Reductase mutants for synthesis of dapoxetine intermediates and use thereof

Abstract

The application relates to the field of biotechnology, in particular to a reductase mutant for synthesizing a dapoxetine intermediate and application, the amino acid sequence of the reductase mutant for synthesizing the dapoxetine intermediate is shown in SEQ ID NO: 13-SEQ ID NO: 15, the stereoselectivity of asymmetric reduction catalysis of the reductase mutant for a dapoxetine intermediate precursor is improved, the R-type dapoxetine intermediate with high chiral purity is obtained, and the synthesis efficiency of the dapoxetine intermediate is improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a reductase mutant for the synthesis of dapoxetine intermediates and its applications. Background Technology

[0002] Dapoxetine hydrochloride, whose scientific name is (+)-(S)-N,N-dimethyl-(α)-[2-(1-naphthoxy)ethyl]benzylamine hydrochloride, has the following chemical structural formula: .

[0003] Dapoxetine hydrochloride was initially used to treat major depressive disorder (MDD), but it was later discovered that the compound could be used to treat premature ejaculation (PE). A key feature of dapoxetine hydrochloride is its short-acting, on-demand pharmacokinetic properties, making it more suitable for targeted treatment of PE than for long-term antidepressant therapy.

[0004] Currently, more and more drugs or intermediates can be synthesized through biocatalysis. (R)-(+)-3-chloro-1-phenyl-1-propanol contains the structural unit of dapoxetine hydrochloride and is a key intermediate in the synthesis of dapoxetine hydrochloride. (R)-(+)-3-chloro-1-phenyl-1-propanol can be obtained by reducing 3-chlorophenylacetone.

[0005] 3-Chlorophenylacetone is a prochiral ketone compound, and (R)-(+)-3-chloro-1-phenyl-1-propanol is an R-type chiral hydroxyl compound. In the background art, the reductase used to synthesize dapoxetine intermediates exhibits low stereoselectivity and catalytic activity in the asymmetric reductive hydrogenation catalysis of the dapoxetine intermediate precursor (3-chlorophenylacetone), which is detrimental to improving the synthesis efficiency of dapoxetine intermediate ((R)-(+)-3-chloro-1-phenyl-1-propanol). Summary of the Invention

[0006] In view of the above problems, this application provides a reductase mutant for synthesizing dapoxetine intermediates and its application, so as to solve the above-mentioned technical problems that are not conducive to improving the synthesis efficiency of dapoxetine intermediates.

[0007] In a first aspect, embodiments of this application provide a reductase mutant for synthesizing dapoxetine intermediates, the amino acid sequence of which is shown in SEQ ID NO: 13 to SEQ ID NO: 15.

[0008] Secondly, embodiments of this application provide a gene for synthesizing dapoxetine intermediates, wherein the gene for synthesizing dapoxetine intermediates encodes the aforementioned reductase mutant for synthesizing dapoxetine intermediates.

[0009] Thirdly, embodiments of this application provide a recombinant vector comprising the aforementioned gene for synthesizing dapoxetine intermediates.

[0010] Fourthly, embodiments of this application provide a recombinant engineered bacterium, which includes the recombinant vector described above.

[0011] Optionally, the recombinant engineered bacteria is *Escherichia coli*, and the *Escherichia coli* is... E. coli BL21(DE3).

[0012] Fifthly, embodiments of this application provide a synthetic system for synthesizing dapoxetine intermediates, the synthetic system comprising recombinant engineered bacteria and isopropanol, wherein the recombinant engineered bacteria is the aforementioned recombinant engineered bacteria.

[0013] Optionally, the volume ratio of isopropanol to the reaction system is 40% to 100%.

[0014] Optionally, the reaction system may further include a buffer solution with a pH value of 5.0 to 7.0.

[0015] Sixthly, embodiments of this application provide a method for the biological preparation of a dapoxetine intermediate, using a dapoxetine intermediate precursor as a substrate, adding the aforementioned recombinant engineered bacteria, and reacting in a reaction system to obtain the dapoxetine intermediate product.

[0016] Seventhly, the embodiments of this application provide the use of the above-mentioned reductase mutant for synthesizing dapoxetine intermediates, or the above-mentioned gene for synthesizing dapoxetine intermediates, or the above-mentioned recombinant vector, or the above-mentioned recombinant engineered bacteria in the preparation of dapoxetine intermediates or in the preparation of dapoxetine.

[0017] The reductase mutant for synthesizing dapoxetine intermediates and its application provided in this application are shown in the embodiments of the present application. The amino acid sequence of the reductase mutant for synthesizing dapoxetine intermediates is shown in SEQ ID NO: 13 to SEQ ID NO: 15. It improves the stereoselectivity of asymmetric reduction catalysis of dapoxetine intermediate precursors, and obtains R-type dapoxetine intermediates with high chiral purity, which is beneficial to improving the synthesis efficiency of dapoxetine intermediates.

[0018] These or other aspects of this application will become more apparent in the following description of the embodiments. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the reaction mechanism for the synthesis system of dapoxetine intermediates.

[0020] Figure 2 A reaction schematic diagram of another embodiment of the synthesis system for dapoxetine intermediates.

[0021] Figure 3 This is a chiral HPLC analysis spectrum of the racemic product standard in the synthesis method of dapoxetine intermediate in this application embodiment.

[0022] Figure 4 The image shows the chiral HPLC analysis spectrum of the standard S configuration in the synthesis method of the dapoxetine intermediate in this application embodiment.

