One-step enzymatic synthesis technology of (2S, 3R)-p-methylsulfonylphenylserine ethyl ester

Abstract

The invention provides a one-step enzymatic synthesis technology of (2S, 3R)-p-methylsulfonyl phenyl serine ethyl ester. Specifically, the invention provides a method for preparing (2S, 3R)-p-methylsulfonyl phenyl serine ethyl ester by a one-step method, and the method comprises the step of obtaining (2S, 3R)-p-methylsulfonyl phenyl serine ethyl ester from p-methylsulfonyl benzaldehyde and L-threonine ethyl ester or a salt thereof under the action of transaldolase in the presence of phosphopyridoxal as a coenzyme.

Description

[0001] This application claims priority to Chinese application No. 202410371510.3, filed on March 28, 2024. The entire text of this priority document is incorporated into this application by reference. Technical Field

[0002] The invention belongs to the field of biopharmaceuticals, and particularly relates to a method for synthesizing (2S, 3R)-p-methylsulfonylphenylserine ethyl ester. Background Art

[0003] Thiamphenicol and its fluorinated derivative, florfenicol, are highly effective broad-spectrum antibiotics against a variety of Gram-positive and Gram-negative bacteria. They have a wide range of applications in clinical medicine and agriculture, and demand for them is increasing annually. Ethyl p-methylsulfonylphenylserine is an important intermediate in the chemical synthesis of thiamphenicol, of which only the (2S,3R)-isomer (L-threo-p-methylsulfonylphenylserine) can be used to prepare biologically active thiamphenicol and its derivatives.

[0004] Currently, there are two methods for synthesizing L-threo-p-methylsulfonylphenylserine ethyl ester: chemical synthesis and enzymatic synthesis. Chemical synthesis primarily uses p-methylsulfonylbenzaldehyde and glycine as raw materials to obtain racemic p-methylsulfonylphenylserine copper salt, which is then subjected to chiral resolution and esterification to obtain L-threo-p-methylsulfonylphenylserine ethyl ester. While the chemical method is mature and widely used in industry, it is subject to significant heavy metal pollution and low resolution yields, leaving limited room for technological advancement and cost reduction. Enzymatic synthesis, on the other hand, eliminates the need for heavy metals and resolution processes, offers mild reaction conditions, and offers significant potential for cost reduction, making it a hot topic of interest in both industry and academia.

[0005] The enzymes used in the currently reported non-split enzymatic synthesis of L-threo-p-methylsulfonylphenylserine ethyl ester (also known as D ester) mainly include aldolase, transaldolase, etc. The reaction catalyzed by transaldolase uses p-methylsulfonylbenzaldehyde and L-threonine as main raw materials. Since the substrate molecule has introduced a chiral carbon atom, the reaction product L-threo-p-methylsulfonylphenylserine has higher ee and de values, and the corresponding industrial application prospects are also better. However, the acetaldehyde produced by the reaction has a very large inhibitory effect on the activity of transaldolase, which causes the reaction to fail to proceed smoothly. In order to remove the inhibitory effect of acetaldehyde, one method is to use alcohol dehydrogenase to convert acetaldehyde into ethanol with relatively less toxicity. The reaction requires NADH as a coenzyme. In order to reduce the amount of coenzyme used and save costs, coenzyme regeneration systems such as glucose dehydrogenase and formate dehydrogenase are usually introduced (see Figure 1 ).

[0006] Another method is to use oxidase to oxidize acetaldehyde into acetic acid, and the hydrogen peroxide produced by the reaction is decomposed into oxygen and water by catalase, ultimately achieving the efficient biosynthesis of L-threo-p-methylsulfonylphenylserine.

[0007] However, the direct products of the above enzymatic synthesis are all L-threo-p-methylsulfonylphenylserine, which needs to be converted into magnesium salt after the reaction, extracted and then subjected to esterification reaction to obtain the required (2S,3R)-p-methylsulfonylphenylserine ethyl ester product. This makes the process more complicated and tedious, and increases the generation of three wastes. Summary of the Invention

[0008] In response to the defects in the above-mentioned prior art, the present invention provides a one-step enzymatic synthesis technology for (2S, 3R)-p-methylsulfonylphenylserine ethyl ester. The method can obtain p-methylsulfonylphenylserine ethyl ester in one step without the need for subsequent separation. At the same time, the reaction process has the advantages of mildness and high selectivity of enzymatic synthesis, and avoids the need for the enzymatic reaction product to further form a magnesium salt and undergo ethyl esterification, thereby reducing the corresponding costs and the generation of three wastes, and has great process advantages and potential.

