Method for synthesizing chiral amine compounds

Abstract

A method for synthesizing chiral amine compounds is provided, in which an aminotransferase is used to carry out an amination reaction on a ketone substrate under the action of an amino donor to obtain a chiral amine compound, and the conservative amino acid sequence region of the aminotransferase includes at least region 1 MAGLWCVN and region 2 YNTFFKT. By employing an aminotransferase having a specific conservative amino acid sequence region to synthesize a highly sterically hindered chiral amine, the enzyme catalysis reaction volume is small, the synthesis route is short, the product yield is high, and there is no need to use expensive precious metal catalysts in the synthesis conditions, which reduces waste liquid, waste gas and solid waste, and saves production costs. [Case 1] TIFF2024506006000027.tif34143

Description

[Technical field]

[0001] The present invention relates to the field of chiral amine synthesis, and in particular to a method for the synthesis of chiral amine compounds. [Background technology]

[0002] Chiral amine compounds with large steric hindrance (in this application, this refers to a type of compound in which the group next to the potential chiral carbonyl group is larger than a methyl group) are an important type of chiral intermediate that are widely used in the synthesis of optically active substances, functional molecules, drugs, and ligands. However, there are few reports on the synthesis of such chiral compounds, and satisfactory results have not been obtained. For example, the selectivity of phenyl-tert-butylamino ester with large steric hindrance on one side of the carbonyl group is 76% (Angew. Chem. Int. Ed. 2007, 46, 4367). Currently, it has been reported that some metal catalysts, such as ruthenium, rhodium, and palladium, all have good stereoselectivity for asymmetric hydrogenation reactions with small steric hindrance (in this application, this refers to a type of compound in which the group next to the potential chiral carbonyl group is H or a methyl group), but the catalytic effect becomes worse as the proximal steric hindrance of the carbonyl group becomes larger.

[0003] Asymmetric hydrogenation is a kind of technology for producing fine chemicals and prodrugs, but this technology has some disadvantages, such as the need to use high pressure hydrogen gas, which poses a big challenge to the safety of production, and the need for expensive transition metal catalysts, which are toxic and need to be carefully handled and removed in the post-processing process. In addition, this process requires a lot of effort and materials to screen the corresponding ligands. In many cases, this process has the problem of low stereoselectivity (Science, 2010, 329, 305). Some reactions also require protection and deprotection of active groups.

[0004] Thus, because the prior art synthetic methods are expensive, require protection and deprotection of active groups, have harsh reaction conditions, and use noble metal catalysts, there is a need to provide new synthetic methods. Summary of the Invention [Problem to be solved by the invention]

[0005] The main objective of the present invention is to provide a method for synthesizing chiral amine compounds, so as to solve the problems of the prior art that the cost of the method for synthesizing chiral amine compounds is high and the reaction conditions are harsh. [Means for solving the problem]

[0006] In order to achieve the above object, according to one aspect of the present invention, there is provided a method for synthesizing a chiral amine compound, the method comprising: employing an aminotransferase to carry out an amino group transfer reaction on a ketone substrate represented by formula I under the action of an amino group donor to obtain a chiral amine compound; [ka] Ar1 represents a substituted or unsubstituted aryl group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heteroarylene group; Ar2 represents a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted linear alkyl group or heteroalkyl group; and optionally R, where R represents a substituted or unsubstituted alkylene group having 1 to 5 carbon atoms, and R is linked to Ar1 to form a ring, and the substituents in the substituted aryl group, the substituted arylene group, the substituted heteroarylene group, and the substituted alkylene group are each independently and each heteroatom in the heteroarylene group and the heteroalkyl group is independently selected from N, O, or S, wherein the aminotransferase is one type of aminotransferase having a conserved amino acid sequence region, the conserved amino acid sequence region includes at least a region 1 and a region 2, the conserved amino acid sequence of the region 1 is MAGLWCVN, and the conserved amino acid sequence of the region 2 is YNTFFKT, and the aminotransferase is a Chromobacterium sp. violaceum and any one of the aminotransferases shown in SEQ ID NO: 2 to 55 in which W and / or C in the conserved amino acid sequence in region 1 is mutated to A, or the aminotransferase is an aminotransferase that is of the same origin as any one of the aminotransferases shown in SEQ ID NO: 1 to 55 and has a homology of 75%, preferably 85%, and more preferably 95% or more, provided that the aminotransferase has region 1 and region 2.