[0023] Figure 5 The chiral HPLC analysis spectrum of the standard R configuration in the synthesis method of the dapoxetine intermediate in this application embodiment is shown.

[0024] Figure 6 This is a chiral HPLC analysis spectrum of the reaction product of an embodiment of this application.

[0025] Figure 7 The figure shows the results of the optimal reaction temperature optimization experiment for the wild-type reductase used to synthesize the dapoxetine intermediate.

[0026] Figure 8 The figure shows the results of the pH optimization experiment for the wild-type reductase used to synthesize the intermediate of dapoxetine.

[0027] Figure 9 This graph shows the changes in product concentration of the wild-type reductase used to synthesize dapoxetine intermediates.

[0028] Figure 10 This is a graph showing the substrate concentration changes of the reductase mutant used to synthesize the dapoxetine intermediate. Detailed Implementation

[0029] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] In this article, the terms "reductase for the synthesis of dapoxetine intermediates" and "reductase mutant for the synthesis of dapoxetine intermediates" refer to enzymes exhibiting asymmetric reduction activity of prochiral ketone compounds, capable of asymmetricly reducing prochiral ketone compounds to chiral hydroxyl compounds. Specifically, the dapoxetine intermediate precursor, serving as a prochiral ketone compound, is asymmetrically reduced to generate the dapoxetine intermediate, which is a chiral hydroxyl compound. The dapoxetine intermediate precursor is 3-chlorophenylacetone, and the dapoxetine intermediate is (R)-(+)-3-chloro-1-phenyl-1-propanol.

[0034] 3-Chloropropiophenone (CAS: 936-59-4).

[0035] (R)-(+)-3-chloro-1-phenyl-1-propanol ((1R)-3-Chloro-1-phenyl-propan-1-ol; CAS: 100306-33-0).

[0036] The reaction principle for converting 3-chlorophenylacetone to (R)-(+)-3-chloro-1-phenyl-1-propanol using the reductase mutant used in the synthesis of dapoxetine intermediates is as follows: .

[0037] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: n". Obviously, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO: n, even with some sequence deletions, modifications, substitutions, conservative substitutions, or additions, can also be used in this application, as long as they exhibit the same or corresponding activity as the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO: n. For example, it is not excluded to add sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conservative substitutions before or after "the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO: n"; and polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO: n, when subjected to the addition of the aforementioned sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conservative substitutions, also fall within the scope of this application, as long as they exhibit the same or corresponding activity as the amino acid sequence shown in SEQ ID NO: n after the addition of the aforementioned sequences, where n is a natural number.

[0038] This application provides a reductase mutant for synthesizing dapoxetine intermediates, the amino acid sequence of which is shown in SEQ ID NO: 13 to SEQ ID NO: 15.

[0039] The amino acid sequence of the wild-type reductase used to synthesize dapoxetine intermediates is shown in SEQ ID NO: 1. The wild-type reductase used to synthesize dapoxetine intermediates exhibits poor stereoselectivity in the asymmetric reduction catalysis of dapoxetine intermediate precursors, and the chiral purity of the R-type dapoxetine intermediates obtained is approximately 73.68%.

[0040] Among them, the amino acid sequence shown in SEQ ID NO: 1 (WP_205145184) is derived from Weissella uvarum The amino acid sequence shown in SEQ ID NO: 1 was used to predict the three-dimensional structure of the protein. Analysis of the predicted three-dimensional structure of the protein revealed that positions 145, 96, 202, 117, 43, 99, 206, 153, 25, 29, 94, 193, 195, 196, 157, 131, 199, 206, 152, 188, 147, 150, 226, and 101 in the amino acid sequence shown in SEQ ID NO: 1 are key catalytic sites.

[0041] During the mutation screening process, it was found that the mutation of glutamic acid E at position 145 to alanine A or glycine G at position 96 to valine V in the amino acid sequence shown in SEQ ID NO: 1 was beneficial to improving stereoselectivity, and the chiral purity of the R-type dapoxetine intermediate was improved compared with the wild type.

[0042] During the mutation screening process, it was found that the glutamic acid E at position 145 of the amino acid sequence shown in SEQ ID NO: 1 was mutated to alanine A and the glutamic acid E at position 202 was mutated to leucine L, which is beneficial to improving stereoselectivity. The chiral purity of the R-type dapoxetine intermediate was increased to over 93%.

[0043] During the mutation screening process, it was found that the glutamic acid E at position 145 of the amino acid sequence shown in SEQ ID NO: 1 was mutated to alanine A and the glycine G at position 96 was mutated to valine V, which is beneficial to improving stereoselectivity. The chiral purity of the R-type dapoxetine intermediate was increased to over 95%.

[0044] During the mutation screening process, it was found that the glutamic acid E at position 145 of the amino acid sequence shown in SEQ ID NO: 1 was mutated to alanine A, the glutamic acid E at position 202 was mutated to leucine L, and the glycine G at position 96 was mutated to valine V (hereinafter referred to as mutant M3), which is beneficial to improving stereoselectivity and the chiral purity of the R-type dapoxetine intermediate was increased to over 95%.

[0045] Based on mutant M3, the serine S at position 117 was mutated to glycine G or the isoleucine I at position 99 was mutated to leucine L, which is beneficial to improving stereoselectivity. The chiral purity of the R-type dapoxetine intermediate was improved compared with mutant M3, and the chiral purity of the R-type dapoxetine intermediate was increased to over 99%.