[0009] In a first aspect, the present invention provides a method for preparing 3-phenyl-p-methylsulfonylphenylserine ethyl ester, comprising reacting p-methylsulfonylbenzaldehyde and L-threonine ethyl ester or its salt with transaldolase to obtain (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0010] The reaction formula for preparing (2S, 3R)-p-methylsulfonylphenylserine ethyl ester according to the present invention is shown in Formula I:

[0011]

[0012] In formula I, p-methylsulfonylbenzaldehyde and L-threonine ethyl ester directly react under the catalysis of transaldolase to generate (2S, 3R)-p-methylsulfonylphenylserine ethyl ester and acetaldehyde.

[0013] In some embodiments, the enzymatic reaction process of the method of the present invention requires the addition of a coenzyme. In some specific embodiments, the enzymatic reaction process of the method of the present invention requires pyridoxal phosphate (PLP) as a coenzyme.

[0014] In a preferred embodiment, the method of the present invention comprises reacting p-methylsulfonylbenzaldehyde with L-threonine ethyl ester in the form of a salt under the action of transaldolase to obtain (2S,3R)-p-methylsulfonylphenylserine ethyl ester. In a specific embodiment, the method of the present invention comprises reacting p-methylsulfonylbenzaldehyde with L-threonine ethyl ester hydrochloride under the action of transaldolase to obtain (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0015] In a preferred embodiment, the transaldolase used in the method of the present invention is L-threonine transaldolase.

[0016] In some embodiments, the transaldolase used in the methods of the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0017] In some embodiments, the transaldolase used in the method of the present invention consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0018] In a more preferred embodiment, the transaldolase used in the method of the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a more preferred embodiment, the transaldolase used in the method of the present invention consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0019] In a specific embodiment, the transaldolase comprises the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4. In a specific embodiment, the transaldolase consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4.

[0020] In a specific preferred embodiment, the transaldolase comprises the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the transaldolase consists of the amino acid sequence of SEQ ID NO: 1.

[0021] In some embodiments, the transaldolase used in the method of the present invention can be a free enzyme. In some embodiments, the transaldolase used in the method of the present invention can be an immobilized enzyme. In some preferred embodiments, the transaldolase is fixed on a resin. In a more preferred embodiment, the transaldolase is fixed on a Ni-IDA (iminodiacetic acid) resin.

[0022] In some embodiments, the enzyme reaction process in the method of the present invention is carried out at 10°C-40°C. In some preferred embodiments, the enzyme reaction process in the method of the present invention is carried out at 15°C-40°C. In some preferred embodiments, the enzyme reaction process in the method of the present invention is carried out at 20°C-40°C. In some preferred embodiments, the enzyme reaction process in the method of the present invention is carried out at 20°C-37°C. In some preferred embodiments, the enzyme reaction process in the method of the present invention is carried out at 25°C-37°C. In some preferred embodiments, the enzyme reaction process in the method of the present invention is carried out at 30°C-37°C.

[0023] In some embodiments, the method of the present invention further comprises purifying the obtained p-methylsulfonylphenylserine ethyl ester. In some embodiments, the purification of the obtained p-methylsulfonylphenylserine ethyl ester comprises the following steps:

[0024] a. The reaction solution was filtered to remove the immobilized resin, the remaining substrate and / or the precipitated (2S, 3R)-p-methylsulfonylphenylserine ester;

[0025] b. Take the supernatant, adjust the pH to 5.0-9.0, and add the organic phase for extraction;

[0026] c. The organic phase obtained in step b is adjusted (preferably with HCl) to a pH of 1.0-4.0, preferably 3.0, to obtain a lower solution;

[0027] d. The pH of the obtained lower solution is adjusted to 8-9 to precipitate (2S, 3R)-methylsulfonylphenylserine ethyl ester, preferably at -20 ° C.

[0028] In some embodiments, in step b of the purification, the pH of the supernatant is adjusted to 5.0-9.0 using hydrochloric acid.

[0029] In some embodiments, in the purification step b, the pH of the supernatant is adjusted to 6.0-8.0. In some embodiments, in the purification step b, the pH of the supernatant is adjusted to 6.5-7.5. In some embodiments, in the purification step b, the pH of the supernatant is adjusted to 6.6-7.4. In some embodiments, in the purification step b, the pH of the supernatant is adjusted to 6.7-7.3. In some embodiments, in the purification step b, the pH of the supernatant is adjusted to 6.8-7.2. In some embodiments, in the purification step b, the pH of the supernatant is adjusted to 6.9-7.1. In a specific embodiment, in the purification step, the pH of the supernatant is adjusted to 7.0.

