CN113881720B - Transaminase and catalytic preparation method using same - Google Patents
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Abstract
The application provides a transaminase and a catalytic preparation method using the same, wherein the transaminase has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3. In addition, the transaminase of the application can also be used to catalyze a process for the preparation of compound C, which comprises the following steps: in a buffer solution with pH=7.5-9.0, in the presence of transaminase, amino donor and pyridoxal phosphate, carrying out a transamination reaction on the compound B to obtain a compound C; the aminotransferase has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3; r is R 1 、R 2 、R 3 、R 4 And R is 5 Are each independently H, halogen, C 1 ‑C 3 Alkyl, C 1 ‑C 3 Alkoxy or CN. The preparation method of the aminotransferase catalysis in the application has the advantages of mild condition, high selectivity, high raw material conversion rate, high yield and high chiral purity of the product.
Description
Technical Field
The application relates to the field of chemical synthesis, in particular to transaminase and a catalytic preparation method using the same.
Background
S-4- (1-aminoethyl) benzoic acid is an important monomer for the synthesis of peptidomimetic drugs (Bioorganic & Medicinal Chemistry Letters,2010, 20 (3): 1225-1228) and is also a common pharmaceutical intermediate for the synthesis of 4- (1-aminoethyl) cyclohexanecarboxylic acid (Chemical and Pharmaceutical Bulletin,1979, 27 (11): 2735-2742).
At present, the preparation of S-4- (1-amino ethyl) benzoic acid is mainly prepared by a chemical method, for example, ISODA SUMIRO and the like takes 4-cyano acetophenone as raw materials, and 4-acetyl benzoic acid is generated by reflux in acetic acid-hydrochloric acid solution; and then dissolving 4-acetylbenzoic acid in 10% ammonia-ethanol solution to carry out ammonia transfer reaction. Finally, 4-acetylbenzoic acid is reduced to 4- (1-aminoethyl) benzoic acid under Raney nickel catalysis (Chemical and Pharmaceutical Bulletin,1979, 27 (11): 2735-2742).
The synthesis method of the S-4- (1-amino ethyl) benzoic acid mainly has the defects of large dosage of organic solvent, low chiral purity and the like, and is an important direction of current research by exploring a synthesis route with simple reaction conditions, environmental protection and easy amplification. Biological enzymes are environmentally friendly catalysts, which have mild reaction conditions and have become an important means for the synthesis of pharmaceutical chemical intermediates or bulk drugs, particularly in asymmetric synthesis to exert this non-substitutable effect (ChemSusChem, 2019, 12:1-24).
Disclosure of Invention
The technical problem to be solved by the application is that the transfer ammonification reaction in the prior art has low yield and low selectivity. Therefore, the application provides the transaminase and the method for catalytic preparation by using the same, and the preparation reaction catalyzed by the transaminase has the advantages of mild condition, high selectivity, high raw material conversion rate, high yield and high chiral purity of the product.
In order to solve the technical problems, the application provides the following technical scheme:
the application provides a preparation method of a compound C, which comprises the following steps:
in a buffer solution with pH=7.5-9.0 (for example, with pH 8.0), compound B is subjected to a transamination reaction in the presence of transaminase, amino donor and pyridoxal phosphate to obtain compound C;
the aminotransferase is a mutant of aminotransferase from rose bacillus (Ruegeria pomeroyi) and has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3;
R 1 、R 2 、R 3 、R 4 and R is 5 Are each independently H, halogen, C 1 -C 3 Alkyl, C 1 -C 3 Alkoxy or CN.
In one embodiment, in the compound B, some groups may be defined as follows, and the remaining groups may be defined as described in any one of the embodiments above: (for this expression, the following abbreviations are in a scheme)
Among the halogens, the halogen is preferably fluorine, chlorine, bromine or iodine, more preferably fluorine or chlorine.
In one embodiment: at C 1 -C 3 In the alkyl group, the C 1 -C 3 The alkyl group is preferably methyl, ethyl, n-propyl or isopropyl, more preferably methyl.
In one embodiment: at C 1 -C 3 In the alkoxy group, the C 1 -C 3 The alkoxy group is preferably methoxy, ethoxy, n-propoxy or isopropoxy, more preferably methoxy.
In one embodiment: r is R 1 H, halogen or CN, preferably H, fluorine, chlorine or CN, more preferably H.
In one embodiment: r is R 2 H, C of a shape of H, C 1 -C 3 Alkyl or CN, preferably H, methyl or CN, more preferably H.
