Process for the synthesis of alpha-hydroxy ketone compounds

Abstract

The application provides a synthesis method of an alpha-hydroxy ketone compound. The synthesis method of the alpha-hydroxy ketone compound comprises the following steps: mixing a substrate and a peroxide enzyme to react, and obtaining the alpha-hydroxy ketone compound; R1, R2 or R3 in the substituent group of the substrate and the alpha-hydroxy ketone compound is independently selected from hydrogen, a substituted or unsubstituted benzyl group, an alkyl group and the like; or R1 and R2 are combined to form a substituted or unsubstituted cycloalkyl group or a benzo-cycloalkyl group, and R3 is selected from hydrogen, a substituted or unsubstituted benzyl group, an alkyl group and the like; or R2 and R3 are combined to form a substituted or unsubstituted cycloalkyl group or a benzo-cycloalkyl group, and R1 is selected from hydrogen, a substituted or unsubstituted benzyl group, an alkyl group and the like. The synthesis method can solve the problem that it is difficult to synthesize the alpha-hydroxy ketone compound at low cost, in a green and efficient manner in the prior art, and is suitable for the field of alpha-hydroxy ketone compound synthesis.

Description

Technical Field

[0001] This invention relates to the field of α-hydroxy ketone compound synthesis, and more specifically, to a method for synthesizing α-hydroxy ketone compounds. Background Technology

[0002] Alpha-hydroxy ketones are a very important and widely used class of compounds, with significant value in the chemical and pharmaceutical industries. For example, they are essential components of many bioactive compounds and synthetic drugs, such as paeonilactone B, daunorubicin, phyllaemblic acid, and doxycycline.

[0003] Over the past few decades, various methods have been developed for the synthesis of α-hydroxycarbonyl compounds, including the use of peroxides, high-valent iodine compounds, metal oxides, and N-sulfonyloxybutanes as oxidants. However, most of these methods have limitations, such as the need for expensive transition metal catalysts or harsh reaction conditions. A method for preparing α-hydroxyketone compounds using oxygen as both the oxidant and oxygen source, cesium carbonate (Cs₂CO₃) as the catalyst, and P(OEt)₃ as an additive in DMSO solvent is reported. While this method offers broad substrate applicability, eliminates the need for transition metal catalysts, and provides mild reaction conditions, the high-boiling-point DMSO solvent makes solvent recovery difficult, product separation after the reaction complex, and is costly. Furthermore, cesium carbonate is very expensive, and its recycling is costly, making it unsuitable for industrial production. Therefore, industry urgently needs a low-cost, green, and efficient method for synthesizing α-hydroxyketone compounds. Summary of the Invention

[0004] The main objective of this invention is to provide a method for synthesizing α-hydroxy ketone compounds, thereby solving the problem of the difficulty in synthesizing α-hydroxy ketone compounds in a low-cost, green, and efficient manner in the prior art.

[0005] To achieve the above objective, according to a first aspect of the present invention, a method for synthesizing an α-hydroxy ketone compound is provided, the method comprising: mixing a substrate and a peroxidase to react and obtain the α-hydroxy ketone compound; the substrate has the following structural formula: The structural formula of the above α-hydroxy ketone compound is as follows: R1, R2, or R3 are each independently selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R1 and R2 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R3 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R2 and R3 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R1 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl.

[0006] Furthermore, the system described above also contains hydrogen peroxide solution.

[0007] Furthermore, the heteroatoms in the aforementioned heteroaryl and heterocyclic groups are selected from one or more of nitrogen, oxygen, or sulfur.

[0008] Furthermore, the peroxidase contains a conserved region sequence, which includes the amino acid sequence shown in SEQ ID NO: 1.

[0009] Furthermore, the above-mentioned conserved region sequence is any one of the amino acid sequences shown in SEQ ID NOs: 2, 3 or 4.

[0010] Further, the above peroxidase is derived from any one or more of the following strains: Pterula gracilis, Pterulicium gracile, Collybiopsis luxurians FD-317 M1, Armillaria solidipes, Dendrothele bispora CBS 962.96, Moniliophthora roreri, Cercospora zeae-maydis, Dothistroma septosporum NZE10, Aspergillus niger CBS 101883, Daldiniacaldariorum, Monilinia fructicola, Fusarium sporotrichioides, Pyriculariaoryzae, Gaeumannomyces tritici R3-111a-1, Pestalotiopsis fici W106-1, Hypoxylonsp. EC38, Aspergillus saccharolyticus JOP 1030-1, Aspergillus nidulans FGSC A4, Truncatella angustata, Penicillium steckii, Aspergillus sydowii CBS 593.65, Madurella mycetomatis, Aspergillus bombycis, Aspergillus sclerotialis, Helicocarpus griseus, Colletotrichum scovillei, Macrophomina phaseolina MS6, Phialocephala subalpina, Aureobasidium pullulans, Aaosphaeria arxii CBS 175.79, Rutstroemia sp. NJR-2017a, Hortaea werneckii, Monilinia fructigena, Hypoxylonsp. CI-4A, Daldinia eschscholtzii, Pestalotiopsis fici W106-1, Viridotheliumvirens, Aspergillus alliaceus, Thermothelomyces thermophilus, Monosporascus sp.mg162, Monosporascus sp. CRB-9-2, Aspergillus brasiliensis CBS 101740, Alternaria tenuissima, Decorospora gaudefroyi, Pyrenophora seminiperda or Fusarium graminearum. .

[0011] Further, the peroxidases described above include: (a) any of the proteins shown in SEQ ID NOs: 5-51; or (b) proteins containing the conserved region sequence described above and having more than 70% homology with the amino acid sequence defined in (a) and having peroxidase activity.