[0046] Based on mutant M3, a further mutation occurred where serine at position 117 (S) was changed to glycine (G), isoleucine at position 99 (I) was changed to leucine (L), and valine at position 43 (V) was changed to arginine (R) (hereinafter referred to as mutant M6). This was beneficial for improving stereoselectivity, and the chiral purity of the R-type dapoxetine intermediate was increased to over 99%.

[0047] Based on mutant M6, a further mutation of serine S at position 206 to valine V (hereinafter referred to as mutant M7) was observed, which is beneficial to improving stereoselectivity, and the chiral purity of the R-type dapoxetine intermediate was increased to over 99.9%.

[0048] Based on the mutant M6, a further mutation of leucine L at position 153 to tyrosine Y was observed, which is beneficial to improving stereoselectivity, and the chiral purity of the R-type dapoxetine intermediate can also reach over 99%.

[0049] In this embodiment, a reductase mutant for synthesizing a dapoxetine intermediate exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: glutamate E at position 145 is mutated to alanine A, glutamate E at position 202 is mutated to leucine L, glycine G at position 96 is mutated to valine V, serine S at position 117 is mutated to glycine G, isoleucine I at position 99 is mutated to leucine L, and valine V at position 43 is mutated to arginine R (SEQ ID NO: 13, M6). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned reductase mutant for synthesizing a dapoxetine intermediate has the amino acid sequence shown in SEQ ID NO: 13.

[0050] In this embodiment, a reductase mutant used for synthesizing a dapoxetine intermediate exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: glutamate E at position 145 is mutated to alanine A, glutamate E at position 202 is mutated to leucine L, glycine G at position 96 is mutated to valine V, serine S at position 117 is mutated to glycine G, isoleucine I at position 99 is mutated to leucine L, valine V at position 43 is mutated to arginine R, and serine S at position 206 is mutated to valine V (SEQ ID NO: 14, M7). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned reductase mutant used for synthesizing a dapoxetine intermediate has the amino acid sequence shown in SEQ ID NO: 14.

[0051] In this embodiment, a reductase mutant for synthesizing a dapoxetine intermediate exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1: glutamate E at position 145 is mutated to alanine A, glutamate E at position 202 is mutated to leucine L, glycine G at position 96 is mutated to valine V, serine S at position 117 is mutated to glycine G, isoleucine I at position 99 is mutated to leucine L, valine V at position 43 is mutated to arginine R, and leucine L at position 153 is mutated to tyrosine Y (SEQ ID NO: 15). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 1 are replaced by other amino acids. The aforementioned reductase mutant for synthesizing a dapoxetine intermediate has the amino acid sequence shown in SEQ ID NO: 15.

[0052] In this embodiment, the amino acid sequence of the reductase mutant used to synthesize the dapoxetine intermediate is shown in SEQ ID NO: 13 to SEQ ID NO: 15. It improves the stereoselectivity of the asymmetric reductive hydrogenation catalysis of the dapoxetine intermediate precursor, and obtains the R-type dapoxetine intermediate with high chiral purity (the chiral purity of the R-type dapoxetine intermediate is above 99.00%), which is beneficial to improving the synthesis efficiency of the dapoxetine intermediate.

[0053] This application provides a gene for synthesizing dapoxetine intermediates, which encodes the aforementioned reductase mutant for synthesizing dapoxetine intermediates.

[0054] The gene used to synthesize the dapoxetine intermediate can be a polynucleotide.

[0055] The polynucleotide has the nucleotide sequences corresponding to SEQ ID NO: 13 to SEQ ID NO: 15.

[0056] The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides.

[0057] The polynucleotide only needs to encode the reductase mutant used to synthesize the dapoxetine intermediate, and any nucleotide in the polynucleotide can be chemically modified.

[0058] This application provides a recombinant vector comprising the aforementioned gene for synthesizing dapoxetine intermediates.

[0059] For example, the recombinant vector includes the polynucleotide described above that encodes the reductase mutant used to synthesize the dapoxetine intermediate.

[0060] The recombinant vector is any natural or artificially constructed expression vector that encodes the reductase mutant nucleic acid molecule used to synthesize the dapoxetine intermediate, which can be catalyzed by cellular transcriptases and / or translatases.

[0061] Specifically, a recombinant vector is a DNA preparation containing a nucleic acid sequence encoding a polynucleotide (Pnucleotide) of a reductase mutant for the synthesis of a dapoxetine intermediate. It may also contain a control sequence. In the recombinant vector, the Pnucleotide sequence encoding the Pnucleotide of the reductase mutant for the synthesis of a dapoxetine intermediate is operatively linked to a suitable control sequence, allowing the reductase mutant for the synthesis of the dapoxetine intermediate to be expressed in a suitable host. Specifically, the control sequence may include, but is not limited to, promoters capable of initiating transcription, arbitrary operon sequences for regulating transcription, suitable mRNA ribosome binding sites, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector can replicate or function independently of the host genome, or it can integrate into the genome itself for replication or function.

[0062] There are no particular restrictions on the type of recombinant vector; any vector known in the art can be used as long as it can replicate in the host cell. Exemplarily, commonly used vectors in the art can include plasmids, granules, viruses, bacteriophages, or transposons in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and the pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, and pET system can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0063] This application provides a recombinant engineered bacterium, which includes the recombinant vector described above.