In one embodiment: r is R 3 Is H, halogen or C 1 -C 3 Alkoxy or CN, preferably H, fluoro, chloro, methoxy or CN, more preferably CN.
In one embodiment: r is R 4 H, C of a shape of H, C 1 -C 3 Alkyl or CN, preferably H, methyl or CN, more preferably H.
In one embodiment: r is R 5 H, halogen or CN, preferably H, fluorine, chlorine or CN, more preferably H.
In one embodiment: r is R 1 And R is 5 H.
In one embodiment: r is R 1 、R 2 、R 4 And R is 5 H.
In one embodiment: the compound B is any one of the following schemes:
scheme one:
R 1 is H, halogen or CN;
R 2 h, C of a shape of H, C 1 -C 3 Alkyl or CN;
R 3 is H, halogen or C 1 -C 3 Alkoxy or CN;
R 4 h, C of a shape of H, C 1 -C 3 Alkyl or CN;
R 5 is H, halogen or CN;
scheme II:
R 1 is H;
R 2 h, C of a shape of H, C 1 -C 3 Alkyl or CN;
R 3 is H, halogen or C 1 -C 3 Alkoxy or CN;
R 4 h, C of a shape of H, C 1 -C 3 Alkyl or CN;
R 5 is H;
scheme III:
R 1 is H;
R 2 is H;
R 3 is H, halogen or C 1 -C 3 Alkoxy or CN;
R 4 is H;
R 5 h.
In one embodiment: the compound B is any one of the following compounds:
in the transamination reaction, the buffer solution can be a buffer solution which is conventional in the field of the transamination reaction, so as not to influence the reaction, for example, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, tris-HCl buffer solution or HEPES buffer solution.
The transamination reaction may further comprise a step of maintaining the pH of the reaction system by adding an acid such as hydrochloric acid, phosphoric acid or sulfuric acid in an amount varying according to the amount of the amino donor so as not to affect the reaction, and maintaining the pH of the reaction system at an amount of 7.5 to 9.0 (e.g., pH 8.0).
In the transamination reaction, the amino donor may be an amino donor conventional in the art for such a transamination reaction, e.g., NH 2 C 1 -C 6 Alkyl, said C 1 -C 6 Alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl, preferably isopropyl.
The transfer ammonification reaction can further comprise a cosolvent, wherein the cosolvent can be a cosolvent conventional in the transfer ammonification reaction, such as a sulfone solvent, preferably dimethyl sulfoxide; the mass to volume ratio of the compound B to the cosolvent can be 0.1 g/mL-1.0 g/mL, for example 0.5g/mL.
In the transamination reaction, the mass ratio of the pyridoxal phosphate to the compound B may be in a ratio conventional in the art for such a transamination reaction, for example 0.01:1 to 0.1:1, for example 0.01:1 or 0.02:1.
in the transamination reaction, the mass ratio of the transaminase to the compound B may be in a ratio conventional in the art for such a transamination reaction, for example 0.1:1 to 1.0:1, for example 0.1:1, 0.2:1 or 0.5:1.
In the described deammonification reaction, the reaction temperature may be a temperature conventional in the art for such deammonification reactions, e.g., 30-45 ℃, e.g., 40 ℃ or 45 ℃.
In the deammonification reaction, the deammonification reaction takes the disappearance or no longer reaction of the compound B as a reaction end point, and the reaction time is, for example, 2 to 80 hours, preferably 4 to 39.5 hours.
In the deammonification reaction, the mass-to-volume ratio of the compound B to the water in the buffer may be a ratio conventional in the art for such deammonification reaction, for example, 1.0g/L to 80g/L, and further, for example, 1.1g/L to 54g/L.
In the deammonification reaction, the deammonification reaction may include the steps of: and (3) adding the amino donor and acid into a buffer solution to adjust the pH to 7.5-9.0 (for example, pH 8.0), dissolving the compound B into a cosolvent, and adding the transaminase and the pyridoxal phosphate for carrying out the transamination reaction. The acid and the co-solvent are as described above.
After the transfer ammoniation reaction is finished, the method can further comprise post-treatment, wherein the post-treatment preferably comprises the following steps: adding alkali, regulating pH to 11-13, layering, extracting water phase with methyltetrahydrofuran, separating organic phase, concentrating to dryness to obtain the compound C.
The application also provides a transaminase which is a mutant of the transaminase from the bacillus roseus (Ruegeria pomeroyi), and has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3.