[0012] Furthermore, the aforementioned substrates include:

[0013] , , , , , , , , , , , , , , , , , , , , , , , or One or more of them.

[0014] Furthermore, the above reaction also contains glucose oxidase and / or glucose.

[0015] Furthermore, the source of the above-mentioned catalase solution includes: mixing the above-mentioned glucose oxidase with the above-mentioned glucose to obtain the above-mentioned hydrogen peroxide solution.

[0016] Furthermore, the reaction concentration of the above substrate is 5-50 mg / mL; the reaction concentration of the above peroxidase is 2-20 mg / mL.

[0017] Furthermore, the reaction concentration of the glucose oxidase is 1-10 eq of the reaction concentration of the substrate; the reaction concentration of the glucose is 0.1-1 mg / mL.

[0018] Furthermore, the temperature of the above reaction is 20-40℃; the reaction time is 6-24h.

[0019] To achieve the above objective, according to a second aspect of the present invention, there is provided an application of a peroxidase in the synthesis of α-hydroxy ketone compounds, wherein the peroxidase contains a conserved region sequence, the conserved region sequence including the amino acid sequence shown in SEQ ID NO: 1.

[0020] Furthermore, the above-mentioned conserved region sequence includes any one of the amino acid sequences shown in SEQ ID NOs: 2, 3 or 4.

[0021] Further, the aforementioned peroxidase is derived from any one or more of the following strains: Pterula gracilis, Pterulicium gracile, Collybiopsis luxurians FD-317 M1, Armillaria solidipes, Dendrothele bispora CBS 962.96, Moniliophthora roreri, Cercospora zeae-maydis, Dothistroma septosporum NZE10, Aspergillus niger CBS 101883, Daldiniacaldariorum, Monilinia fructicola, Fusarium sporotrichioides, Pyriculariaoryzae, Gaeumannomyces tritici R3-111a-1, Pestalotiopsis fici W106-1, Hypoxylonsp. EC38, Aspergillus saccharolyticus JOP 1030-1, Aspergillus nidulans FGSC A4, Truncatella angustata, Penicillium steckii, Aspergillus sydowii CBS 593.65, Madurella mycetomatis, Aspergillus bombycis, Aspergillus sclerotialis, Helicocarpus griseus, Colletotrichum scovillei, Macrophomina phaseolina MS6, Phialocephala subalpina, Aureobasidium pullulans, Aaosphaeria arxii CBS 175.79, Rutstroemia sp. NJR-2017a, Hortaea werneckii, Monilinia fructigena, Hypoxylonsp. CI-4A, Daldinia eschscholtzii, Pestalotiopsis fici W106-1, Viridotheliumvirens, Aspergillus alliaceus, Thermothelomyces thermophilus, Monosporascus sp.mg162, Monosporascus sp. CRB-9-2, Aspergillus brasiliensis CBS 101740, Alternaria tenuissima, Decorospora gaudefroyi, Pyrenophora seminiperda or Fusarium graminearum. .

[0022] Further, the peroxidases described above include: (a) any of the proteins shown in SEQ ID NOs: 5-51; or (b) proteins containing the conserved region sequence described above and having more than 70% homology with the amino acid sequence defined in (a) and having peroxidase activity.

[0023] Furthermore, the above applications include: using the above-mentioned peroxidase to catalyze the reaction of the substrate to obtain the above-mentioned α-hydroxy ketone compound; the structural formula of the above-mentioned substrate is as follows: The structural formula of the above α-hydroxy ketone compound is as follows: R1, R2, or R3 are each independently selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R1 and R2 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R3 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R2 and R3 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R1 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl.

[0024] By applying the technical solution of this invention, the target product, an α-hydroxy ketone compound, can be obtained by reacting the substrate and peroxidase of this application in a system containing hydrogen peroxide. Compared with existing synthetic methods, the synthetic method of this application is simple in steps, mild in reaction conditions, and uses readily available and low-cost raw materials and enzymes. Furthermore, compared with traditional synthetic methods for α-hydroxy ketone compounds, it has advantages such as low cost, environmental friendliness, and high efficiency. Therefore, the method of this application is more suitable for the industrial production of α-hydroxy ketone compounds. Attached Figure Description

[0025] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0026] Figure 1 A schematic diagram of the synthesis of α-hydroxyketone compounds according to an embodiment of the present invention is shown. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0028] As mentioned in the background section, existing methods for synthesizing α-hydroxy ketone compounds, particularly chemical synthesis methods, suffer from low yields, use of toxic reagents, and the use of expensive and air-sensitive organometallic catalysts. Therefore, these methods are unsuitable for industrial-scale production. In this application, the inventors attempt to develop a novel method for synthesizing α-hydroxy ketone compounds. This method features a simple synthesis process, mild reaction conditions, readily available and low-cost raw materials. Compared to existing synthetic routes, this method is simpler, more environmentally friendly, and more efficient, making it suitable for industrial-scale production. Therefore, this application proposes a series of protective measures.

[0029] In this application, the term "substituted or unsubstituted" refers to the substitution of one or more hydrogen atoms on the original group by one or more substituents selected from the following: deuterium; halogen group; nitrile group; nitro group; hydroxyl group; acetyl group; carbonyl group; ester group; imide group; amino group; phosphine oxide group; alkoxy group; aryloxy group; alkylthio group; arylthio group; alkylsulfonyl group; silyl group; boron group; alkyl group; cycloalkyl group; alkenyl group; aryl group; aralkyl group; arylenyl group; alkylamine group; aralkylamine group; heteroarylamine group; arylamine group; arylphosphine group; or a heterocyclic group containing at least one of N, O, and S, or without substituents, or substituted by substituents linked by two or more substituents of the exemplified substituents, or without substituents. For example, the term "substituents linked by two or more substituents" can be biphenyl. That is, biphenyl can be an aromatic group, or can be interpreted as substituents linked by two phenyl groups.