[0030] In some embodiments, in the purification step b, after adjusting the supernatant to 5.0-9.0, the supernatant is evaporated before adding the organic phase for extraction. In a specific embodiment, the supernatant is subjected to rotary evaporation.

[0031] In some embodiments, in the purification step b, the organic phase used for extraction is butyl acetate or ethyl acetate. In a preferred embodiment, in the purification step b, the organic phase used for extraction is ethyl acetate.

[0032] In some embodiments, in the purification step b, after extraction with the organic phase, the obtained organic phase is evaporated. In some embodiments, rotary evaporation is used to evaporate the organic phase.

[0033] In some embodiments, in the purification step c, the pH of the organic phase layer obtained in step b is adjusted to 1.0-5.0 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 1.5-4.5 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 2-4 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 2.5-3.5 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 2.6-3.4 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 2.7-3.3 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the reaction solution is adjusted to 2.8-3.2 after the organic phase is evaporated to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 2.9-3.1 to obtain a lower layer solution. In some embodiments, in the purification step c, the pH of the organic phase obtained in step b is adjusted to 3.0 to obtain a lower layer solution.

[0034] In some embodiments, in the purification step d, the pH of the obtained lower layer solution is adjusted to 8.0-9.5, and (2S,3R)-p-methylsulfonylphenylserine ethyl ester is precipitated. In some embodiments, in the purification step d, the pH of the obtained lower layer solution is adjusted to 8.0-9.4, and (2S,3R)-p-methylsulfonylphenylserine ethyl ester is precipitated. In some embodiments, in the purification step d, the pH of the obtained lower layer solution is adjusted to 8.0-9.3, and (2S,3R)-p-methylsulfonylphenylserine ethyl ester is precipitated. In some embodiments, in the purification step d, the pH of the obtained lower layer solution is adjusted to 8.0-9.2, and (2S,3R)-p-methylsulfonylphenylserine ethyl ester is precipitated. In some embodiments, in the purification step d, the pH of the obtained lower layer solution is adjusted to 8.0-9.1, and (2S,3R)-p-methylsulfonylphenylserine ethyl ester is precipitated. In some embodiments, in the purification step d, the pH of the obtained lower layer solution is adjusted to 8.0-9.0 to precipitate (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0035] In some embodiments, in the purification step d, (2S,3R)-p-methylsulfonylphenylserine ethyl ester is precipitated at -20°C.

[0036] In a second aspect, the present invention provides the use of L-threonine aldolase in the synthesis of florfenicol chiral intermediates, characterized in that L-threonine aldolase asymmetrically catalyzes the reaction of synthesizing (2S, 3R)-p-methylsulfonylphenylserine ethyl ester, which comprises reacting p-methylsulfonylbenzaldehyde and L-threonine ethyl ester or a salt thereof under the action of L-threonine aldolase to obtain (2S, 3R)-p-methylsulfonylphenylserine ethyl ester.

[0037] In some embodiments, the enzymatic reaction process of the method of the present invention requires the addition of a coenzyme. In some specific embodiments, the enzymatic reaction process of the method of the present invention requires pyridoxal phosphate (PLP) as a coenzyme.

[0038] In a preferred embodiment, the method of the present invention comprises reacting p-methylsulfonylbenzaldehyde with L-threonine ethyl ester in the form of a salt under the action of transaldolase to obtain (2S,3R)-p-methylsulfonylphenylserine ethyl ester. In a specific embodiment, the method of the present invention comprises reacting p-methylsulfonylbenzaldehyde with L-threonine ethyl ester hydrochloride under the action of transaldolase to obtain (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0039] In some embodiments, the L-threonine transaldolase used in the method of the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0040] In some embodiments, the L-threonine transaldolase used in the method of the present invention consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0041] In a more preferred embodiment, the L-threonine transaldolase used in the method of the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a more preferred embodiment, the L-threonine transaldolase used in the method of the present invention consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0042] In a specific embodiment, the transaldolase comprises the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4. In a specific embodiment, the transaldolase consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4.

[0043] In a specific preferred embodiment, the transaldolase comprises the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the transaldolase consists of the amino acid sequence of SEQ ID NO: 1.

[0044] In some embodiments, the said L-threonine transaldolase that uses in the method for the present invention can be a free enzyme. In some embodiments, the said transaldolase that uses in the method for the present invention can be an immobilized enzyme. In some preferred embodiments, said transaldolase is fixed on the resin. In a more preferred embodiment, said transaldolase is fixed on the Ni-IDA (iminodiacetic acid) resin. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a route for preparing L-threo-p-methylsulfonylphenylserine and its esters using a coenzyme regeneration system.