The application also provides application of the aminotransferase in preparing the compound C and/or the compound E, wherein the aminotransferase has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3;
R 1 、R 2 、R 3 、R 4 and R is 5 Is defined as above;
compound C is preferablyCompound E is preferably->
In the preparation method of the compound C, when the compound B isWhen (1):
the compound B is obtained by cyanation reaction of bromoacetophenone (compound A) as follows;
the cyanation reaction preferably comprises the steps of:
in [ Pd (C) 3 H 5 )Cl] 2 And (3) in the presence of triphenylphosphine, a solvent and a cyanation reagent, performing the cyanation reaction on bromoacetophenone (compound A) to obtain a compound B.
In the cyanation reaction, the cyanation reagent may be a reagent conventionally used in such cyanation reaction in the art, for example, K 4 [Fe(CN) 6 ]。
In the cyanation reaction, the solvent may be a solvent conventional in the cyanation reaction such as an amide-based solvent such as dimethylacetamide and/or water.
In the cyanation reaction, the [ Pd (C) 3 H 5 )Cl] 2 The molar ratio to the compound A may be in a proportion conventional in the art for such cyanation reactions, for example from 0.001:1 to 0.01:1, for example from 0.005:1 to 0.01:1.
In the cyanation reaction, the molar ratio of triphenylphosphine to compound A may be in a ratio conventional in the art for such cyanation reactions, e.g., 0.01:1 to 0.1:1, and still more e.g., 0.02:1 to 0.03:1.
In the cyanation reaction, the molar ratio of the cyanation reagent to the compound A may be in a ratio conventional in the art for such cyanation reactions, for example, 0.4:1 to 1:1, and further for example, 0.5:1.
In the cyanation reaction, the concentration of the compound A in the solvent may be a concentration conventional in the cyanation reaction of this type, for example, 0.1mol/L to 1mol/L, and further, for example, 0.2mol/L to 0.5mol/L.
In the cyanation reaction, the reaction temperature may be a temperature conventional in the cyanation reaction of this type in the art, for example, 60 to 95℃and further, for example, 85 to 95 ℃.
In the cyanation reaction, the reaction progress of the cyanation reaction takes the disappearance or no longer reaction of the compound A as the reaction end point, and the reaction time is, for example, 15 to 40 hours, and further, for example, 18 to 25 hours.
The application also provides a preparation method of the S-4- (1-amino ethyl) benzoic acid (compound D), which comprises the following steps:
when the compound C isWhen (1):
the compound C is subjected to hydrolysis reaction shown below to obtain S-4- (1-amino ethyl) benzoic acid (compound D);
the preparation method of the compound C is as described above;
the hydrolysis reaction preferably comprises the steps of:
in a buffer solution with pH=6.5-8.0, in the presence of cyano hydrolase, performing the hydrolysis reaction on the compound C to obtain S-4- (1-amino ethyl) benzoic acid (compound D);
the cyano hydrolase is a cyano hydrolase of Burkholderia neoplasia (Paraburkholderia phymatum) and has an amino acid sequence shown as SEQ ID NO. 4.
In the hydrolysis reaction, the buffer may be a buffer which is conventional in the art of hydrolysis reaction, so as not to affect the reaction, for example, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, tris-HCl buffer or HEPES buffer.
In the hydrolysis reaction, the mass ratio of the cyano hydrolase to the compound C may be a ratio conventional in this type of hydrolysis reaction in the art, for example, 0.05:1 to 0.3:1, for example 0.08:1 to 0.1:1.
In the hydrolysis reaction, the mass to volume ratio of the compound C to the water in the buffer may be a ratio conventional in the hydrolysis reaction of this type in the art, for example, 0.1g/L to 1.0g/L, and still more for example, 0.6g/L to 1.0g/L.
The hydrolysis reaction may further comprise a cosolvent, which may be a cosolvent conventional in the art for such hydrolysis reaction, for example, a sulfone solvent, and further for example, dimethyl sulfoxide; the mass to volume ratio of the compound C to the cosolvent can be 0.5 g/mL-2.0 g/mL, for example 1.2 g/mL-1.5 g/mL.
The hydrolysis reaction may further comprise a step of adding an acid such as hydrochloric acid, phosphoric acid or sulfuric acid to maintain the pH of the reaction system so as not to affect the reaction, the amount of the acid being varied depending on the amount of the compound, and the pH of the reaction system being maintained at an amount of 6.5 to 8.0.
In the hydrolysis reaction, the reaction temperature may be a temperature conventional in the art for such hydrolysis reaction, for example, 10 to 40℃and further, for example, 25 to 30 ℃.