[0030] In this application, "aromatic group" refers to aryl, (aryeinyl)aryl, or heteroaryl, where "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon derived from aromatic hydrocarbons, including phenyl, biphenyl, triphenyl, naphthyl, binatyl, phenylnaphthyl, naphthylphenyl, geniyl, phenylgeniyl, benzo[a]geniyl, dibenzo[a]geniyl, phenanthrene, phenylphenanthrene, anthracene, indole, triphenylene, pyrene, fused tetraphenyl, peryl, kinel, naphthanoyl, and propadienylgeniyl.

[0031] In this application, "heteroaryl" refers to a heteroaryl or (sub)heteroaryl group, which is an aryl group containing at least one heteroatom selected from the group consisting of N, O, and S in the ring backbone. It can be a monocyclic ring such as furanyl, thiopheneyl, pyrroleyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, triazinyl, tetraazinyl, triazolyl, tetrazolyl, furazolidyl, or pyridine. It can be a group consisting of pyrazinyl, pyrimidinyl, pyridazinyl, etc., or a fused ring condensed with at least one benzene ring, such as benzofuranyl, benzothiopheneyl, isobenzofuranyl, dibenzofuranyl, dibenzothiopheneyl, benzimidazolyl, benzothiazolyl, benzoisothiazolyl, benzoisooxazolyl, quinolinyl, isoquinolinyl, cinolinyl, quinazolinyl, quinoxolinyl, carbazole, phenanthreneoxazolyl, phenadiazolyl, benzodiazepine, dihydroacridyl, etc.

[0032] In this application, "alkyl" includes straight-chain alkyl and branched-chain alkyl. C1 to C20 alkyl includes, but is not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, n-pentyl, sec-pentyl, neopentyl, n-hexyl, neohexyl, n-heptyl, n-octyl, 2-ethylhexyl, trifluoromethyl, pentafluoroethyl, 2,2,2-trifluoroethyl, etc.

[0033] In this application, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, etc.

[0034] In this application, "heterocyclic group" refers to a heterocyclic group or (sub)heterocyclic group, which is a cycloalkyl group containing a ring backbone atom selected from the group consisting of N, O, and S, and can be a monocyclic ring such as tetrahydrofuran. In this application, "alkenyl" includes, but is not limited to, vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, isopropenyl, cyclopentenyl, cyclohexenyl, propenidyl, or butadienyl.

[0035] In this application, "alkoxy" refers to a group composed of the aforementioned chain alkyl group and oxygen, or a group composed of the aforementioned cycloalkyl group and oxygen, including but not limited to methoxy, ethoxy, or propoxy.

[0036] In a first typical embodiment of this application, a method for synthesizing an α-hydroxy ketone compound is provided. The method includes: mixing a substrate and a peroxidase to react and obtain the α-hydroxy ketone compound; the structural formula of the substrate is as follows: The structural formula of the above α-hydroxy ketone compound is as follows: R1, R2, or R3 are each independently selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R1 and R2 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R3 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R2 and R3 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R1 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl.

[0037] In this application, benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl may optionally contain one or more substitutions, i.e., substituted "benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl"; or may not contain any substituents, i.e., unsubstituted "benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl".

[0038] In a preferred embodiment, the system of the above reaction also contains a hydrogen peroxide solution.

[0039] In a preferred embodiment, the heteroatoms in the heteroaryl group and the heterocyclic group are selected from one or more of nitrogen, oxygen or sulfur.

[0040] In a preferred embodiment, R1, R2, or R3 are each independently selected from... , , , , , , or One or more of them.

[0041] In a preferred embodiment, the combination of R1 and R2 to form substituted or unsubstituted cycloalkyl or benzocycloalkyl groups includes: , or One or more of them.

[0042] In a preferred embodiment, the combination of R1 and R2 to form substituted or unsubstituted cycloalkyl or benzocycloalkyl groups includes: or .

[0043] In the synthetic method of this application, under the catalysis of peroxidase, such substrates react in a system containing hydrogen peroxide in an aqueous or two-phase medium to generate α-hydroxy ketone compounds. The reaction route of this application is as follows:

[0044] .

[0045] In existing synthetic routes for α-hydroxy ketones, various methods have been developed for synthesizing α-hydroxy carbonyl compounds, including those using oxidants such as peroxides, high-valent iodine compounds, metal oxides, and N-sulfonyloxybutanes. However, most of these methods have certain limitations, such as requiring expensive transition metal catalysts or demanding reaction conditions. The recovery of reagents and solvents is difficult, the separation of post-reaction products is complex and costly, and recycling is also expensive, making them unsuitable for industrial production.

[0046] The synthetic method presented in this application is simple and economical, capable of directly synthesizing a variety of α-hydroxy ketone compounds. Furthermore, the substrate + peroxidase synthetic route in this application, which includes a hydrogen peroxide system, broadens the range of products, making it suitable for the synthesis of α-hydroxy ketone compounds with various structures. In addition, the reaction conditions are mild, the raw materials and catalysts are readily available and inexpensive, and it is environmentally friendly. Compared to existing synthetic methods, the α-hydroxy ketone compound synthesis method presented in this application has a wider substrate range, enabling the synthesis of more target products. Moreover, the reaction conditions are easily controlled, and the cost is lower, making it more suitable for industrial-scale production.

[0047] In a preferred embodiment, the peroxidase contains a conserved region sequence, which includes the amino acid sequence shown in SEQ ID NO: 1.

[0048] SEQ ID NO: 1: NXLANH.

[0049] In the sequence SEQ ID NO:1, “X” represents threonine (Thr; T), serine (Ser; S), or alanine (Ala; A).