[0046] Figure 2 is Mass spectrometry results of the purified (2S,3R)-methylsulfonylphenylserine ethyl ester product.

[0047] Figure 3This is a comparison of the H NMR spectra of the purified (2S,3R)-p-methylsulfonylphenylserine ethyl ester and the standard (2S,3R)-p-methylsulfonylphenylserine ethyl ester. Figure 3 A is the hydrogen spectrum of (2S, 3R)-p-methylsulfonylphenylserine ethyl ester prepared and purified by the present invention. Figure 3 B is the hydrogen spectrum of the standard substance (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0048] Figure 4 This is a comparison of the NMR carbon spectra of the purified (2S,3R)-p-methylsulfonylphenylserine ethyl ester and the standard (2S,3R)-p-methylsulfonylphenylserine ethyl ester. Figure 4 A is the carbon spectrum of (2S, 3R)-p-methylsulfonylphenylserine ethyl ester prepared and purified by the present invention. Figure 4 B is the carbon spectrum of the standard substance (2S,3R)-p-methylsulfonylphenylserine ethyl ester. DETAILED DESCRIPTION

[0049] The present invention will be further described in detail below with reference to specific examples, but the present invention is not limited to the following examples.

[0050] definition

[0051] In order to make the present disclosure more easily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0052] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" includes a plurality of vectors, and the like.

[0053] Unless otherwise stated or defined, the term “comprise” and variations such as “include” and “comprising” will be understood to imply the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps.

[0054] L-Threonine transaldolase (LTTA) is a specific serine hydroxymethyltransferase (SHMT) that catalyzes the trans-aldehyde reaction of L-threonine and aldehydes to produce L-threo-β-hydroxy-α-amino acids with excellent stereoselectivity.

[0055] Immobilized enzyme refers to a water-soluble enzyme that has been treated by physical or chemical methods to make it insoluble in water, but still has enzymatic activity and can be used repeatedly and continuously.

[0056] Free enzyme refers to a water-soluble enzyme extracted from microbial cells that reacts with the substrate only once and is not fixed by a carrier.

[0057] 1 U / ml is the amount of enzyme that catalyzes the production of 1 micromole of D-ester in 1 minute.

[0058] As used herein, the term "sequence identity" refers to the degree to which two sequences (amino acids) have identical residues at the same position after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the percent identity of the amino acid sequence to SEQ ID NO: Y and is stated as X% of the residues in the amino acid sequence being identical to the residues in the sequence disclosed in SEQ ID NO: Y. Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0059] In addition, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of a similar chemical structure, which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example WO 04 / 037999, GB-A-2 357 768, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and (preferably) the type and / or combination of these substitutions can be selected according to the relevant teachings from WO 04 / 037999 and WO 98 / 49185 and the further references cited therein.

[0060] Such conservative substitutions are preferably substitutions in which an amino acid from the following groups (a) to (e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0061] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0062] Transaldolase

[0063] In this article, the transaldolase is an L-threonine transaldolase. In some embodiments, the L-threonine transaldolase is derived from a prokaryotic organism. In some embodiments, the L-threonine transaldolase is from Pseudomonas or Chitinophilus. In some specific embodiments, the L-threonine transaldolase is from Pseudomonas fluorescens, Pseudomonas strain 34E7 (Pseudomonas sp.34E 7), Pseudomonas strain (Pseudomonas sp.Irchel s3a18) or Chitiniphilus shinanonensis. In some embodiments, the L-threonine transaldolase is an engineered L-threonine transaldolase.

[0064] In some embodiments, the L-threonine transaldolase used in the method of the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0065] In some embodiments, the L-threonine transaldolase used in the method of the present invention consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

[0066] In a more preferred embodiment, the L-threonine transaldolase used in the method of the present invention comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a more preferred embodiment, the L-threonine transaldolase used in the method of the present invention consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0067] In a specific embodiment, the transaldolase comprises the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4. In a specific embodiment, the transaldolase consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4.

[0068] In a specific preferred embodiment, the transaldolase comprises the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the transaldolase consists of the amino acid sequence of SEQ ID NO: 1.

[0069] Reduce the inhibitory effect of acetaldehyde

[0070] The generated acetaldehyde has a significant inhibitory effect on the reaction and can be removed using methods known in the art to reduce the inhibition, accelerate the reaction process or improve the reaction efficiency.