[0064] In this embodiment, the recombinant engineered bacteria serves as the host cell. The recombinant vector described above is transformed into the host cell, enabling the synthesis of the reductase mutant used for the synthesis of dapoxetine intermediates within the host cell. The recombinant vector is introduced into the host cell, and the polynucleotide encoding the reductase mutant for the synthesis of dapoxetine intermediates in the recombinant vector is expressed in the host cell, allowing the host cell to synthesize the aforementioned reductase mutant for the synthesis of dapoxetine intermediates. This polynucleotide can be inserted into the host cell's chromosome, located outside the host cell's chromosome, or simultaneously inserted into the host cell's chromosome and located outside the chromosome. The polynucleotide can be DNA or RNA, as long as it can be expressed in the host cell. Exemplarily, the recombinant vector can be an expression cassette, including a promoter, transcription termination element, ribosomal domain, and translation termination element operatively linked to the polynucleotide.

[0065] The host cell can be a eukaryotic cell or a prokaryotic cell, and further, the prokaryotic cell can be a bacterial cell.

[0066] As one implementation method, the host cell can be Escherichia coli (Escherichia coli). Escherichia ) genus, Erwinia ( Erwinia ) genus, Serratia ( Serratia ) genus, Providencia ( Providencia ) genus, Corynebacterium ( Corynebacterium ) genus or short bacilli ( Brevibacterium ) genus; for example, the host cell can be *Escherichia coli* (E. coli). Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) or Aspergillus oryzae ( Aspergillus oryzae ).

[0067] For example, the recombinant engineered bacteria is *Escherichia coli*, which is... E. coli BL21(DE3).

[0068] This application provides a synthesis system for dapoxetine intermediates, comprising recombinant engineered bacteria and a co-substrate. The recombinant engineered bacteria is a recombinant engineered bacteria containing a reductase mutant encoding gene for the synthesis of dapoxetine intermediates. The amino acid sequence of the reductase mutant for the synthesis of dapoxetine intermediates is shown in SEQ ID NO: 13 to SEQ ID NO: 15.

[0069] 3-Chlorophenylacetone (a precursor to dapoxetine intermediate) is a prochiral ketone compound. 3-Chlorophenylacetone (a precursor to dapoxetine intermediate) is used as a substrate. A reductase mutant used to synthesize the dapoxetine intermediate is used as a catalyst. Utilizing the asymmetric reduction catalytic activity of the reductase mutant used to synthesize the dapoxetine intermediate, the substrate 3-chlorophenylacetone is converted to (R)-(+)-3-chloro-1-phenyl-1-propanol (dapoxetine intermediate), which is then used as the product.

[0070] The catalytic reaction for asymmetric reduction of the reductase mutant used to synthesize the dapoxetine intermediate requires the presence of a coenzyme.

[0071] Coenzymes can be NADP + / NADPH, NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate, which structurally contains an additional hydride (i.e., a negatively charged hydrogen atom); NADP + It is the oxidized state of nicotinamide adenine dinucleotide phosphate, which does not have an additional hydride (i.e., a negatively charged hydrogen atom), and therefore exhibits a positive charge. NADP + It mainly acts as an electron acceptor in cells, participating in a variety of redox reactions.

[0072] like Figure 1 As shown, in the asymmetric reductive hydrogenation reaction of the substrate 3-chlorophenylacetone catalyzed by the reductase mutant used to synthesize the dapoxetine intermediate to generate (R)-(+)-3-chloro-1-phenyl-1-propanol (dapoxetine intermediate), NADPH is converted to NADP. + .

[0073] In the embodiments of this application, both the reductase mutant and the wild-type reductase used for synthesizing dapoxetine intermediates are short-chain dehydrogenases. In addition to the aforementioned asymmetric reduction catalytic activity, they also possess oxidation catalytic activity, capable of oxidizing the co-substrate to form byproducts. During the oxidation of the co-substrate to form byproducts, NADP... + It is converted into NADPH.

[0074] In the asymmetric reduction catalytic reaction, NADPH is converted to NADP. + NADP in oxidative catalysis + It is converted into NADPH, coenzyme NADP + / NADPH is recycled.

[0075] The recombinant engineered bacteria contain coenzymes within their cells, so the synthesis system for this dapoxetine intermediate does not require the addition of additional coenzymes.

[0076] In this embodiment, the reductase mutant synthesized by recombinant engineered bacteria for the synthesis of dapoxetine intermediates efficiently catalyzes the asymmetric reduction of prochiral ketone compounds in a system without the addition of any coenzymes, generating chiral hydroxyl compounds with high optical purity (ee > 99.0%), which has good prospects for industrial application. In this embodiment, the reductase mutant for the synthesis of dapoxetine intermediates has the characteristics of high conversion rate and high chiral selectivity for 3-chlorophenylacetone (dapoxetine intermediate precursor).

[0077] As one implementation method, please refer to Figure 2 As shown, the co-substrate can be isopropanol, which is used to oxidize the reductase mutant in the synthesis of dapoxetine intermediates to form acetone.

[0078] Specifically, the recombinant engineered bacteria can be the engineered bacteria described in the above embodiments. The construction of the recombinant engineered bacteria can be referred to the description of the above engineered bacteria embodiments, which will not be repeated here.

[0079] In some embodiments, the recombinant engineered bacteria used in the synthesis system can be wet cells obtained by inducing and culturing recombinant engineered bacteria.

[0080] In some embodiments, the recombinant engineered bacteria is Escherichia coli. E. coli BL21(DE3).

[0081] In some embodiments, the recombinant engineered bacteria used in the synthesis system may be the crude enzyme solution obtained by breaking down the wet bacterial cells, or the immobilized cells prepared from the wet bacterial cells.