[0050] The peroxidase described in this application exhibits high catalytic activity and good substrate compatibility when applied to the synthesis of α-hydroxy ketone compounds via the aforementioned routes, successfully synthesizing the target α-hydroxy ketone compound products. Conserved regions refer to DNA, RNA, or protein sequence fragments that have remained unchanged or changed very little during evolution. These conserved regions typically have important biological functions, such as participating in specific protein folding, catalytic activity, ligand binding, or signal transduction. Therefore, even if the overall sequence differs significantly between different species, specific functional regions can exhibit a high degree of similarity or identity.

[0051] In enzymology research and protein engineering, conserved regions are often used as important markers for identifying enzymes of the same family or function. In this application, the conserved region sequence of the peroxidase is SEQ ID NO: 1. In the peroxidase of this application, the conserved region sequence is crucial for enzyme activity or substrate recognition. The peroxidase containing the above-mentioned SEQ ID NO: 1 can catalyze the above-mentioned reaction to generate the target α-hydroxy ketone compound product.

[0052] In a preferred embodiment, the above-mentioned conserved region sequence is any one of the amino acid sequences shown in SEQ ID NOs: 2, 3 or 4 (including but not limited to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4).

[0053] SEQ ID NO: 2: NTLANH.

[0054] SEQ ID NO: 3: NSLANH.

[0055] SEQ ID NO: 4: NALANH.

[0056] In a preferred embodiment, the peroxidase is derived from any one or more of the following strains: *Pterula gracilis*, *Pterulicium gracile*, *Collybiopsis luxurians* FD-317 M1, *Armillaria solidipes*, *Dendrothele bispora* CBS 962.96, *Moniliophthora roreri*, *Cercospora zeae-maydis*, *Dothistroma septosporum* NZE10, *Aspergillus niger* CBS101883, *Daldinia caldariorum*, *Monilinia fructicola*, *Fusarium sporotrichioides*, *Pyricularia oryzae*, *Gaeumannomyces tritici* R3-111a-1, *Pestalotiopsis fici* W106-1, *Hypoxylon sp. EC38*, *Aspergillus saccharolyticus* JOP 1030-1, and *Aspergillus nidulans*. FGSC A4, Truncatella angustata, Penicillium steckii, Aspergillussydowii CBS 593.65, Madurella mycetomatis, Aspergillus bombycis, Aspergillussclerotialis, Helicocarpus griseus, Colletotrichum scovillei, Macrophominaphaseolina MS6, Phialocephala subalpina, Aureobasidium pullulans, Aaosphaeriaarxii CBS 175.79, Rutstroemia sp. NJR-2017a, Hortaea werneckii, Moniliniafructigena, Hypoxylon sp. CI-4A, Daldinia eschscholtzii, Pestalotiopsis ficiW106-1, Viridothelium virens, Aspergillus alliaceus, Thermothelomycesthermophilus, Monosporascus sp.mg162, Monosporascus sp. CRB-9-2, Aspergillusbrasiliensis CBS 101740, Alternaria tenuissima, Decorospora gaudefroyi, Pyrenophora seminiperda or Fusarium graminearum. .

[0057] In this application, Gaeumannomyces tritici R3-111a-1 includes the protein shown in SEQ ID NOs: 18 or SEQ ID NOs: 19.

[0058] In a preferred embodiment, the peroxidase comprises: (a) any of the proteins shown in SEQ ID NOs: 5-51; or (b) a protein containing the conserved region sequence described above and having more than 70% homology with the amino acid sequence defined in (a) and having peroxidase activity.

[0059] Peroxidases with more than 70% homology, including but not limited to peroxidases with homology of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even more than 99.9%, and with the same function.

[0060] In this specification, homology refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0061] The proteins that share 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even 99.9% or more) homology with the proteins provided by the above-mentioned amino acid sequences and have the same function, are likely to have the same active sites, active pockets, active mechanisms, and protein structures as the proteins provided by the above-mentioned sequences, and are homologous proteins obtained through amino acid mutation.

[0062] Obtaining sequences with the aforementioned homology can be achieved through amino acid substitution and replacement. Generally, substitutions between amino acids with similar properties produce similar effects. For ease of description, the abbreviations of the amino acid residues are listed below: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0063] Amino acid substitutions or replacements, for example, can occur in the aforementioned homologous proteins, where conserved amino acid substitutions may take place. "Conserved amino acid substitutions" include, but are not limited to:

[0064] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0065] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0066] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0067] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0068] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0069] Using the 47 peroxidases from different sources mentioned above, all of which were able to catalyze the substrate to complete the above reaction in the specific embodiments of this application, the target α-hydroxy ketone compound product was successfully synthesized.

[0070] This application discovers that the peroxidases mentioned above, including but not limited to (a) proteins with any sequence in SEQ ID NOs: 5-51; or (b) proteins containing the above-mentioned conserved region sequence and having more than 70% homology with the amino acid sequence defined in (a) and possessing peroxidase activity, exhibit high activity and catalytic efficiency in the synthesis of α-hydroxy ketone compounds, enabling the successful synthesis of the target α-hydroxy ketone compound. The conserved region sequence of the above-mentioned peroxidase, SEQ ID NO: 1, has a significant impact on the catalytic activity of the enzyme and the conversion rate of the substrate during the reaction. The discovery of these enzymes is beneficial for further exploring synthetic routes for α-hydroxy ketone compounds, pushing the boundaries of α-hydroxy ketone compound synthesis technology, and has broad application prospects in the field of α-hydroxy ketone compound synthesis.

[0071] In a preferred embodiment, the substrate includes: , , , , , , , , , , , , , , , , , , , , , , , or One or more of them.