[0071] In order to relieve the inhibitory effect of acetaldehyde, one method is to use alcohol dehydrogenase to convert acetaldehyde into ethanol, which is relatively less toxic. This reaction requires NADH as a coenzyme. In order to reduce the amount of coenzyme and save costs, coenzyme regeneration systems such as glucose dehydrogenase and formate dehydrogenase are usually introduced (see Figure 1 ).

[0072] Another method is to use oxidase to oxidize acetaldehyde into acetic acid, and the hydrogen peroxide produced by the reaction is decomposed into oxygen and water by catalase, ultimately achieving the efficient biosynthesis of L-threo-p-methylsulfonylphenylserine.

[0073] Purification of (2S,3R)-p-Methylsulfonylphenylserine Ethyl Ester

[0074] The (2S,3R)-p-methylsulfonylphenylserine ethyl ester produced by the one-step enzymatic method of the present invention has the advantage of low solubility and can be precipitated during the reaction. By optimizing the reaction conditions, a large amount of (2S,3R)-p-methylsulfonylphenylserine ethyl ester is produced during the reaction, and precipitation of (2S,3R)-p-methylsulfonylphenylserine ethyl ester can be achieved during the reaction, thereby obtaining precipitated (2S,3R)-p-methylsulfonylphenylserine ethyl ester. This avoids the need for subsequent purification steps.

[0075] If the reaction produces less (2S,3R)-p-methylsulfonylphenylserine ethyl ester, the ester can be separated and purified using various techniques, such as precipitation separation or extraction and back extraction, by taking advantage of its varying lipid and water solubility at different pH values. In contrast, (2S,3R)-p-methylsulfonylphenylserine is too water-soluble to be directly extracted, requiring separation and extraction in the form of a magnesium salt, which must then be removed during the subsequent ethylation reaction, increasing the complexity of the process and the difficulty of treating the three wastes.

[0076] Example

[0077] Example 1. Reaction of the free enzyme of L-threonine transaldolase in patent CN110951799A

[0078] To a clean reaction flask, add 1.5 g of p-methylsulfonylbenzaldehyde, 2 g of L-threonine ethyl ester hydrochloride, 0.05 g of pyridoxal phosphate, 10 mL of L-threonine transaldolase (amino acid sequence of SEQ ID NO: 1) (enzyme activity 83.1 U / mL), and 40 mL of ddH2O, for a total volume of 50 mL. The reaction was adjusted to pH 7.0 and the temperature was maintained at 35°C for 22 h. High-performance liquid chromatography (HPLC) determined the concentration of the generated (2S,3R)-p-methylsulfonylphenylserine ethyl ester to be 8.4 g / L. HPLC conditions were: detection wavelength 220 nm, mobile phase acetonitrile:0.1% phosphoric acid (50:50), column temperature 35°C, flow rate 1 mL / min, injection volume 5 mL, column type: Hedera NH2, column length 4.6 × 200 mm.

[0079] Example 2. Reaction of free transaldolase LF140 enzyme

[0080] To a clean reaction flask, add 3 g of p-methylsulfonylbenzaldehyde, 3.5 g of L-threonine ethyl ester hydrochloride, 0.05 g of pyridoxal phosphate, 10 mL of crude transaldolase LF145 (from Taizhou Lingfeng Biotechnology Co., Ltd.) (enzyme activity 67.8 U / mL), and 40 mL of ddH2O, for a total volume of 50 mL. 15 mL of ddH2O was then added, and the pH of the reaction system was adjusted to 7.0. The reaction temperature was controlled at 35°C and the reaction was incubated for 23 h. The concentration of the generated (2S,3R)-p-methylsulfonylphenylserine ethyl ester was 2.8 g / L as determined by high-performance liquid chromatography. HPLC conditions were: detection wavelength 220 nm, mobile phase acetonitrile:0.1% phosphoric acid (50:50), column temperature 35°C, flow rate 1 mL / min, injection volume 5 mL, column type: Hedera NH2, column length 4.6 × 200 mm.

[0081] Example 3. Reaction of free transaldolase LF145 enzyme

[0082] To a clean reaction flask, add 1.5 g of p-methylsulfonylbenzaldehyde, 2 g of L-threonine ethyl ester hydrochloride, 0.05 g of pyridoxal phosphate, 10 mL of crude LF140 enzyme (from Taizhou Lingfeng Biotechnology Co., Ltd.) (enzyme activity 64.3 U / mL), and 40 mL of ddH2O, for a total volume of 50 mL. The reaction was adjusted to pH 7.0 and maintained at 35°C for 21 h. High-performance liquid chromatography (HPLC) determined the concentration of the generated (2S,3R)-p-methylsulfonylphenylserine ethyl ester to be 6.2 g / L. HPLC conditions were: detection wavelength 220 nm, mobile phase acetonitrile:0.1% phosphoric acid (50:50), column temperature 35°C, flow rate 1 mL / min, injection volume 5 mL, column type: Hedera NH2, column length 4.6 × 200 mm.