In the hydrolysis reaction, the reaction is terminated by the disappearance or absence of the compound C, for example, for 20 to 40 hours, for example, for 23 to 30 hours.
In the hydrolysis reaction, the hydrolysis reaction may include the steps of: adding a compound C dissolved in a cosolvent into a buffer solution, adding acid to adjust the pH to 6.5-8.0, and then adding cyano hydrolase to carry out hydrolysis reaction to obtain the compound D.
The synthetic process route of the application is as follows:
the above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present application can be obtained.
The reagents and materials used in the present application are commercially available.
The application has the positive progress effects that: compared with the existing transaminase-catalyzed transamination reaction, the transaminase-catalyzed transamination reaction provided by the application has the advantages of high selectivity, high raw material conversion rate, high yield and high chiral purity of the product. In addition, the application also provides a preparation method of the S-4- (1-aminoethyl) benzoic acid, the reaction adopts an enzyme catalytic conversion method, the selectivity is high, the reaction condition is mild, the use of a large amount of organic reagents is avoided, the product conversion rate is high, and the feasibility of industrialized amplification is ensured.
Drawings
FIG. 1 is a high performance liquid chromatography of the reaction mixture after completion of the reaction of transaminase 1 (SEQ ID NO: 1) in example 2.
FIG. 2 is a graph showing the chiral purity of transaminase 1 (SEQ ID NO: 1) in example 2.
FIG. 3 is a high performance liquid chromatography of the reaction mixture after completion of the reaction of transaminase 2 (SEQ ID NO: 2) in example 2.
FIG. 4 is a graph showing the chiral purity of transaminase 2 (SEQ ID NO: 2) in example 2.
FIG. 5 is a high performance liquid chromatography of the reaction mixture after completion of the reaction of transaminase 2 (SEQ ID NO: 2) in example 3.
FIG. 6 is a high performance liquid chromatography of the reaction mixture after completion of the reaction of transaminase 3 (SEQ ID NO: 3) in example 3
FIG. 7 is a high performance liquid chromatography of the reaction mixture after the completion of the reaction in example 4
FIG. 8 is a high performance liquid chromatography of the reaction solution after the completion of the reaction in example 5
FIG. 9 is a mass spectrum of the product after the end of the reaction in example 5
Detailed Description
The following description of the present application will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1
Preparation of 4-cyanoacetophenone by using bromoacetophenone as raw material
In a 700. Mu.L reaction flask, 0.0457mg (0.125. Mu. Mol,0.005 eq.) of a metallic palladium catalyst such as [ Pd (C) 3 H 5 )Cl] 2 50. Mu.L (10 vol.) of dimethylacetamide solution containing 0.1311mg (0.5. Mu. Mol,0.02 eq.) of triphenylphosphine, and further 50. Mu.L (10 vol.) of dimethylacetamide solution containing 4.98mg (25. Mu. Mol,1.0 eq.) of p-bromoacetophenone, containing 5.28mg of K4[ Fe (CN) 6]·3H 2 30 mu L of O aqueous solution, adjusting and controlling the temperature of the reaction system to be 85-95 ℃ and continuously stirring for 18 hours. The detection result shows that the process conversion rate of the reaction is 100%.
Example 2
SEQ ID NO 1-3 aminotransferase was synthesized entirely and used in the following experiments.
Comparison of transaminase 2 (SEQ ID NO: 2) with transaminase 1 (SEQ ID NO: 1) for the catalytic synthesis of S-4-cyanophenyl-ethylamine from 4-cyanoacetophenone
Potassium dihydrogen phosphate (2.55 g), dipotassium hydrogen phosphate trihydrate (6.3 g), 472mL isopropyl amine, 210mL 85% phosphoric acid were dissolved in 900mL deionized water, and a buffer (pH 8.0) containing an amino donor was prepared. 250 mL jacketed bottles A, B were taken and 30mL of the above buffer was added, respectively. 20mg pyridoxal phosphate was added, respectively, and the solution was stirred. Then, 0.5g of aminotransferase 1 (having the amino acid sequence of SEQ ID NO: 1) was added to the A flask, and 0.5g of aminotransferase 2 (having the amino acid sequence of SEQ ID NO: 2) was added to the B flask, and the mixture was dissolved by stirring. Then 1.0g of 4-cyanoacetophenone was dissolved in 2mL of DMSO, transferred to A and B, and reacted at 45℃for 4h. After completion of the reaction, 200. Mu.L of the reaction mixture was dissolved in 1mL of methanol. After filtration, liquid chromatography detection (conversion) and SFC detection (chiral purity) were performed. The results showed that the conversion rate of mutant aminotransferase 2 was 100% (see FIG. 3 for details), the chiral purity was 100% (see FIG. 4 for details, the peak position of isomer was 2.4min, the peak position of cyanoacetophenone was 1.4 min), the conversion rate of wild type aminotransferase 1 was 75.5% (see FIG. 1 for details), and the chiral purity was 100% (see FIG. 2 for details). It is thus shown that the transaminase 2 reported in this patent is significantly better than the wild-type transaminase 1 reported in the literature. The high performance liquid chromatogram and the chiral purity map in the application are both compared with standard substances.