[0072] In this application, the inventors discovered that by using the above-mentioned peroxidase to catalyze the reaction of a substrate having the above-mentioned structure in a system containing hydrogen peroxide solution, the target product α-hydroxy ketone compound can be prepared effectively, and this method is well applicable to the industrial-scale production of α-hydroxy ketone compounds. In this application, the specific substrates and the structures of the corresponding α-hydroxy ketone compounds are shown in Table 1 below.

[0073] Table 1

[0074]

[0075] In a preferred embodiment, the reaction solution also contains glucose oxidase and / or glucose.

[0076] In a preferred embodiment, the source of the hydrogen peroxide solution includes: mixing the glucose oxidase with the glucose to obtain the hydrogen peroxide solution.

[0077] In this application, the inventors discovered that when the above-mentioned reaction solution contains, but is not limited to, glucose oxidase and / or glucose, hydrogen peroxide can be stably provided in the reaction of the above-mentioned peroxidase catalyzing the substrate to produce the target product α-hydroxy ketone compound, thereby achieving the technical effect of improving the reaction efficiency of α-hydroxy ketone compound preparation. Therefore, in this application, the inventors preferably use the reaction of glucose oxidase with glucose to prepare hydrogen peroxide.

[0078] In a preferred embodiment, the reaction concentration of the above-mentioned substrate is 5-50 mg / mL (including but not limited to 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL). mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL or 50 mg / mL; the reaction concentration of the above peroxidase is 2-20 mg / mL (including but not limited to 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL or 20 mg / mL).

[0079] In a preferred embodiment, the reaction concentration of the glucose oxidase is 1-10 eq (including but not limited to 1 eq, 2 eq, 3 eq, 4 eq, 5 eq, 6 eq, 7 eq, 8 eq, 9 eq or 10 eq) of the reaction concentration of the substrate; and the reaction concentration of the glucose is 0.1-1 mg / mL (including but not limited to 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL).

[0080] In a preferred embodiment, the temperature of the above reaction is 20-40°C (including but not limited to 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C); the reaction time is 6-24 hours (including but not limited to 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours).

[0081] Under the aforementioned control conditions, the parameters in the reaction system of this application can promote the forward reaction and synthesize the corresponding products. Those skilled in the art can adjust the parameters in the reaction system according to actual production needs and substrate properties to improve reaction efficiency.

[0082] In a preferred embodiment, the method for synthesizing the α-hydroxy ketone compound of this application further includes: mixing a substrate, an auxiliary agent, a peroxidase, and a reaction solution to react and obtain the α-hydroxy ketone compound.

[0083] The aforementioned auxiliaries include, but are not limited to, one or more of the following known to those skilled in the art: solubilizers (including but not limited to DMSO, DMF, or THF), surfactants (including but not limited to one or more of the Tween series, Span series, or Pluronic F-68), buffers (including but not limited to one or more of Tris-HCl buffer, phosphate buffer, and HEPES buffer), or chelating agents (including but not limited to EDTA). Substrate containing a pyridine ring exhibits good solubility and is soluble in ammonia solution; however, some substrates have poor solubility, and the addition of the aforementioned solubilizers known to those skilled in the art is beneficial to the reaction. Those skilled in the art can flexibly select and adjust the auxiliaries according to the actual properties of the substrate and production conditions.

[0084] In a second typical embodiment of this application, an application of a peroxidase in the synthesis of α-hydroxy ketone compounds is provided. The peroxidase contains a conserved region sequence, which includes the amino acid sequence shown in SEQ ID NO: 1.

[0085] The peroxidase described in this application exhibits high catalytic activity and good substrate compatibility when applied to the synthesis of α-hydroxy ketone compounds via the aforementioned routes, successfully synthesizing the target α-hydroxy ketone compound products. Conserved regions refer to DNA, RNA, or protein sequence fragments that have remained unchanged or changed very little during evolution. These conserved regions typically have important biological functions, such as participating in specific protein folding, catalytic activity, ligand binding, or signal transduction. Therefore, even if the overall sequence differs significantly between different species, specific functional regions can exhibit a high degree of similarity or identity.

[0086] In enzymology research and protein engineering, conserved regions are often used as important markers for identifying enzymes of the same family or function. In this application, the conserved region sequence of the peroxidase is SEQ ID NO: 1. In the peroxidase of this application, the conserved region sequence is crucial for enzyme activity or substrate recognition. The peroxidase containing the above-mentioned SEQ ID NO: 1 can catalyze the above-mentioned reaction to generate the target α-hydroxy ketone compound product.

[0087] In a preferred embodiment, the conserved region sequence includes any one of the amino acid sequences shown in SEQ ID NOs: 2, 3 or 4 (including but not limited to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4). In a preferred embodiment, the peroxidase is derived from any one or more of the following strains: *Pterula gracilis*, *Pterulicium gracile*, *Collybiopsis luxurians* FD-317 M1, *Armillaria solidipes*, *Dendrothele bispora* CBS 962.96, *Moniliophthora roreri*, *Cercospora zeae-maydis*, *Dothistroma septosporum* NZE10, *Aspergillus niger* CBS 101883, *Daldinia caldariorum*, *Monilinia fructicola*, *Fusarium sporotrichioides*, *Pyricularia oryzae*, *Gaeumannomyces tritici* R3-111a-1, *Pestalotiopsis fici* W106-1, *Hypoxylonsp. EC38*, *Aspergillus saccharolyticus* JOP 1030-1, and *Aspergillus nidulans*. FGSC A4, Truncatella angustata, Penicillium steckii, Aspergillus sydowii CBS 593.65, Madurella mycetomatis, Aspergillus bombycis, Aspergillus sclerotialis, Helicocarpus griseus, Colletotrichum scovillei, Macrophomina phaseolina MS6, Phialocephala subalpina, Aureobasidium pullulans, Aaosphaeria arxii CBS 175.79, Rutstroemia sp. NJR-2017a, Hortaea werneckii, Monilinia fructigena, Hypoxylonsp.CI-4A, Daldinia eschscholtzii, Pestalotiopsis fici W106-1, Viridotheliumvirens, Aspergillus alliaceus, Thermothelomyces thermophilus, Monosporascus sp.mg162, Monosporascus sp. CRB-9-2, Aspergillus brasiliensis CBS 101740, Alternaria tenuissima, Decorospora gaudefroyi, Pyrenophora seminiperda or Fusarium graminearum. .