[0083] Example 4. Reaction of free transaldolase LF160 enzyme

[0084] To a clean reaction flask, add 1 g of p-methylsulfonylbenzaldehyde, 2 g of L-threonine ethyl ester hydrochloride, 0.05 g of pyridoxal phosphate, 15 mL of crude transaldolase LF160 (from Taizhou Lingfeng Biotechnology Co., Ltd.) (LF160 crude enzyme activity: 71.6 U / mL), and 35 mL of ddH2O, for a total volume of 50 mL. Adjust the reaction pH to 7.0 and maintain the reaction temperature at 35°C for 24 h. High-performance liquid chromatography (HPLC) determined the concentration of the generated (2S,3R)-p-methylsulfonylphenylserine ethyl ester to be 6.3 g / L. HPLC conditions were: detection wavelength 220 nm, mobile phase acetonitrile:0.1% phosphoric acid (50:50), column temperature 35°C, flow rate 1 mL / min, injection volume 5 mL, column type: Hedera NH2, column length 4.6 × 200 mm.

[0085] Example 5. Scale-up reaction of free transaldolase LF160 enzyme

[0086] To a clean reaction flask, add 6 g of p-methylsulfonylbenzaldehyde, 10 g of L-threonine ethyl ester hydrochloride, 0.2 g of pyridoxal phosphate, 15 mL of crude LF160 enzyme (LF160 crude enzyme activity 71.6 U / mL), and 180 mL of ddH2O, for a total volume of 200 mL. Adjust the reaction pH to 7.0 and maintain the reaction temperature at 35°C for 21 hours. High-performance liquid chromatography (HPLC) determined the concentration of the generated (2S,3R)-p-methylsulfonylphenylserine ethyl ester to be 6.7 g / L. HPLC conditions were: detection wavelength 220 nm, mobile phase acetonitrile:0.1% phosphoric acid (50:50), column temperature 35°C, flow rate 1 mL / min, injection volume 5 mL, column type: HederaNH2, column length 4.6 × 200 mm.

[0087] The reaction solution was adjusted to pH 3.0 with HCl, and the supernatant was collected by centrifugation and concentrated to 25 mL in a rotary evaporator. The solution was allowed to stand at 4°C for 4 h and then filtered. The filtrate was adjusted to pH 8-9 with NaOH, allowed to stand at 4°C overnight, and the precipitate was collected by centrifugation to obtain a crude product.

[0088] Example 6. Reaction of immobilized transaldolase LF160 enzyme

[0089] To a clean reaction flask, add 5 g of p-methylsulfonylbenzaldehyde, 10 g of L-threonine ethyl ester hydrochloride, 0.2 g of pyridoxal phosphate, 10 g of Ni-IDA immobilized transaldolase LF160 (IDA resin purchased from Gaoling Lanxiao Technology New Materials Co., Ltd., catalog number LXTE-800) (immobilized enzyme activity 107.7 U / g), and 180 mL of ddH2O, for a total volume of 200 mL. Adjust the pH of the reaction system to 7.0 and maintain the reaction temperature at 35°C for 18.5 hours. High-performance liquid chromatography (HPLC) analysis revealed a concentration of 7.1 g / L for the resulting (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0090] Example 7. Determination of conversion rate of LF160 immobilized enzyme reaction

[0091] To a 50 mL centrifuge tube, add 50 mg of p-methylsulfonylbenzaldehyde, 15 mg of L-threonine ethyl ester hydrochloride, 10 mg of pyridoxal phosphate, and 50 mg of immobilized transaldolase LF160 Ni-IDA (IDA resin purchased from Gaoling Lanxiao Technology New Materials Co., Ltd., catalog number LXTE-800) (immobilized enzyme activity: 107.7 U / g). Add ddH2O to 10 mL of the solution, adjust the pH of the reaction system to 7.0, and maintain the reaction temperature at 35°C for 225 min. High-performance liquid chromatography (HPLC) analysis revealed a concentration of 1.89 g / L for the produced (2S,3R)-p-methylsulfonylphenylserine ethyl ester, yielding 80.69% of the substrate L-threonine ethyl ester.