Example 3
Comparison of transaminase 2 (SEQ ID NO: 2) with transaminase 3 (SEQ ID NO: 3) for the catalytic synthesis of S-4-cyanophenyl-ethylamine from 4-cyanoacetophenone
Potassium dihydrogen phosphate (2.55 g), dipotassium hydrogen phosphate trihydrate (6.3 g), 472mL isopropyl amine, 210mL 85% phosphoric acid were dissolved in 900mL deionized water, and a buffer (pH 8.0) containing an amino donor was prepared. 250 mL jacketed bottles A, B were taken and 30mL of the above buffer was added, respectively. 20mg pyridoxal phosphate was added, respectively, and the solution was stirred. Then, 0.5g of aminotransferase 2 (having the amino acid sequence of SEQ ID NO: 2) was added to the A flask, and 0.2g of aminotransferase 3 (having the amino acid sequence of SEQ ID NO: 3) was added to the B flask, and the mixture was dissolved by stirring. Then 1.0g of 4-cyanoacetophenone was dissolved in 2mL of DMSO, transferred to A and B, and reacted at 45℃for 4h. After completion of the reaction, 200. Mu.L of the reaction mixture was dissolved in 1mL of methanol. After filtration, liquid chromatography detection was performed. The results showed that the conversion of transaminase 2 was 97.7% (see FIG. 5 for details) and that of transaminase 3 was 96.7% (see FIG. 6 for details). It is thus shown that the aminotransferase 3 and aminotransferase 2 reported in this patent have close enzymatic effects and are superior to the wild-type aminotransferase 1.
Example 4
Process amplification for synthesizing S-4-cyanophenyl ethylamine by catalyzing 4-cyanoacetophenone with aminotransferase 2 (SEQ ID NO: 2)
Potassium dihydrogen phosphate (2.55 g), dipotassium hydrogen phosphate trihydrate (6.3 g), 472mL isopropyl amine, 210mL 85% phosphoric acid were dissolved in 900mL deionized water, and a buffer (pH 8.0) containing an amino donor was prepared. After 4-cyanoacetophenone (50 g) was dissolved in dimethyl sulfoxide (100 mL), it was added to the above-prepared buffer along with 500mg of pyridoxal phosphate, 5g of transaminase 2 (having the amino acid sequence of SEQ ID NO: 2). The mixed reaction system is placed in a water bath at 40 ℃ for reaction for 39.5h. After completion of the reaction, 200. Mu.L of the reaction mixture was dissolved in 1mL of methanol. After filtration, liquid chromatography detection was performed. The high performance liquid chromatography determination method of the substrate conversion rate comprises the following steps: chromatographic column CHIRALPAK IG-3 (250 x 4.6mm,3 μm) showed a conversion of 4-cyanoacetophenone of 96.1% and a chiral purity of 100% for the product in the transaminase catalytic step. Mobile phase a:0.05% aqueous trifluoroacetic acid, mobile phase B:0.05% acetonitrile trifluoroacetate solution, detection wavelength 230nm, test result shown in FIG. 7, and retention time of S-4-cyanophenyl ethylamine was 1.868min. 150g of potassium hydroxide was added to the reaction solution, and the pH was adjusted to 12 to 13. And (3) standing the uniformly stirred reaction solution for 1-2 hours, and dividing the reaction solution into two layers. The lower aqueous phase was mixed with 2-methyltetrahydrofuran (300 mL) and filtered through celite. The filtered solution was also allowed to stand for 1 to 2 hours, after which it was extracted once with 2-methyltetrahydrofuran (300 mL). The organic phase was concentrated to give 63g of S-4-cyanophenethylamine with 20% DMSO.