[0088] In a preferred embodiment, the peroxidase comprises (a) any of the proteins shown in SEQ ID NOs: 5-51; or (b) a protein containing the conserved region sequence described above and having more than 70% homology with the amino acid sequence defined in (a) and having peroxidase activity.

[0089] This application discovers that the peroxidases mentioned above, including but not limited to (a) proteins with any sequence in SEQ ID NOs: 5-51; or (b) proteins containing the above-mentioned conserved region sequence and having more than 70% homology with the amino acid sequence defined in (a) and possessing peroxidase activity, exhibit high activity and catalytic efficiency in the synthesis of α-hydroxy ketone compounds, enabling the successful synthesis of the target α-hydroxy ketone compound. The conserved region sequence of the above-mentioned peroxidase, SEQ ID NO: 1, has a significant impact on the catalytic activity of the enzyme and the conversion rate of the substrate during the reaction. The discovery of these enzymes is beneficial for further exploring synthetic routes for α-hydroxy ketone compounds, pushing the boundaries of α-hydroxy ketone compound synthesis technology, and has broad application prospects in the field of α-hydroxy ketone compound synthesis.

[0090] In a preferred embodiment, the substrate includes: , , , , , , , , , , , , , , , , , , , , , , , or One or more of them.

[0091] In this application, the inventors discovered that by using the above-mentioned peroxidase to catalyze the reaction of a substrate having the above-mentioned structure in a system containing hydrogen peroxide solution, the target product α-hydroxy ketone compound can be prepared effectively, and this method is well applicable to the industrial-scale production of α-hydroxy ketone compounds. In this application, the specific substrates and the structures of the corresponding α-hydroxy ketone compounds are shown in Table 1.

[0092] In a preferred embodiment, the application includes: using the peroxidase to catalyze a reaction of the substrate to obtain the α-hydroxy ketone compound; the structural formula of the substrate is as follows: The structural formula of the above α-hydroxy ketone compound is as follows: R1, R2, or R3 are each independently selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R1 and R2 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R3 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl; or R2 and R3 are combined to form substituted or unsubstituted cycloalkyl or benzocycloalkyl, and R1 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl.

[0093] In a preferred embodiment, the system of the above reaction also contains a hydrogen peroxide solution.

[0094] In a preferred embodiment, the heteroatoms in the heteroaryl group and the heterocyclic group are selected from one or more of nitrogen, oxygen or sulfur.

[0095] In a preferred embodiment, R1, R2, or R3 are each independently selected from... , , , , , , or One or more of them.

[0096] In a preferred embodiment, the combination of R1 and R2 to form substituted or unsubstituted cycloalkyl or benzocycloalkyl groups includes: , or One or more of them.

[0097] In a preferred embodiment, the combination of R1 and R2 to form substituted or unsubstituted cycloalkyl or benzocycloalkyl groups includes: or .

[0098] In the synthetic method of this application, under the catalysis of peroxidase, such substrates react in a system containing hydrogen peroxide in an aqueous or two-phase medium to generate α-hydroxy ketone compounds. The reaction route of this application is as follows:

[0099] .

[0100] In existing synthetic routes for α-hydroxy ketones, various methods have been developed for synthesizing α-hydroxy carbonyl compounds, including those using oxidants such as peroxides, high-valent iodine compounds, metal oxides, and N-sulfonyloxybutanes. However, most of these methods have certain limitations, such as requiring expensive transition metal catalysts or demanding reaction conditions. The recovery of reagents and solvents is difficult, the separation of post-reaction products is complex and costly, and recycling is also expensive, making them unsuitable for industrial production.

[0101] The synthetic method presented in this application is simple and economical, capable of directly synthesizing a variety of α-hydroxy ketone compounds. Furthermore, the substrate + peroxidase synthetic route in this application, which includes a hydrogen peroxide system, broadens the range of products, making it suitable for the synthesis of α-hydroxy ketone compounds with various structures. In addition, the reaction conditions are mild, the raw materials and catalysts are readily available and inexpensive, and it is environmentally friendly. Compared to existing synthetic methods, the α-hydroxy ketone compound synthesis method presented in this application has a wider substrate range, enabling the synthesis of more target products. Moreover, the reaction conditions are easily controlled, and the cost is lower, making it more suitable for industrial-scale production.

[0102] This application discovers that peroxidases from the aforementioned sources, including but not limited to (a) proteins with any sequence in SEQ ID NOs: 5-51; or (b) proteins containing the aforementioned conserved region sequence and having more than 70% homology with the amino acid sequence defined in (a) and possessing peroxidase activity, exhibit high activity and catalytic efficiency in the synthesis of α-hydroxy ketone compounds, enabling successful synthesis of the target α-hydroxy ketone compound. The conserved region sequence of the aforementioned peroxidase, SEQ ID NO: 1, has a significant impact on the enzyme's catalytic activity and substrate conversion rate during the reaction. The discovery of these enzymes facilitates further exploration of synthetic routes for α-hydroxy ketone compounds, pushes the boundaries of α-hydroxy ketone compound synthesis technology, and has broad application prospects in the field of α-hydroxy ketone compound synthesis.