[0007] Further, the aminotransferase is any one of the aminotransferases set forth in SEQ ID NO:1 to SEQ ID NO:55.

[0008] Further, the substituent in the substituted aryl or arylene group is a halogen or -S-CH3, and the halogen or -S-CH3 is located at any one or more of the ortho, meta or para positions of the aryl or arylene group, and preferably the halogen is F, Cl or Br.

[0009] Furthermore, Ar1 represents a substituted or unsubstituted aryl group or arylene group, Ar2 represents an unsubstituted aryl group, R represents an unsubstituted alkylene group having 1 to 5 C atoms, and R is linked to Ar1 to form a ring.

[0010] Furthermore, Ar1 represents an unsubstituted heteroarylene group, Ar2 represents an aryl group substituted with a halogen, R represents an alkylene group having 1 to 5 carbon atoms substituted with a hydroxyl group, and R is linked to Ar1 to form a ring.

[0011] Furthermore, Ar1 represents an arylene group substituted with a halogen and an amino group, and Ar2 is a cycloalkyl group having 3 to 8 carbon atoms.

[0012] Furthermore, Ar1 represents an unsubstituted cycloalkyl group or an aryl group, and Ar2 represents a substituted or unsubstituted cycloalkyl group or an aryl group.

[0013] Furthermore, Ar1 represents a cycloalkyl group or an aryl group substituted with a hydroxyl group, a methyl group, an ethyl group, or a -S-CH3 group, and Ar2 is an unsubstituted aryl group or an unsubstituted alkyl group.

[0014] Further, the ketone substrate is as follows: [ka]

[0015] Further, the amino group donor is isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine or aniline. Effect of the Invention

[0016] By applying the technical solution of the present invention, an aminotransferase having a specific conservative amino acid sequence region is adopted to synthesize chiral amines with large steric hindrance, which reduces the enzyme catalysis reaction volume, shortens the synthesis route, and increases the product yield. It is not necessary to use expensive precious metal catalysts in the synthesis conditions, and the waste liquid, waste gas and solid waste are greatly reduced, thereby saving production costs. [Brief description of the drawings]

[0017] The accompanying drawings, which form a part of this application, are intended to provide a further understanding of the present invention, and the illustrative embodiments of the present invention and the description thereof are intended to interpret the present invention and are not intended to unduly limit the present invention. The drawings are as follows: [Figure 1] 1 shows a schematic diagram of partial results of a multiple sequence (SEQ ID NO: 1 and SEQ ID NO: 2 to SEQ ID NO: 19) alignment according to an embodiment of the present invention. [Diagram 2] 1 shows a schematic diagram of partial results of a multiple sequence (SEQ ID NO: 19 to SEQ ID NO: 37) alignment according to an embodiment of the present invention. [Diagram 3] 1 shows a schematic diagram of partial results of a multiple sequence (SEQ ID NO: 38 to SEQ ID NO: 55) alignment according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In addition, the embodiments and features of the embodiments in the present application can be combined with each other as long as they are not inconsistent. The present invention will be described in detail below with reference to examples.

[0019] In this application, in order to solve the problems of the prior art that the synthesis method of chiral amines with large steric hindrance is expensive and the reaction conditions are harsh, the inventors have attempted to adopt a bioenzyme method to synthesize them, but since aminotransferases naturally have two pockets, large and small, structurally, there is a significant limit to the substrates that can be converted, and no aminotransferases with catalytic activity for chiral amine compounds with large steric hindrance have been reported in the prior art. In order to improve this situation, the inventors have screened a large number of existing reported aminotransferases and screened more than several thousand wild-type aminotransferases, and although most of them have no catalytic activity (i.e., the target product is not detected at all), some aminotransferases that can catalyze the synthesis of such chiral amine compounds with large steric hindrance have been found, which, although their activity is low, provide at least a potential basis for improvement.

[0020] These aminotransferases are the wild-type CvTA aminotransferase of Chromobaterium violaceum (SEQ ID NO: 1), the congener and heterogeneous aminotransferase of the wild-type CvTA aminotransferase, and the heterogeneous aminotransferase of the wild-type CvTA aminotransferase. Whether the wild-type aminotransferase is congener and heterogeneous with the CvTA aminotransferase (shown in Table 1) or heterogeneous with the CvTA aminotransferase (shown in Table 2), these wild-type aminotransferases have a certain activity to catalyze the conversion of a highly sterically hindered substrate to a chiral amine compound, although the activity is low (less than 1%).

