112] 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.

[0113] Example 1: Preparation of transketolase mutant

[0114] (1) Preparation of transketolase mutants

[0115] Mutations were introduced at six sites—I189G, G67H, E468W, T469P, F437W, and I247A—into the transketolase EcTK_M6, resulting in the final mutant EcTK_M12.

[0116] The starting sequence of transketolase EcTK_M6 is:

[0117] The nucleotide sequence of EcTK_M6:

[0118] The specific modification process is as follows: The nucleotide sequence encoding the transketolase EcTK_M6 is ligated into the pET28a vector to construct a recombinant plasmid expressing EcTK_M6. Primer pairs targeting the I189G mutation of EcTK_M6 are designed, and Overlap PCR is performed using the recombinant plasmid expressing EcTK_M6 as a template to mutate the 189th amino acid from isoleucine to glycine.

[0119] 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.

[0120] 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. Finally, extend for 5 min and store the PCR amplification product at 4℃.

[0121] 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.

[0122] 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 at 37°C in a shaker 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 h. The clones that grew were confirmed to be correct by sequencing and were identified as single clones containing the target mutation.

[0123] Construction of the EcTK_M8 mutant: Using the successfully sequenced EcTK_M7 plasmid as a template, PCR amplification was performed using primers targeting the G67H mutation in EcTK_M7. Following the same procedure, glycine at position 67 was mutated to histidine, yielding EcTK_M8. This iterative mutagenesis was continued until a single clone of EcTK_M12 containing six mutation sites (I189G / G67H / E468W / T469P / F437W / I247A) was obtained.

[0124] Single clones of EcTK_M12 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 with a shaker at 220 rpm until OD500. 600 =0.6; Add 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) for induction at 20℃. After induction for 16 h, centrifuge at 8,000 rpm for 10 min to obtain bacterial cells that can efficiently express EcTK_M12. Resuspend the collected bacterial cells in phosphate buffer (50 mM, pH 7.5) and sonicate (280 W, 2 sec on, 2 sec off, 10 min) until the cells are clear, or use a high-pressure cell disruptor to disrupt the cells by pressure. Centrifuge at 10,000 rpm for 30 min; the supernatant is the crude EcTK_M12 enzyme solution.

[0125] 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 EcTK_M12 enzyme.

[0126] The amino acid sequence of EcTK_M12:

[0127] Example 2: Preparation of transaminase mutants

[0128] Mutations were introduced at four sites (F113I, V148F, Q143N, and I146R) on the wild-type transaminase TA24, and the resulting mutant was named TA24_M4.

[0129] The wild-type sequence of transaminase TA24 is:

[0130] The nucleotide sequence of transaminase TA24 is as follows:

[0131] 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 F113I 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 isoleucine.

[0132] 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, the plasmid was extracted and iteratively mutated until a single clone of TA24_M4 containing four mutation sites, F113I / V148F / Q143N / I146R, was prepared.

[0133] Pick a single colony containing TA24_M4 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 sonication followed by a 2 s pause, for a total of 10 min. The lysed mixture is then centrifuged at 4℃ and 12000 rpm for 40 min, and the resulting supernatant is the TA24_M4 cell lysis buffer.

[0134] 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_M4 enzyme.

[0135] The amino acid sequence of TA24_M4:

[0136] Example 3: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using acetone

[0137] In 50 mL of 100 mM phosphate buffer (pH 7.5), the concentrations of p-methylsulfonylbenzaldehyde were 50 mM, acetone 1 mM, D-serine 1 M, transketolase EcTK_M12 0.1 U / mL, transaminase TA24_M4 0.2 U / mL, thiamine pyrophosphate 0.1 mM, magnesium chloride 0.5 mM, and pyridoxal phosphate 0.1 mM. The reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0138] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 82%, and the stereoselectivity of the product was 95% de and >99% ee.

[0139] Example 4: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using sodium pyruvate

[0140] In 50 mL of 100 mM phosphate buffer (pH 7.5), the concentrations of p-methylsulfonylbenzaldehyde were 50 mM, sodium pyruvate 1 mM, D-serine 1 M, transketolase EcTK_M12 0.1 U / mL, transaminase TA24_M4 0.2 U / mL, thiamine pyrophosphate 0.1 mM, magnesium chloride 0.5 mM, and pyridoxal phosphate 0.1 mM. The reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0141] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 83%, and the stereoselectivity of the product was 95% de and >99% ee.

[0142] Example 5: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine using other mutants (TA24_M2)

[0143] Based on the wild-type transaminase TA24, mutations were introduced at two sites, F113I and V148F, and the resulting mutant was named TA24_M2. For the specific preparation method, please refer to Example 2.

[0144] In 50 mL of 100 mM phosphate buffer (pH 7.5), the concentrations of p-methylsulfonylbenzaldehyde were 50 mM, sodium pyruvate 1 mM, D-serine 1 M, transketolase EcTK_M12 0.1 U / mL, transaminase TA24_M2 0.2 U / mL, thiamine pyrophosphate 0.1 mM, magnesium chloride 0.5 mM, and pyridoxal phosphate 0.1 mM. The reaction was carried out at 30 °C for 18 h to catalyze the synthesis of (1R,2R)-p-methylsulfonylbenzaldehyde.