[0103] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0104] Example 1

[0105] Screening for wild-type peroxidases:

[0106] Select wild-type peroxidases from the enzyme library and test their effects on... The catalytic activity of substrate 1 was investigated to synthesize the corresponding α-hydroxy ketone compound. The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 1, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30 °C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC (Gas Chromatography). The results showed that 47 wild-type peroxidases in the enzyme library exhibited activity against substrate 1, while the remaining wild-type peroxidases in the library showed no activity or very low activity against substrate 1 (some are shown below). The added mass of peroxidase and glucose oxidase refers to the added mass of pure enzymes; the conversion rate in Table 2 represents the activity against substrate 1.

[0107] In this application, 10 eq glucose means that the concentration of glucose is 10 times the reaction concentration of the substrate.

[0108] In this application, the conversion rate is calculated as: (mass of product / mass of added substrate) × 100%.

[0109] The specific results are shown in Table 2 below.

[0110] Table 2

[0111]

[0112] In Table 2, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, conversion rates of 80-90% are recorded as +++, and conversion rates of 0-0.01% (excluding the endpoint value of 0.01%) are recorded as -.

[0113] The wild-type peroxidases whose conversion rates are marked as "-" in Table 2 are enzymes recorded in the NCBI database. The numbers in parentheses are the enzyme numbers in the NCBI database.

[0114] Example 2

[0115] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 2):

[0116] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 2, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0117] The specific experimental results are shown in Table 3.

[0118] Table 3

[0119]

[0120] In Table 3, conversion rates of 50-70% (excluding the endpoint value of 70%) are marked as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are marked as ++, and conversion rates of 80-90% are marked as +++.

[0121] Example 3

[0122] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 3):

[0123] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 3, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0124] The specific experimental results are shown in Table 4 below.

[0125] Table 4

[0126]

[0127] In Table 4, conversion rates of 50-70% (excluding the endpoint value of 70%) are marked as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are marked as ++, and conversion rates of 80-90% are marked as +++.

[0128] Example 4

[0129] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 4):

[0130] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 4, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0131] The specific experimental results are shown in Table 5 below.

[0132] Table 5

[0133]

[0134] In Table 5, conversion rates of 50-70% (excluding the endpoint value of 70%) are marked as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are marked as ++, and conversion rates of 80-90% are marked as +++.

[0135] Example 5

[0136] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 5).

[0137] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 5, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0138] The specific experimental results are shown in Table 6 below.

[0139] Table 6

[0140]

[0141] In Table 6, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0142] Example 6

[0143] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 6):

[0144] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 6, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0145] The specific experimental results are shown in Table 7 below.

[0146] Table 7

[0147]

[0148] In Table 7, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0149] Example 7

[0150] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 7):

[0151] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 7, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0152] The specific experimental results are shown in Table 8 below.

[0153] Table 8

[0154]

[0155] In Table 8, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0156] Example 8

[0157] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 8):

[0158] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 8, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0159] The specific experimental results are shown in Table 9 below.

[0160] Table 9

[0161]

[0162] In Table 9, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0163] Example 9

[0164] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 9):

[0165] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 9, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0166] The specific experimental results are shown in Table 10 below.

[0167] Table 10

[0168]

[0169] In Table 10, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0170] Example 10

[0171] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 10):

[0172] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 10, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0173] The specific experimental results are shown in Table 11 below.

[0174] Table 11

[0175]

[0176] In Table 11, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0177] Example 11

[0178] The catalytic activity of the peroxidase screened in Example 1 on substrate 11 was tested:

[0179] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 11, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0180] The specific experimental results are shown in Table 12 below.

[0181] Table 12

[0182]

[0183] In Table 12, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0184] Example 12

[0185] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 12):

[0186] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 12, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0187] The specific experimental results are shown in Table 13 below.

[0188] Table 13

[0189]

[0190] In Table 13, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0191] Example 13

[0192] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 13):

[0193] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 13, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0194] The specific experimental results are shown in Table 14 below.

[0195] Table 14

[0196]

[0197] In Table 14, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0198] Example 14

[0199] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 14):

[0200] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 14, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0201] The specific experimental results are shown in Table 15 below.

[0202] Table 15

[0203]

[0204] In Table 15, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0205] Example 15

[0206] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 15):

[0207] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 15, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0208] The specific experimental results are shown in Table 16 below.

[0209] Table 16

[0210]

[0211] In Table 16, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0212] Example 16

[0213] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 16):

[0214] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 16, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0215] The specific experimental results are shown in Table 17 below.

[0216] Table 17

[0217]

[0218] In Table 17, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0219] Example 17

[0220] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 17):

[0221] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 17, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0222] The specific experimental results are shown in Table 18 below.

[0223] Table 18

[0224]

[0225] In Table 18, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0226] Example 18

[0227] Using the peroxidase screened in Example 1, the effects on... Catalytic activity of (substrate 18):

[0228] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 18, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0229] The specific experimental results are shown in Table 19 below.

[0230] Table 19

[0231]

[0232] In Table 19, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0233] Example 19

[0234] The catalytic activity of the peroxidase screened in Example 1 on substrate 19 was tested:

[0235] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 19, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0236] The specific experimental results are shown in Table 20 below.

[0237] Table 20

[0238]

[0239] In Table 20, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0240] Example 20

[0241] The catalytic activity of the peroxidase screened in Example 1 on substrate 20 was tested:

[0242] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 20, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0243] The specific experimental results are shown in Table 21 below.