[0092] Example 8. Product purification

[0093] 188 mL of the immobilized enzyme reaction solution was filtered to remove the immobilized resin, residual substrate, and a small amount of precipitated (2S,3R)-p-methylsulfonylphenylserine ethyl ester. The supernatant was adjusted to a neutral pH of 7.0 with HCl and placed in a rotary evaporator. The volume was reduced to 25 mL using a rotary evaporator. 160 mL of ethyl acetate was added and extracted under neutral conditions to obtain the organic phase. The organic phase was further rotary evaporated to a remaining 8 mL. A small amount of ddH2O was added, and the pH was adjusted to 3.0 with HCl. After thorough vortexing, the lower layer was removed and the pH was adjusted to 8-9 with NaOH. HPLC analysis of the (2S,3R)-p-methylsulfonylphenylserine ethyl ester concentration was 230.34 g / L. The solution was placed in a -20°C refrigerator to allow precipitation. HPLC analysis of the precipitated material revealed a peak area ratio of 94.4%. The solution was then dried in an oven at 45°C. After drying, HPLC analysis revealed a chromatographic purity of 97.48%. The mass spectrometry analysis result of the product showed a molecular weight of 287.33. Figure 2 Mass spectrometry results.

[0094] The product was subjected to NMR analysis. Figure 3 A and Figure 3B shows the comparison of the H NMR spectrum of the purified product and the standard (2S,3R)-methylsulfonylphenylserine ethyl ester. Figure 4 A and Figure 4 B shows a comparison of the NMR carbon spectra of the purified product and the standard (2S,3R)-p-methylsulfonylphenylserine ethyl ester. The results show that the obtained product is (2S,3R)-p-methylsulfonylphenylserine ethyl ester.

[0095] The above examples are merely illustrative of the fact that, through this technical solution, L-threonine ethyl ester and (2S,3R)-p-methylsulfonylbenzaldehyde can be directly enzymatically reacted to produce (2S,3R)-p-methylsulfonylphenylserine ethyl ester, which can then be further extracted and purified to obtain the (2S,3R)-p-methylsulfonylphenylserine ethyl ester product. These examples are not intended to limit the methods of increasing the yield and production of (2S,3R)-p-methylsulfonylphenylserine ethyl ester by methods well known in the art, such as adjusting the product ratio, adjusting the reaction temperature and pH, and modifying the enzyme structure to enhance the catalytic activity of the enzyme.

[0096] sequence

[0097] >SEQ ID.1

[0098] MSNVKQQTAQIVDWLSSTLGKDHQYREDSLSLTANENYPSALVRLTSGSTAGAFYHCSFPFEVPAGEWHFPEPGHMNAIADQVRDLGKTLIGAQAFDWRPNGGSTAEQAL MLAACKPGEGFVHFAHRDGGHFALESLAQKMGIEIFHLPVNPTSLLIDVAKLDEMVRRNPHIRIVILDQSFKLRWQPLAEIRSVLPDSCTTLTYDMSHDGGLIMGGVFDSP LSCGADIVHGNTHKTIPGPQKGYIGFKSAQHPLLVDTSLWVCPHLQSNCHAEQLPPMWVAFKEMELFGRDYAAQIVSNAKTLARHLHELGLDVTGESFGFTQTHQVHFAV GDLQKALDLCVNSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMKEKDFEVVARFIADLYFKKTEPAKVAQQIKEFLQAFPLAPLAYSFDNYLDEELLAAVYQGAQR

[0099] >SEQ ID.2

[0100] MSNVKQQTAQIVDWLSSTLGKDHQYREDSLSLTANENYPSALVRLTSGSTAGAFYHCSFPFEVPAGEWHFPEPGHMNAIADQVRDLGKTLIGAQAFDWRPNGGSTAEQALMLAACKPGEGFVHFAHRDGGHFALESLAQKMGIEIFHLPVNPISLLIDVAKLDEMVRRNPHIRIVILDQSFKLRWQPLAEIRSVLPDSCTLTYDMSHDGGLIMGGVFDSPLSCGADIVHGNTHKTIPGPQKGYIGFKSAQHPLLVDTSLWVCPHLQSNCHAEQLPPMWVAFKEMELFGRDYAAQIVSNAKTLARHLHELGLDVTGESFGFTQTHQVHFAVGDLQKALDLCVNSLHAGGIRSTNIEIPGKPGVHGIRLGVQAMTRRGMKEKDFEVVARFIADLYFKKTEPAKVAQQIKEFLQAFPLAPLAYSFDNYLDDELLAAVYQGAQR