[0145] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 57%, and the stereoselectivity of the product was 88% de and >99% ee.

[0146] The amino acid sequence of TA24_M2:

[0147] The gene sequence of TA24_M2:

[0148] Example 6: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine from 0.2mM thiamine pyrophosphate

[0149] In 50 mL of 100 mM phosphate buffer (pH 7.5), the concentrations of p-methylsulfonylbenzaldehyde were 50 mM, D-serine was 1 M, transketolase EcTK_M12 was 0.1 U / mL, transaminase TA24_M4 was 0.2 U / mL, magnesium chloride was 0.5 mM, pyridoxal phosphate was 0.1 mM, sodium pyruvate was 1 mM, and thiamine pyrophosphate was 0.2 mM. 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 82-83%, and the stereoselectivity of the product was 94-95% de and >99% ee.

[0151] Example 7: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine from 2mM sodium pyruvate

[0152] In 50 mL of 100 mM phosphate buffer (pH 7.5), the concentrations of p-methylsulfonylbenzaldehyde were 50 mM, D-serine was 1 M, transketolase EcTK_M12 was 0.1 U / mL, transaminase TA24_M4 was 0.2 U / mL, magnesium chloride was 0.5 mM, pyridoxal phosphate was 0.1 mM, sodium pyruvate was 2 mM, and thiamine pyrophosphate was 0.1 mM. The reaction was carried out at 30 °C for 18 h to catalyze the reaction of the substrate p-methylsulfonylbenzaldehyde to synthesize (1R,2R)-p-methylsulfonylbenzylserine.

[0153] After the reaction was completed, the reaction solution was collected and analyzed. The yield of the target product (1R,2R)-p-methylsulfonylbenzylamine was 82-83%, and the stereoselectivity of the product was 94-95% de and >99% ee.

[0154] Comparative Example 1: Synthesis of (1R,2R)-p-methylsulfonylbenzylamine without the addition of acetone or sodium pyruvate

[0155] The concentration of p-methylsulfonylbenzaldehyde was 50 mM, without the addition of acetone or sodium pyruvate; the concentration of D-serine was 1 M; the concentration of transketolase EcTK_M12 was 0.1 U / mL; the concentration of transaminase TA24_M4 was 0.2 U / mL; the concentration of thiamine pyrophosphate was 0.1 mM; the concentration of magnesium chloride was 0.5 mM; and the concentration of pyridoxal phosphate was 0.1 mM. The reaction was carried out in 50 mL of 100 mM phosphate buffer (pH 7.5) at 30 °C for 18 h, catalyzing the reaction of the substrate p-methylsulfonylbenzaldehyde to synthesize (1R,2R)-p-methylsulfonylbenzylserine.

[0156] After the reaction was completed, the reaction solution was collected and tested. The amount of byproducts increased, resulting in a yield of 48% for the target product (1R,2R)-p-methylsulfonylbenzylamine. The stereoselectivity of the product was 84% ​​de and >99% ee.

Claims

1. A method for preparing (1R,2R)-p-methylsulfonylbenzylaminoethanol, characterized in that, In a system containing aldehydes and / or ketones, (1R,2R)-p-methylsulfonylbenzaldehyde is synthesized by enzyme catalysis. Preferably, the enzyme catalyst comprises transketase and transaminase; Preferably, the system further comprises an amino donor; 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; Optionally, 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, The method includes the following steps: Step 1: Under the action of transaminase, amino donors and ketones and / or aldehydes are converted into hydroxypyruvate; Step 2: Under the action of transketolase, p-methylsulfonylbenzaldehyde and the hydroxypyruvate obtained in Step 1 are catalyzed to form the compound shown in Formula 1. Step 3: Under the action of transaminase, the compound shown in Formula 1 is catalyzed to (1R,2R)-p-methylsulfonylbenzylamine and hydroxypyruvate; Optionally, the amino donor includes serine, isopropylamine, and α-phenylethylamine; The preferred form is serine; D-serine is preferred; Preferably, the hydroxypyruvate is β-hydroxypyruvate.

3. The method according to claim 1 or 2, characterized in that, The aldehydes and / or ketones are compounds containing a carbonyl group; The aldehyde compounds include aliphatic aldehydes and aromatic aldehydes; optionally, the aldehyde compounds include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and benzaldehyde; The ketone compounds include aliphatic ketones and aromatic ketones; optionally, the ketone compounds include acetone, pyruvate, cyclohexanone and phenylmethyl ketone.

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 enzyme, crude enzyme, or cell lysate; 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 cofactor includes 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. Alternatively, the pH can be maintained at 7.5 ± 0.5 using Tris-HCl or phosphate buffer.

7. A transaminase, characterized in that, The transaminase is used in the method according to any one of claims 1 to 6, wherein 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.

8. A transketolase, characterized in that, The transketolase is used in the method according to any one of claims 1 to 6, 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.

9. A product for preparing (1R,2R)-p-methylsulfonylbenzylaminoethanol, characterized in that, It contains the transaminase as described in claim 7 and the transketase as described in claim 8.

10. The product according to claim 9, characterized in that, The product also contains auxiliary factors; Optionally, the cofactor includes Mg 2+ Thiamine pyrophosphate and pyridoxal phosphate.

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