[0082] In some embodiments, the reaction system further includes a buffer solution, wherein the volume percentage of isopropanol in the reaction system is 40% to 100%. Specifically, the reaction system includes isopropanol, a buffer solution, a substrate, and recombinant engineered bacteria, wherein isopropanol and the buffer solution are in liquid form, and the volume of the reaction system is mainly determined by the volume of isopropanol and the volume of the buffer solution. The volume of the reaction system can be understood as the sum of the volumes of isopropanol and the buffer solution.

[0083] In some embodiments, the reaction system may not contain a buffer solution and may include isopropanol, substrate, and recombinant engineered bacteria, with isopropanol comprising 100% by volume.

[0084] In some implementations, the buffer is a PBS buffer, and the pH of the reaction system is 5.0 to 7.0.

[0085] This application provides a method for the biological preparation of dapoxetine intermediates, using dapoxetine intermediate precursors as substrates, adding the aforementioned recombinant engineered bacteria, and reacting in a reaction system to obtain dapoxetine intermediate products.

[0086] Among them, 3-chlorophenylacetone (dapoxetine intermediate precursor) is a prochiral ketone compound. 3-chlorophenylacetone (dapoxetine intermediate precursor) is used as a substrate, and a reductase mutant used to synthesize dapoxetine intermediate is used as a catalyst. Utilizing the catalytic activity of the asymmetric reduction of the reductase mutant used to synthesize dapoxetine intermediate, the substrate 3-chlorophenylacetone is converted into (R)-(+)-3-chloro-1-phenyl-1-propanol (dapoxetine intermediate), and (R)-(+)-3-chloro-1-phenyl-1-propanol (dapoxetine intermediate) is the product.

[0087] In one embodiment, in a reaction system containing isopropanol, 3-chlorophenylacetone (a precursor of dapoxetine intermediate) is converted to (R)-(+)-3-chloro-1-phenyl-1-propanol (dapoxetine intermediate) using a recombinant engineered bacterium containing a reductase mutant for the synthesis of dapoxetine intermediates.

[0088] In this embodiment, the reductase mutant synthesized by recombinant engineered bacteria for the synthesis of dapoxetine intermediates efficiently catalyzes the asymmetric reduction of prochiral ketone compounds in a system without the addition of any coenzymes, generating chiral hydroxyl compounds with high optical purity (ee > 99.0%), which has good prospects for industrial application. In this embodiment, the reductase mutant for the synthesis of dapoxetine intermediates has the characteristics of high conversion rate and high chiral selectivity for 3-chlorophenylacetone (dapoxetine intermediate precursor).

[0089] Specifically, the recombinant engineered bacteria can be the engineered bacteria described in the above embodiments. The construction of the recombinant engineered bacteria can be referred to the description of the above engineered bacteria embodiments, which will not be repeated here.

[0090] In some embodiments, the recombinant engineered bacteria can be wet cells obtained by inducing and culturing recombinant engineered bacteria.

[0091] In some embodiments, the recombinant engineered bacteria is Escherichia coli. E. coli BL21(DE3).

[0092] In some embodiments, the recombinant engineered bacteria may also be the crude enzyme solution obtained by breaking down the above-mentioned wet bacterial cells, or the immobilized cells prepared from the above-mentioned wet bacterial cells.

[0093] In some embodiments, the reaction system includes isopropanol and a buffer solution, with the volume percentage of isopropanol in the reaction system being 40% to 100%. Specifically, the reaction system includes isopropanol, a buffer solution, a substrate, and recombinant engineered bacteria, wherein isopropanol and the buffer solution are in liquid form, and the volume of the reaction system is mainly determined by the volume of isopropanol and the volume of the buffer solution. The volume of the reaction system can be understood as the sum of the volumes of isopropanol and the buffer solution.

[0094] In some embodiments, the reaction system may not contain a buffer solution and may include isopropanol, substrate, and recombinant engineered bacteria, with isopropanol comprising 100% by volume.

[0095] In some embodiments, the buffer solution is a PBS buffer, and the pH of the reaction system is 5.0 to 7.0. Exemplarily, the reaction pH can be 5.0, 5.5, 5.7, 6.0, 6.3, 6.5, 6.8, or 7.0.

[0096] In some embodiments, the reaction temperature can be 35°C to 45°C. For example, the reaction temperature can be 35°C, 37°C, 39°C, 40°C, 42°C, 44°C, or 45°C.

[0097] This application provides the use of the above-described reductase mutant for synthesizing dapoxetine intermediates, the above-described gene for synthesizing dapoxetine intermediates, the above-described recombinant vector, the above-described recombinant engineered bacteria, or the above-described synthetic system in the preparation of dapoxetine or in the preparation of dapoxetine.

[0098] Preparation of reductase mutants for the synthesis of dapoxetine intermediates The reductase mutant used to synthesize dapoxetine intermediates and the gene encoding the reductase used to synthesize dapoxetine intermediates can be referred to as the KRED gene. The KRED gene contains the nucleotide sequence corresponding to the amino acid sequence of the aforementioned reductase mutant or reductase used to synthesize dapoxetine intermediates. For example, the KRED gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 15. The KRED gene is constructed in the pET-28a plasmid to obtain a recombinant vector.

[0099] The recombinant vector is a pET-28a plasmid containing the KRED gene, hereinafter referred to as pET-28a-KRED; the host cell used in the various embodiments and comparative examples of this application is Escherichia coli. The structure and sequence of the pET-28a plasmid can be found in CN202410706921.3. The nucleotide sequences corresponding to the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 15 in the various embodiments of this application and Comparative Example 1 are then used as the target expression genes to construct the pET-28a plasmid.