[0244] Table 21

[0245]

[0246] In Table 21, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0247] Example 21

[0248] Using the peroxidases screened in Example 1, the catalytic activity against substrates 21, 22, 23, 24, or 25 was tested:

[0249] The reaction system and conditions were as follows: 1 mL of the system contained 5 mg of substrate 21, 22, 23, 24, or 25, 20 mg of peroxidase, 10 eq of glucose, 0.1 mg of glucose oxidase, and 100 mM phosphate buffer. The reaction was carried out at 30°C for 16 h. After extraction with ethyl acetate, the ethyl acetate solution was analyzed by GC. The added mass of peroxidase and glucose oxidase refers to the mass of pure enzymes.

[0250] The specific experimental results are shown in Table 22 below.

[0251] Table 22

[0252]

[0253] In Table 22, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0254] Example 22

[0255] Reaction system optimization:

[0256] In Examples 1 to 21, the reaction systems used a glucose oxidase + glucose oxygen supply method. The core function was the in-situ, continuous generation of hydrogen peroxide (H₂O₂) to provide a stable oxygen donor for UPO (Unspecific Peroxygenase), with hydrogen peroxide being the direct oxygen donor. This study attempted to replace the glucose oxidase + glucose oxygen supply method by directly adding hydrogen peroxide to the reaction system. The conditions were as follows: 1 mL of the system contained 5 mg of substrate 1, 20 mg of peroxidase, 10 eq of glucose, and 100 mM phosphate buffer. Hydrogen peroxide was added to the system at a rate of 0.1-1 eq (substrate molar equivalents) / h, and the reaction was carried out at 30°C for 16 h. The reaction system was extracted with ethyl acetate and then analyzed by GC. The results showed that adding hydrogen peroxide in a fed-batch manner, instead of the glucose oxidase + glucose oxygen supply method, still maintained the same catalytic effect. The mass of peroxidase added refers to the mass of pure enzyme added.

[0257] Since the principle is the same, it can be inferred that the process of adding hydrogen peroxide can also be used for other substrate reactions.

[0258] The specific experimental results are shown in Table 23 below.

[0259] Table 23

[0260]

[0261] In Table 23, conversion rates of 50-70% (excluding the endpoint value of 70%) are recorded as +, conversion rates of 70-80% (excluding the endpoint value of 80%) are recorded as ++, and conversion rates of 80-90% are recorded as +++.

[0262] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0263] By utilizing the peroxidase and its mutants of this application, it is possible to react the substrate under mild conditions in a system containing hydrogen peroxide solution to generate α-hydroxy ketone compounds. Furthermore, the substrate, enzyme, and reaction reagents of this application are readily available and inexpensive. The synthesis of α-hydroxy ketone compounds can be achieved through simple mixing, resulting in improved substrate conversion. Compared to existing synthetic routes and methods for α-hydroxy ketone compounds, the synthetic method of this application is simpler, has higher yields, lower costs, and produces less solvent that is easier to handle, making it more suitable for industrial-scale production.

[0264] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing an α-hydroxy ketone compound, characterized in that, The synthesis method includes: The substrate and peroxidase are mixed and reacted to obtain the α-hydroxy ketone compound; The structural formula of the substrate is as follows: ； The structural formula of the α-hydroxy ketone compound is as follows: ； Wherein, R1, R2, or R3 are each independently selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl groups; or R1 and R2 combine to form a substituted or unsubstituted cycloalkyl or benzocycloalkyl group, and R3 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl groups; or R2 and R3 combine to form a substituted or unsubstituted cycloalkyl or benzocycloalkyl group, where R1 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl groups; The substrate is: , , , , , , , , , , , , , , , , , , , , , , , or One or more of the following; The peroxidase contains a conserved region sequence, which is the amino acid sequence shown in SEQ ID NO: 1; The conserved region sequence is any one of the amino acid sequences shown in SEQ ID NOs: 2, 3 or 4; The peroxidase is any one of the proteins shown in SEQ ID NOs: 5-51; The reaction system also contains hydrogen peroxide solution.

2. The synthesis method according to claim 1, characterized in that, The reaction also contains glucose oxidase and / or glucose.

3. The synthesis method according to claim 2, characterized in that, The hydrogen peroxide solution is obtained by mixing the glucose oxidase with the glucose.

4. The synthesis method according to claim 1, characterized in that, The reaction concentration of the substrate is 5-50 mg / mL; The reaction concentration of the peroxidase is 2-20 mg / mL.

5. The synthesis method according to claim 3, characterized in that, The reaction concentration of glucose oxidase is 1-10 eq of the reaction concentration of the substrate; The concentration of glucose in the reaction is 0.1-1 mg / mL.

6. The synthesis method according to claim 1, characterized in that, The reaction temperature is 20-40℃; The reaction time is 6-24 hours.

7. The application of peroxidase in the synthesis of α-hydroxyketone compounds, characterized in that, The peroxidase contains a conserved region sequence, which includes the amino acid sequence shown in SEQ ID NO: 1; The application includes: using the peroxidase to catalyze a reaction of a substrate to obtain the α-hydroxy ketone compound; The structural formula of the substrate is as follows: ； The structural formula of the α-hydroxy ketone compound is as follows: ； Wherein, R1, R2, or R3 are each independently selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkenyl, alkoxy, cycloalkyl, heterocyclic, aromatic, or heteroaryl groups; or R1 and R2 combine to form a substituted or unsubstituted cycloalkyl or benzocycloalkyl group, and R3 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl groups; or R2 and R3 combine to form a substituted or unsubstituted cycloalkyl or benzocycloalkyl group, where R1 is selected from hydrogen, substituted or unsubstituted benzyl, alkyl, alkoxy, cycloalkyl, heterocyclic, aromatic or heteroaryl groups; The substrate is: , , , , , , , , , , , , , , , , , , , , , , , or One or more of the following; The conserved region sequence is any one of the amino acid sequences shown in SEQ ID NOs: 2, 3 or 4; The peroxidase includes any of the proteins shown in SEQ ID NOs: 5-51.

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