Example 5: synthesis of S-4- (1-aminoethyl) benzoic acid by catalysis of S-4-cyanophenylethylamine by cyano hydrolase (SEQ ID NO: 4)
SEQ ID NO. 4 cyano hydrolase was synthesized in a fully artificial manner and used in the following experiments.
Sodium dihydrogen phosphate (2.54 g) and disodium hydrogen phosphate (57.6 g) were dissolved in 2L deionized water to prepare a buffer solution having a pH of 8.0. After S-4-cyanophenyl ethylamine (1.2 g) containing 20% DMSO obtained in the previous step was dissolved in dimethyl sulfoxide (1 mL), it was dissolved in 50mL of a buffer, and the pH of the solution was adjusted to 8.0 with 2M hydrochloric acid. Then, 0.1g of cyano hydrolase (having the amino acid sequence of SEQ ID NO: 4) was added thereto, and the mixed reaction system was placed in a water bath at 30℃for reaction for 23 hours. After completion of the reaction, 200. Mu.L of the reaction mixture was dissolved in 1mL of methanol. After filtration, liquid chromatography detection was performed. The results showed that the conversion of S-4-cyanophenyl ethylamine in the cyanohydrolase catalytic step was 98.3% (see FIG. 8 for details) and the mass spectrum was shown in FIG. 9, [ M+H ]] + =165.8. The high performance liquid chromatography determination method of the substrate conversion rate comprises the following steps: chromatographic column xbridge@c18 (150×4.6mm,3.5 um), mobile phase a:0.05% aqueous trifluoroacetic acid, mobile phase B:0.05% trifluoroacetic acid acetonitrile solution, detection wavelength 230nm, the test results are shown in FIG. 6, and the retention time of S-4- (1-aminoethyl) benzoic acid is 2.226min.
The sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 in the present application are shown as follows:
SEQ ID NO. 1 (transaminase 1)
MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDAMAGLWCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDLNHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGMAGMHAQSGLIPDVHHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQGAGGVIVAPDSYWPEIQRICDKYDILLIADEVICGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFNHGYTYSGHPVAAAVALENLRILEEENILDHVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMRHVGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSEGHHHHHH
SEQ ID NO. 2 (transaminase 2)
MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDAMAGLFCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDLNHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGMAGMHAQSGLIPDVHHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQGAGGVIVAPDSYWPEIQRICDKYDILLIADEVICGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFNHGYTYSGHPVAAAVALENLRILEEENILDHVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMRHVGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSEGHHHHHH
SEQ ID NO. 3 (transaminase 3)
MSLATITNHMPTAELQALDAAHHLHPFSANNALGEEGTRVITRARGVWLNDSEGEEILDAMAGLMCVNIGYGRDELAEVAARQMRELPYYNTFFKTTHVPAIALAQKLAELAPGDLNHVFFAGGGSEANDTNIRMVRTYWQNKGQPEKTVIISRKNAYHGSTVASSALGGMAGMHAQSGLIPDVHHINQPNWWAEGGDMDPEEFGLARARELEEAILELGENRVAAFIAEPVQGAGGVIVAPDSYWPEIQRICDKYDILLIADEVICGFGRTGNWFGTQTMGIRPHIMTIAKGLSSGYAPIGGSIVCDEVAHVIGKDEFNHGYTYSGHPVAAAVALENLRILEEENILDHVRNVAAPYLKEKWEALTDHPLVGEAKIVGMMASIALTPNKASRAKFASEPGTIGYICRERCFANNLIMRHVGDRMIISPPLVITPAEIDEMFVRIRKSLDEAQAEIEKQGLMKSEGHHHHHH
SEQ ID NO. 4 (NIT, cyano hydrolase)
MSDQRVIRAAAVQIAPDFERPGGTLDRVCAAIDEAASKGVQLIVFPETFVPYYPYFSFVRPPVASGADHMRLYEQAVVVPGPVTHAVSERARRHAMVVVLGVNERDHGSLYNTQLVFDIDGCQVLKRRKITPTFHERMIWGQGDAAGLKVARTGIARVGALACWEHYNPLARYALMTQHEEIHCSQFPGSLVGPIFAEQIEVTIRHHALESGCFVVNSTGWLSDAQIESVTTDPKLQKALRGGCMTAIVSPEGQHLAEPLREGEGMVVADLDMALITKRKRMMDSVGHYARPELLSLAINDRPAMPVVPMSMSFERAGADVAPEIISGGQDECQHEPVAG
In summary, the above embodiments are only preferred embodiments of the present application, and are not intended to limit the scope of the present application, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present application should be included in the scope of the present application.