[0100] Expression of the gene encoding the reductase mutant used to synthesize dapoxetine intermediates a. Transformation of pET-28a-KRED into E. coli E. coli In BL21(DE3), select a single clone of pET-28a-KRED or a strain preserved at -80℃, streak it onto the surface of LB solid medium containing the corresponding antibiotic (such as kanamycin, final concentration 50 μg / mL) in a clean bench, and then place it in a 37 ℃ constant temperature incubator for 12 h until a clear single colony is formed.

[0101] b. Use a sterile inoculation loop to pick a single colony from a fresh plate and inoculate it into 10 mL of LB liquid medium containing the same antibiotic. Incubate at 37 ℃ and 200 rpm for 12 h with shaking to obtain a seed culture with OD600≈3.0, ensuring that the bacteria are in the logarithmic growth phase.

[0102] c. Transfer the seed culture at an inoculum of 1% (v / v) to an Erlenmeyer flask containing 50 mL of LB liquid medium (kanamycin, final concentration 50-100 μg / mL), and culture with shaking at 37 ℃ and 200 rpm for 2 h. Monitor the cell growth until the OD600 is approximately 0.6-0.8, providing highly active cells for subsequent catalytic reactions.

[0103] d. Lower the temperature of the shaker to 16 ℃-18 ℃. After the temperature of the cultured bacterial solution has decreased, add isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 h.

[0104] e. After expression is complete, collect the above culture solution into a bottle, pre-cool the centrifuge to 4°C, and centrifuge at 5500 rpm for 10 min.

[0105] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0106] g. Obtaining whole-cell catalyst: Centrifuge the resuspended bacterial cells again at 5500 rpm for 10 min, discard the supernatant, and collect the wet bacterial cells as the whole-cell catalyst. This wet bacterial cell can be used directly as a reductase mutant catalyst for the synthesis of dapoxetine intermediates.

[0107] h. Preservation of whole-cell catalyst: Add wet bacterial cells to 30 mL of protein purification buffer, vortex the bacterial cells (there should be no solid particles), pour into a 50 mL centrifuge tube, and store at -80 ℃.

[0108] Purification of reductase mutant protein used for the synthesis of dapoxetine intermediates a. Preparation of crude enzyme solution: 1.0 g of collected wet bacterial cells (whole-cell catalyst) were added to 20 mL of equilibration buffer for resuspending. The resuspended cells were then disrupted using a cell disruptor set to 300 W to prevent excessive temperature from affecting enzyme activity. The disruption program was set to run for 1 second and pause for 3 seconds. The disruption solution was continuously cooled with an ice-water mixture until the suspension became clear and transparent. The disruption solution was then centrifuged at 4 ℃ and 12000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 µm filter membrane to obtain the crude enzyme solution. This crude enzyme solution can be used directly as a catalyst for the synthesis of reductase mutants of dapoxetine intermediates.

[0109] b. Ion exchange chromatography column regeneration and equilibration: Protein purification was performed using a DEAE Sepharose Fast Flow anion exchange column. The column was washed with high-salt buffer (containing 1–2 M NaCl) at a flow rate of 1 mL / min for 3–5 column volumes, followed by washing with 0.1 M NaOH for 3–5 column volumes, then washing with elution buffer for 3–5 column volumes. Equilibration buffer was then used until the detector OD value reached 3–5 column volumes. 280 The signals of parameters such as conductivity and pH value are stable.

[0110] c. Loading and elution of crude enzyme solution: Load the prepared crude enzyme solution at a loading rate of 0.5 mL / min, with a loading volume of 20 mL. After loading, wash with equilibration buffer for 3-5 column volumes, then elute using an increasing salt concentration gradient with elution buffer. Collect each fraction and confirm by protein electrophoresis. If the purification effect is unsatisfactory, this step can be repeated, or purification can be performed again using agarose gel G75 FF.

[0111] d. Protein concentration: The collected target protein was concentrated using ultrafiltration membrane concentration method. The concentration was carried out using a 10 kDa protein concentration tube and centrifuged at 4 ℃ and 5000 rpm for 30 min.

[0112] e. Protein desalting: Dilute the concentrated protein with an appropriate amount of PBS buffer (20 mM, pH 7.0) and place it in a dialysis bag (molecular weight cutoff 8~14 kDa). Use 20 mM, pH 7.0 PBS dialysate and let it stand overnight at 4 ℃. The dialysate needs to be changed once during the process.

[0113] f. Storage of ion exchange chromatography columns: After use, the ion exchange chromatography column should be rinsed with 1 M NaOH for 3-5 column volumes, then rinsed with 20% ethanol, and stored in a refrigerator at 4 ℃.

[0114] Electrophoretic analysis of reductase mutant protein used in the synthesis of dapoxetine intermediates a. Protein sample preparation: Add the purified protein solution and 5× loading buffer at a ratio of 4:1 (v / v), heat in boiling water for 10 min, and set aside for later use.

[0115] b. Sample loading and electrophoresis: Place the precast protein gel (Genscript, SurePAGE, 4%~20%) in the electrophoresis tank, and add the protein sample and marker to the sample wells of the protein gel using a pipette.

[0116] c. Staining and destaining: Remove the outer shell of the pre-cast gel after electrophoresis, and automatically destain and stain using a protein staining and destaining instrument for 15 minutes.