[0101] >SEQ ID.3

[0102] MKQDESNVGPVIDWLAQTLGQDYKYRQDTLSLTANENYPSELVRLTSGSTAGAFYHCSFPFPVPLGEWHFPEPGQMNEIADDLRGLAKRMMGAQAFDWRPNGGSPAEQALMLAACKQGEGFVHFAHRDGGHFALEQLATKMGIEIFHLPVDPQSLLIDVAKLDDMVRRNPHIRIVILDQSFKLRWQPLAEIRAILPDSCTLTYDMSHDGGLILGGVFDSPLACGADIAHGNTHKTIPGPQKGFIAFKSAQHPLLVETSLWVCPHLQSNCHAELLPSMWAAFKEMEAFGPAYAHQMVRNAKALANQLHELGLNVSGESFGFTETHQVHFAVGDLQQALSMCVDSLHAGGIRSTNIEIPGKPGMHGIRLGVQAMTRRGMKEDDFRRVAGLIADLYFKRTEPARVASKVKELLGDFPLAPLAYSFDQQIDESRRRLLERGIQR

[0103] >SEQ ID.4

[0104] MTRTTPQARHVVERLNSVLGQDYRYREDCLSLTANENYPSALVRLAGSATAGAFYHCSFPFEVPPGEWYFPESGRMGELAQQLNELGRSLLGAGTFDWRPNGGSPAEQALMLAACKHGEGMVHFAHRDGGHFALENLAQKAGIDIFHLPVDPQTLLIDVARLDELVRRNPQIRIVILDQSFKLRWQPLAAIRKVLPPSCTLTYDTSHDGGLIMGGVFDSPLHCGADVIHGNTHKTVPGPQKGYIAFKSAEHPLLVDTSLWLCPHLQSNCHAELLPPMWVAFKEMEAFGHDYAPQVARNAKALAGHLHRLGFEVSGEAFGFTETHQVHFAVGDLQQALDLCMNTLHRGGIRSTNIEIPGKPGIQGIRLGVQAMTRRGLREDDFEQVARFIADLHFRKADPAGVAAQVAEFLRAFPLAPLHYSFDQELDHELLQSLIGEALR。

Claims

1. A method for preparing (2S, 3R)-p-methylsulfonylphenylserine ethyl ester, comprising reacting p-methylsulfonylbenzaldehyde and L-threonine ethyl ester or a salt thereof with pyridoxal phosphate as a coenzyme under the action of transaldolase to obtain (2S, 3R)-p-methylsulfonylphenylserine ethyl ester.

2. The method of claim 1, wherein the salt of L-threonine ethyl ester is in the form of L-threonine ethyl ester hydrochloride.

3. The method according to claim 1 or 2, wherein the transaldolase is L-threonine transaldolase.

4. The method of claim 3, wherein the transaldolase comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4. 5 . The method according to claim 4 , wherein the transaldolase comprises the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4, preferably, the transaldolase consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO:

4.

6. The method according to any one of claims 1 to 5, wherein the transaldolase is a free enzyme.

7. The method according to any one of claims 1 to 5, wherein the transaldolase is an immobilized enzyme, preferably, the transaldolase is immobilized on a resin, more preferably, the transaldolase is immobilized on a Ni-IDA (iminodiacetic acid) resin.

8. The method according to any one of claims 1 to 7, wherein the enzyme reaction process is carried out at 10°C to 40°C, preferably 30°C to 37°C.

9. The method according to any one of claims 1 to 8, further comprising purifying the obtained p-methylsulfonylphenylserine ethyl ester.

10. Use of L-threonine transaldolase in the synthesis of florfenicol chiral intermediates, characterized in that: L-threonine aldolase asymmetric catalyzes the reaction for synthesizing (2S,3R)-p-methylsulfonylphenylserine ethyl ester, which comprises the steps of: using p-methylsulfonylbenzaldehyde and L-threonine ethyl ester or its salt in the presence of pyridoxal phosphate as a coenzyme to obtain (2S,3R)-p-methylsulfonylphenylserine ethyl ester under the action of L-threonine aldolase.

11. The method according to claim 10, wherein the salt of L-threonine ethyl ester is in the form of L-threonine ethyl ester hydrochloride.

12. The use of claim 10 or 11, wherein the transaldolase comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1-4.

13. The use according to claim 12, wherein the transaldolase comprises the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 4, preferably, the transaldolase consists of the amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO:

4.

14. The use according to any one of claims 10 to 13, wherein the enzyme reaction process is carried out at 20°C to 40°C, preferably 30°C to 37°C.

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