[0021] The sequence of SEQ ID NO:1 is as follows (459 amino acids): [ka]

[0022] The inventors further analyzed the sequences of aminotransferases having the activity of the relevant portion and discovered that the aminotransferases of the relevant portion have multiple highly conserved amino acid regions, and as shown in Figures 1, 2 and 3, the sequence homology of these conserved regions is 95% or more, in some cases 98% or more, and in some cases even reaching 100%.

[0023] Furthermore, the inventors have found through experiments that these conserved amino acid regions are common to all aminotransferases in the corresponding regions, which all have catalytic activity for catalyzing the synthesis of chiral amines with large steric hindrance. To further verify this hypothesis, the inventors have found that by selecting and substituting one or several amino acids from the above common conserved amino acid regions and mutating these conserved amino acid regions, aminotransferase mutants with improved catalytic activity can be obtained. Thus, the inventors have proven that they have found an aminotransferase that catalyzes the synthesis of chiral amines with large steric hindrance.

[0024] Based on the above research results, the applicant put forward the technical solution of the present application. In a preferred embodiment of the present application, a method for synthesizing chiral amine compounds is provided, which comprises using the above modified aminotransferase mutant of the present application to carry out amination reaction with a ketone substrate represented by formula I under the action of an amino donor to obtain a chiral amine compound; [ka] Ar1 represents a substituted or unsubstituted aryl group, a substituted or unsubstituted arylene group, or a substituted or unsubstituted heteroarylene group; Ar2 represents a substituted or unsubstituted aryl group, a substituted or unsubstituted cycloalkyl group, or a substituted or unsubstituted linear alkyl group or heteroalkyl group; and R is an optional group, R represents a substituted or unsubstituted alkylene group having 1 to 5 carbon atoms, R is linked to Ar1 to form a ring, wherein the substituents in the substituted aryl group, the substituted arylene group, the substituted heteroarylene group, and the substituted alkylene group are each independently selected from a halogen, a hydroxyl group, an amino group, and -S-CH3, and the heteroatoms in the heteroarylene group and the heteroalkyl group are each independently selected from N, O, or S, and wherein the aminotransferase is one type of aminotransferase having a conserved amino acid sequence region, the conserved amino acid sequence region includes at least region 1 and region 2, the conserved amino acid sequence of region 1 is MAGLWCVN, and the conserved amino acid sequence of region 2 is YNTFFKT.

[0025] As mentioned above, the aminotransferase having a certain catalytic activity for such a substrate with large steric hindrance has multiple conserved amino acid regions, and the above-mentioned two conserved amino acid sequence regions have 100% homology in all the detected aminotransferases with a certain catalytic activity. And, it has been proved by a preliminary test that such conserved amino acid sequence regions are candidate regions for increasing the catalytic activity for such a substrate, so that the aminotransferase having the above conserved amino acid sequence regions is desired to be modified into an aminotransferase having a catalytic activity for catalyzing a substrate with large steric hindrance and having a higher activity, which further improves the synthesis efficiency of such a chiral amine compound with large steric hindrance, reduces the production cost, and reduces waste liquid, waste gas and solid waste.

[0026] As is apparent from Figures 1 to 3, such an aminotransferase has another conserved amino acid sequence region in addition to the above two conserved amino acid sequence regions. Thus, in a preferred embodiment, the aminotransferase further includes region 3, and the conserved amino acid sequence of region 3 is RWNGYHGST.

[0027] In a preferred embodiment, the aminotransferase is a mutant aminotransferase in which W and / or C in the conserved amino acid sequence of region 1 of the aminotransferase from Chromobaterium violaceum shown in SEQ ID NO: 1 and any one of SEQ ID NO: 2 to SEQ ID NO: 55 in the table below has been mutated to A, or the aminotransferase has 75%, preferably 85%, more preferably 95% or more homology with the aminotransferase from Chromobaterium violaceum shown in SEQ ID NO: 1 and any one of SEQ ID NO: 2 to SEQ ID NO: 55 in Tables 1 and 2, and has region 1 and region 2.

[0028] In a more preferred embodiment, the aminotransferase is the aminotransferase derived from Chromobaterium violaceum and shown in SEQ ID NO: 1, or any one of the aminotransferases shown in SEQ ID NO: 2 to SEQ ID NO: 55 in Tables 1 and 2.