[0117] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0118] Enzyme-catalyzed reactions In vitro enzyme catalytic reaction conditions: Buffer for the reaction (may not include buffer): PBS buffer at concentrations of 100mM, 200mM, and 300mM; Reaction pH: pH5, pH6, pH7; The concentrations of 3-chlorophenylacetone are 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, and 300 g / L. The volume concentrations of isopropanol are 4 mL / 10 mL, 5 mL / 10 mL, 6 mL / 10 mL, 7 mL / 10 mL, 8 mL / 10 mL, 9 mL / 10 mL, and 10 mL / 10 mL. The amount of *E. coli* expressing the reductase mutant encoding gene for the synthesis of dapoxetine intermediate (the whole-cell catalyst obtained in the above steps) added was 50 g / L and 100 g / L. Reaction temperatures: 35℃, 40℃, 45℃; Reaction time: 2h, 3.5h, 4h, 6h, 8h, 10h, 12h, 15h, 16h, 18h, 20h, 22h, or 24h; The total volume of the conversion reaction is 10 mL.

[0119] Detection of (R)-(+)-3-chloro-1-phenyl-1-propanol Detection method: OD-H chiral column; Mobile phase: n-hexane:isopropanol 95:5 Detector: 2998 PDA; in, Figure 3 , Figure 4 and Figure 5 Spectral analysis was performed on the purchased standard samples for result comparison. Figure 3 This is the chiral HPLC chromatogram of the racemic mixture of the product standard, used to compare the chiral differences between the reaction product and the racemic mixture; Figure 4 The chiral HPLC chromatogram of the S-configuration standard is used to identify the reaction byproducts of the S-configuration and to compare the chiral characteristics of the reaction products. Figure 5 This is the chiral HPLC chromatogram of the standard R configuration, used to confirm that the reaction product is of the R configuration; according to Figure 3 , Figure 4 and Figure 5 The comparison confirms the reaction product of the R configuration, which elutes at approximately 12.4 min, while the reaction byproduct of the S configuration elutes at approximately 10.4 min. Figure 6 Here is an example of a chiral HPLC analysis spectrum of the reaction products in one embodiment. The R-configuration reaction product elutes at approximately 12.4 min, and the S-configuration reaction byproduct elutes at approximately 10.4 min. The chiral purity of the R-configuration can be calculated by using the concentrations of the R-configuration reaction product and the S-configuration reaction byproduct from the chiral HPLC analysis spectrum and their respective proportions.

[0120] Substrate detection Detection method: Gas chromatography, BGB-174 column; Mobile phase: Nitrogen; Detector: FID; Temperature program: 100℃ for 1 min, 25℃ / min, increase to 160℃, hold for 1 min, 5℃ / min, increase to 210℃, hold for 5 min; The substrate elutes at approximately 11.5 min, and the product elutes at approximately 12.3 min. The concentration of the substrate can be obtained from the gas phase spectrum.

[0121] It should be noted that in the following examples and comparative examples, the product specifically refers to the R-configuration reaction product ((R)-(+)-3-chloro-1-phenyl-1-propanol), and the substrate specifically refers to 3-chlorophenylacetone. During the measurement of product and substrate concentrations, dilution of the reaction solution and liquid-phase or gas-phase detection can introduce detection errors, leading to differences in the same concentration at different measurement points in the product or substrate concentration change curves.

[0122] Calculation of relative enzyme activity Relative enzyme activity = (activity of the tested enzyme / activity of the standard enzyme) × 100% = (product production of the tested enzyme / product production of the standard enzyme) × 100%.

[0123] Catalytic conditions of wild-type reductase used to synthesize dapoxetine intermediates a) Wild-type reaction temperature comparison experiment The reaction temperatures were 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. Other reaction conditions are as follows: The reaction buffer was a 100 mM PBS buffer with a volume concentration of 1 mL / 10 mL. Reaction pH: pH 6; The initial concentration of 3-chlorophenylacetone was 50 g / L; The volume concentration of isopropanol is 9 mL / 10 mL; The amount of *E. coli* expressing the reductase encoding gene for the synthesis of dapoxetine intermediates (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 1) added was 50 g / L; Reaction time: 0.5 h; The reaction system is 10 mL.

[0124] Please see Figure 7 As shown, the reductase used to synthesize the dapoxetine intermediate exhibits relatively good activity at reaction temperatures ranging from 30°C to 45°C, with the activity being optimal at 35°C.

[0125] b) Wild-type pH comparison experiment The reaction buffers and their pH values ​​were: pH 4.0 (acetic acid-sodium acetate buffer), pH 5.0 (acetic acid-sodium acetate buffer), pH 5.0 (PBS buffer), pH 6.0 (PBS buffer), pH 7.0 (PBS buffer), pH 8.0 (PBS buffer), pH 8.0 (Tris-HCl buffer), and pH 9.0 (Tris-HCl buffer). Other reaction conditions are as follows: The initial concentration of 3-chlorophenylacetone was 50 g / L; The volume concentration of isopropanol is 9 mL / 10 mL; The buffer concentration is 1 mL / 10 mL; The amount of *E. coli* expressing the reductase encoding gene for the synthesis of dapoxetine intermediates (the whole-cell catalyst obtained in the above steps, with the amino acid sequence shown in SEQ ID NO: 1) added was 50 g / L; Reaction temperature: 35℃; Reaction time: 0.5 h; The reaction system is 10 mL.

[0126] Please see Figure 8 As shown, the reductase used to synthesize the dapoxetine intermediate exhibits relatively good activity in PBS buffer at a pH of 6.0.