[0029] The large sterically hindered substrate referred to in this application refers to a type of compound in which the group next to the potential chiral carbonyl group is larger than a methyl group, and any compound that fits the above definition can be used as a substrate catalyzed by such aminotransferases. In a preferred embodiment, the substituent in the substituted aryl or arylene group is a halogen or -S-CH3, and the halogen or -S-CH3 is located at one or more of the ortho, meta, or para positions of the aryl or arylene group, and preferably the halogen is F, Cl, or Br.

[0030] In a preferred embodiment, Ar1 represents a substituted or unsubstituted aryl group or arylene group, Ar2 represents an unsubstituted aryl group, R represents an unsubstituted alkylene group having 1 to 5 C atoms, and R is linked to Ar1 to form a ring.

[0031] In a preferred embodiment, Ar1 represents an unsubstituted heteroarylene group, Ar2 represents an aryl group substituted with a halogen, R represents an alkylene group having 1 to 5 carbon atoms substituted with a hydroxyl group, and R is linked to Ar1 to form a ring.

[0032] In a preferred embodiment, Ar1 represents an arylene group substituted with a halogen and an amino group, and Ar2 is a cycloalkyl group having 3 to 8 carbon atoms.

[0033] In a preferred embodiment, Ar1 represents an unsubstituted cycloalkyl group or an aryl group, and Ar2 is a substituted or unsubstituted cycloalkyl group or an aryl group.

[0034] In a preferred embodiment, Ar1 represents a cycloalkyl group or an aryl group substituted with a hydroxyl group, a methyl group, an ethyl group, or a -S-CH3 group, and Ar2 represents an unsubstituted aryl group or an alkyl group.

[0035] In a preferred embodiment, the ketone substrate is: [ka]

[0036] In preferred embodiments, the amino group donor is isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine or aniline.

[0037] The above technical solutions and technical effects will be described below with reference to specific examples. [ka]

[0038] (1) The first part involves screening and validating the catalytic activity of wild-type aminotransferases from different species towards such highly sterically hindered substrates.

[0039] Example 1: Screening for wild-type aminotransferases In this example, an aminotransferase derived from Chromobaterium violaceum (the sequence is shown in SEQ ID NO: 1, and is referred to as CvTA wild-type aminotransferase) was first selected, and its catalytic activity for catalyzing the synthesis of the target substrate with large steric hindrance (substrate 1) was tested. The detailed process of the CvTA wild-type aminotransferase catalyzing substrate 1 was as follows: 1 mg of substrate 1, 0.1 mg of PLP, 1 mg of isopropylamine hydrochloride, 50 mg of enzyme powder, and 100 mM phosphate buffer at pH 8.0 were added to a 1 mL system, and the reaction was carried out at 30°C for 40 hours.

[0040] As a result, the efficiency of the wild-type CvTA aminotransferase in catalyzing the synthesis of substrate 1 was 0.56%.

[0041] Next, wild-type aminotransferases (specifically, see Table 1) that are of the same genus but different species as the above-mentioned CvTA wild-type aminotransferase and have an amino acid sequence with 82% or more homology compared with SEQ ID NO: 1 were selected, and their catalytic activity was tested, and the catalytic process was the same as above. As a result, similar to the CvTA wild-type aminotransferase, the efficiency of these wild-type aminotransferase species in catalyzing the synthesis of the target substrate (substrate 1) with a large steric hindrance was 0.01% to 1%. Specific results are shown in Table 1. [Table 1] 10 * Enzyme number 10 does not belong to the genus Chromobacterium, but is listed in the table because of its high homology. In the above table, + indicates a conversion rate of 0.01% to 0.1%, ++ indicates a conversion rate of 0.1 to 0.5%, and +++ indicates a conversion rate of 0.5 to 1%.

[0042] Example 2 In addition to the aminotransferase from Chromobacterium, the present application also tested aminotransferases from other sources with 69% to 87% homology with Chromobacterium violaceum. Specific information is shown in Table 2.

[0043] In this example, wild-type aminotransferases from different origins showed high conservation of the amino acid sequences from positions 54 to 63 and from positions 84 to 96. Tests showed that these wild-type aminotransferases had catalytic activity of 0.01% to 1% even for the target substrate (substrate 1) with a large steric hindrance (see Table 2). [Table 2] In the above table, + indicates a conversion rate of 0.01% to 0.1%, ++ indicates a conversion rate of 0.1 to 0.5%, and +++ indicates a conversion rate of 0.5 to 1%.

[0044] Furthermore, the sequences of different aminotransferase strains as shown in Tables 1 and 2 were aligned, and the alignment results are shown in Figures 1 to 3. From the results of the multiple sequence alignment, it was found that, with reference to the sequence of wild-type Chromobaterium violaceum aminotransferase (i.e., SEQ ID NO: 1), the two conserved amino acid sequence regions from positions 56 to 63 and from positions 84 to 96 are highly conserved in the aminotransferases of the listed species, are structurally located close to the substrate binding site, and are relatively important amino acids. Therefore, it is speculated that these two conserved amino acid regions improve the affinity for substrates with large steric hindrances and contribute to further improvement of catalytic activity.

[0045] Example 3 In order to further confirm the effect of the aminotransferase having such a conserved amino acid region on the catalytic activity of a substrate with a large steric hindrance, the inventors have identified the conserved amino acid region 1 (specifically, the sequence is DGMAGL W.C. We preliminary performed A mutations on the amino acids W and C in (VNVGYGR) and found that the catalytic efficiency for substrate 1 was improved after mutation of these conservative amino acids, with the conversion rate after WA mutation increasing to 5-10% and after CA mutation increasing to 1-5% (the specific reaction process is the same as that described in Example 1).

[0046] In order to confirm that the conversion rate to the substrate is improved after the same mutation occurs at the conserved site in aminotransferases from other species, the inventors also performed the same mutations as above on aminotransferases from other species, and the detection results are shown in the table below. [Table 3] In the above table, ++++ indicates a conversion rate of 1% to 5%, and +++++ indicates a conversion rate of 5 to 10%.

[0047] Therefore, by further recognizing the conserved amino acid site in the conserved amino acid sequence region and making it a candidate for a signature sequence that improves catalytic activity, the signature sequence is further introduced into all wild-type strains. The above data show that after the signature sequence is introduced, all wild-type aminotransferases have significantly improved catalytic activity against the target highly sterically hindered substrate (Substrate 1).

[0048] Example 4 The inventors further detected the catalytic activity towards different highly sterically hindered substrates (specifically see Substrate 2 to Substrate 13 below), and employed 55 wild-type aminotransferases of the present invention and the corresponding mutants after WA and CA mutations, and detected the catalytic activity towards the following different substrates according to the similar reaction conditions as in Example 1. [ka]

[0049] [Table 4]

[0050] [Table 5]

[0051] [Table 6]

[0052]

Table 7

[0053]

Table 8

[0054]

Table 9

[0055]

Table 10

[0056]

Table 11

[0057]

Table 12

[0058]

Table 13

[0059]

Table 14

[0060]

Table 15

[0061] As is apparent from the above description, the above embodiments of the present invention achieve the following technical effects: 1) the wild-type aminotransferases having the above-mentioned multiple different origin conservative regions are used to perform biocatalysis, which eliminates the need for heavy metal catalysts and realizes green chemistry; 2) compared with heavy metal catalysts, the aminotransferases are used to catalyze the reaction, which shortens the synthesis route, contributes to reducing waste liquids, waste gases and solid waste, and saves production costs; 3) the aminotransferase mutants using different origin aminotransferases and conservative site mutations can improve the bioconversion reaction catalyzing ketone substrates with large steric hindrance; and 4) the substrate type is widely applicable.

[0062] The above is merely a preferred embodiment of the present invention, and is not intended to limit the present invention. Various modifications and variations of the present invention are possible for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. made within the scope of the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for synthesizing a chiral amine compound, comprising: employing an aminotransferase to carry out a transamination reaction on a ketone substrate represented by formula I under the action of an amino donor to obtain the chiral amine compound; 【Chemistry 1】 wherein the aminotransferase is an aminotransferase having one type of conserved amino acid sequence region, the conserved amino acid sequence region includes at least a region 1 and a region 2, the conserved amino acid sequence of the region 1 is MAGLWCVN, and the conserved amino acid sequence of the region 2 is YNTFFKT; The aminotransferase is a mutant aminotransferase in which W and / or C in the conserved amino acid sequence in the region 1 of any one of the aminotransferases shown in SEQ ID NO: 2 to SEQ ID NO: 55 in the table below has been mutated to A, or the aminotransferase is any one of the aminotransferases shown in SEQ ID NO: 2 to SEQ ID NO: 55, 【Table 1】 The method for synthesizing a chiral amine compound, wherein the ketone substrate is as follows: 【Chemistry 2】

2. 2. The method according to claim 1, wherein the amino group donor is isopropylamine, isopropylamine hydrochloride, alanine, n-butylamine or aniline.

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