[0127] C) Wild-type product concentration change experiment The initial concentration of 3-chlorophenylacetone was 100 g / L; The volume concentration of isopropanol is 10 mL / 10 mL; The amount of wild-type reductase (whole-cell catalyst) used to synthesize dapoxetine intermediates was added at 50 g / L; Reaction temperature: 35℃; Reaction time: 24 hours; The reaction system is 10 mL.

[0128] Samples were taken at reaction times of 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours for chiral HPLC analysis to measure product concentration and gas chromatography analysis to measure substrate concentration. The results are as follows: Figure 9 As shown. Within 0 to 2 hours of the reaction, the substrate is consumed at an extremely rapid rate, and the product increases at an extremely rapid rate; within 2 to 8 hours of the reaction, the substrate is consumed at a relatively rapid rate, and the product increases at a relatively rapid rate; within 8 to 24 hours of the reaction, the substrate is consumed at a relatively slow rate, and the product increases at a relatively slow rate.

[0129] Screening experiments on mutant enzyme activity and R conformation chiral purity The amino acid sequences of the reductase mutants used to synthesize dapoxetine intermediates in Examples 1 to 14 are shown in SEQ ID NO: 2 to SEQ ID NO: 15, and the amino acid sequence of the reductase used to synthesize dapoxetine intermediates in Comparative Example 1 is shown in SEQ ID NO: 1.

[0130] Catalytic reaction conditions of Examples 1 to 14 and Comparative Example 1 The initial concentration of 3-chlorophenylacetone was 200 g / L; The volume concentration of isopropanol is 10 mL / 10 mL; The amount of reductase mutant (whole-cell catalyst) used to synthesize the dapoxetine intermediate was added at 50 g / L; Reaction temperature: 35℃; Reaction time: 0.5 h; The reaction system is 10 mL.

[0131] Samples were taken from Examples 1 to 14 and Comparative Example 1 after a reaction time of 0.5 hours. Chiral HPLC analysis was performed to measure the relative enzyme activity and R-configuration chiral purity of Examples 1 to 14 and Comparative Example 1. The results are shown in Table 1.

[0132] Table 1 Parameters of each embodiment The results of Examples 1 to 14 and Comparative Example 1 are shown in Table 1. In Comparative Example 1, the relative activity of the reductase used to synthesize the dapoxetine intermediate is 1. The relative activities of the reductase mutants used to synthesize the dapoxetine intermediate in Examples 1 to 14 are shown based on Comparative Example 1.

[0133] Table 1 Parameters of Examples 1 to 14 and Comparative Example 1 As shown in Table 1, the chiral purity of the R configuration of the mutants in Examples 12 to 14 is above 99%, and the enzyme activity of the mutants in Examples 12 to 14 is significantly improved compared with that of Comparative Example 1.

[0134] Mutant substrate concentration change experiment Catalytic reaction conditions in Examples 15-18 The initial concentrations of 3-chlorophenylacetone differ; The volume concentration of isopropanol is 10 mL / 10 mL; The amount of reductase mutant (whole-cell catalyst) used to synthesize the dapoxetine intermediate was added at 50 g / L. Reaction temperature: 35℃; Reaction time: 0.5 h; The reaction system is 10 mL.

[0135] Table 2 Initial substrate concentrations for each example Samples from Examples 15, 17, 18, and 18 were taken at reaction times of 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours, respectively, and chiral HPLC analysis was performed to measure the product mass concentration. The results are as follows: Figure 10 As shown.

[0136] like Figure 10 As shown, for Examples 15 to 18, during the entire reaction process, the substrate consumption rate was extremely rapid during the first 0 to 2 hours, and relatively slowed down during the second to 8 hours. The substrate change curves of Examples 15 to 18 showed basically the same trend.

[0137] The above description is merely an embodiment of this application. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of this application, but these improvements all fall within the protection scope of this application.

Claims

1. A reductase mutant for synthesizing dapoxetine intermediates, characterized in that, The amino acid sequence of the reductase mutant used to synthesize the dapoxetine intermediate is shown in SEQ ID NO: 13 to SEQ ID NO:

15.

2. A gene for synthesizing dapoxetine intermediates, characterized in that, The gene used to synthesize the dapoxetine intermediate encodes the reductase mutant for synthesizing the dapoxetine intermediate as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector includes the gene for synthesizing dapoxetine intermediates as described in claim 2.

4. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria include the recombinant vector as described in claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The recombinant engineered bacteria is *Escherichia coli*, and the *Escherichia coli* is... E. coli BL21(DE3).

6. A synthetic system for synthesizing dapoxetine intermediates, characterized in that, The synthetic system for synthesizing dapoxetine intermediates includes recombinant engineered bacteria and isopropanol, wherein the recombinant engineered bacteria is the recombinant engineered bacteria as described in claim 4.

7. The synthetic system for synthesizing dapoxetine intermediates according to claim 6, characterized in that, The volume ratio of isopropanol to the reaction system is 40% to 100%.

8. The synthetic system for synthesizing dapoxetine intermediates according to claim 7, characterized in that, The reaction system also includes a buffer solution with a pH value of 5.0 to 7.

0.

9. A method for the biological preparation of a dapoxetine intermediate, characterized in that, Using the dapoxetine intermediate precursor as a substrate, the recombinant engineered bacteria as described in claim 4 were added to the reaction system to obtain the dapoxetine intermediate product.

10. The use of the reductase mutant for synthesizing dapoxetine intermediates as described in claim 1, the gene for synthesizing dapoxetine intermediates as described in claim 2, the recombinant vector as described in claim 3, or the recombinant engineered bacteria as described in claim 4 in the preparation of dapoxetine intermediates or in the preparation of dapoxetine.

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