SEQUENCE LISTING
<110> Shanghai full pharmaceutical research and development Co., ltd
SHANGHAI SYNTHEALL PHARMACEUTICALS Co.,Ltd.
<120> a transaminase and a catalytic preparation method using the same
<130> P20017063C
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 472
<212> PRT
<213> Ruegeria pomeroyi
<400> 1
Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln
1 5 10 15
Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala
20 25 30
Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp
35 40 45
Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu
50 55 60
Trp Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala
65 70 75 80
Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr
85 90 95
Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala
100 105 110
Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala
115 120 125
Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly
130 135 140
Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly
145 150 155 160
Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala
165 170 175
Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp
180 185 190
Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg
195 200 205
Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val
210 215 220
Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val
225 230 235 240
Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr
245 250 255
Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr
260 265 270
Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met
275 280 285
Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser
290 295 300
Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn
305 310 315 320
His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg
340 345 350
Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp
355 360 365
His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile
370 375 380
Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro
385 390 395 400
Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu
405 410 415
Ile Met Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val
420 425 430
Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser
435 440 445
Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser
450 455 460
Glu Gly His His His His His His
465 470
<210> 2
<211> 472
<212> PRT
<213> Artificial Sequence
<220>
<223> transaminase 2
<400> 2
Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln
1 5 10 15
Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala
20 25 30
Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp
35 40 45
Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu
50 55 60
Phe Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala
65 70 75 80
Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr
85 90 95
Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala
100 105 110
Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala
115 120 125
Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly
130 135 140
Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly
145 150 155 160
Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala
165 170 175
Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp
180 185 190
Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg
195 200 205
Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val
210 215 220
Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val
225 230 235 240
Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr
245 250 255
Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr
260 265 270
Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met
275 280 285
Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser
290 295 300
Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn
305 310 315 320
His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg
340 345 350
Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp
355 360 365
His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile
370 375 380
Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro
385 390 395 400
Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu
405 410 415
Ile Met Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val
420 425 430
Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser
435 440 445
Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser
450 455 460
Glu Gly His His His His His His
465 470
<210> 3
<211> 472
<212> PRT
<213> Artificial Sequence
<220>
<223> transaminase 3
<400> 3
Met Ser Leu Ala Thr Ile Thr Asn His Met Pro Thr Ala Glu Leu Gln
1 5 10 15
Ala Leu Asp Ala Ala His His Leu His Pro Phe Ser Ala Asn Asn Ala
20 25 30
Leu Gly Glu Glu Gly Thr Arg Val Ile Thr Arg Ala Arg Gly Val Trp
35 40 45
Leu Asn Asp Ser Glu Gly Glu Glu Ile Leu Asp Ala Met Ala Gly Leu
50 55 60
Met Cys Val Asn Ile Gly Tyr Gly Arg Asp Glu Leu Ala Glu Val Ala
65 70 75 80
Ala Arg Gln Met Arg Glu Leu Pro Tyr Tyr Asn Thr Phe Phe Lys Thr
85 90 95
Thr His Val Pro Ala Ile Ala Leu Ala Gln Lys Leu Ala Glu Leu Ala
100 105 110
Pro Gly Asp Leu Asn His Val Phe Phe Ala Gly Gly Gly Ser Glu Ala
115 120 125
Asn Asp Thr Asn Ile Arg Met Val Arg Thr Tyr Trp Gln Asn Lys Gly
130 135 140
Gln Pro Glu Lys Thr Val Ile Ile Ser Arg Lys Asn Ala Tyr His Gly
145 150 155 160
Ser Thr Val Ala Ser Ser Ala Leu Gly Gly Met Ala Gly Met His Ala
165 170 175
Gln Ser Gly Leu Ile Pro Asp Val His His Ile Asn Gln Pro Asn Trp
180 185 190
Trp Ala Glu Gly Gly Asp Met Asp Pro Glu Glu Phe Gly Leu Ala Arg
195 200 205
Ala Arg Glu Leu Glu Glu Ala Ile Leu Glu Leu Gly Glu Asn Arg Val
210 215 220
Ala Ala Phe Ile Ala Glu Pro Val Gln Gly Ala Gly Gly Val Ile Val
225 230 235 240
Ala Pro Asp Ser Tyr Trp Pro Glu Ile Gln Arg Ile Cys Asp Lys Tyr
245 250 255
Asp Ile Leu Leu Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr
260 265 270
Gly Asn Trp Phe Gly Thr Gln Thr Met Gly Ile Arg Pro His Ile Met
275 280 285
Thr Ile Ala Lys Gly Leu Ser Ser Gly Tyr Ala Pro Ile Gly Gly Ser
290 295 300
Ile Val Cys Asp Glu Val Ala His Val Ile Gly Lys Asp Glu Phe Asn
305 310 315 320
His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu
325 330 335
Glu Asn Leu Arg Ile Leu Glu Glu Glu Asn Ile Leu Asp His Val Arg
340 345 350
Asn Val Ala Ala Pro Tyr Leu Lys Glu Lys Trp Glu Ala Leu Thr Asp
355 360 365
His Pro Leu Val Gly Glu Ala Lys Ile Val Gly Met Met Ala Ser Ile
370 375 380
Ala Leu Thr Pro Asn Lys Ala Ser Arg Ala Lys Phe Ala Ser Glu Pro
385 390 395 400
Gly Thr Ile Gly Tyr Ile Cys Arg Glu Arg Cys Phe Ala Asn Asn Leu
405 410 415
Ile Met Arg His Val Gly Asp Arg Met Ile Ile Ser Pro Pro Leu Val
420 425 430
Ile Thr Pro Ala Glu Ile Asp Glu Met Phe Val Arg Ile Arg Lys Ser
435 440 445
Leu Asp Glu Ala Gln Ala Glu Ile Glu Lys Gln Gly Leu Met Lys Ser
450 455 460
Glu Gly His His His His His His
465 470
<210> 4
<211> 340
<212> PRT
<213> Paraburkholderia phymatum
<400> 4
Met Ser Asp Gln Arg Val Ile Arg Ala Ala Ala Val Gln Ile Ala Pro
1 5 10 15
Asp Phe Glu Arg Pro Gly Gly Thr Leu Asp Arg Val Cys Ala Ala Ile
20 25 30
Asp Glu Ala Ala Ser Lys Gly Val Gln Leu Ile Val Phe Pro Glu Thr
35 40 45
Phe Val Pro Tyr Tyr Pro Tyr Phe Ser Phe Val Arg Pro Pro Val Ala
50 55 60
Ser Gly Ala Asp His Met Arg Leu Tyr Glu Gln Ala Val Val Val Pro
65 70 75 80
Gly Pro Val Thr His Ala Val Ser Glu Arg Ala Arg Arg His Ala Met
85 90 95
Val Val Val Leu Gly Val Asn Glu Arg Asp His Gly Ser Leu Tyr Asn
100 105 110
Thr Gln Leu Val Phe Asp Ile Asp Gly Cys Gln Val Leu Lys Arg Arg
115 120 125
Lys Ile Thr Pro Thr Phe His Glu Arg Met Ile Trp Gly Gln Gly Asp
130 135 140
Ala Ala Gly Leu Lys Val Ala Arg Thr Gly Ile Ala Arg Val Gly Ala
145 150 155 160
Leu Ala Cys Trp Glu His Tyr Asn Pro Leu Ala Arg Tyr Ala Leu Met
165 170 175
Thr Gln His Glu Glu Ile His Cys Ser Gln Phe Pro Gly Ser Leu Val
180 185 190
Gly Pro Ile Phe Ala Glu Gln Ile Glu Val Thr Ile Arg His His Ala
195 200 205
Leu Glu Ser Gly Cys Phe Val Val Asn Ser Thr Gly Trp Leu Ser Asp
210 215 220
Ala Gln Ile Glu Ser Val Thr Thr Asp Pro Lys Leu Gln Lys Ala Leu
225 230 235 240
Arg Gly Gly Cys Met Thr Ala Ile Val Ser Pro Glu Gly Gln His Leu
245 250 255
Ala Glu Pro Leu Arg Glu Gly Glu Gly Met Val Val Ala Asp Leu Asp
260 265 270
Met Ala Leu Ile Thr Lys Arg Lys Arg Met Met Asp Ser Val Gly His
275 280 285
Tyr Ala Arg Pro Glu Leu Leu Ser Leu Ala Ile Asn Asp Arg Pro Ala
290 295 300
Met Pro Val Val Pro Met Ser Met Ser Phe Glu Arg Ala Gly Ala Asp
305 310 315 320
Val Ala Pro Glu Ile Ile Ser Gly Gly Gln Asp Glu Cys Gln His Glu
325 330 335
Pro Val Ala Gly
340
Claims (21)
1. A process for the preparation of compound C, characterized in that it comprises the steps of:
in a buffer solution with pH=7.5-9.0, in the presence of transaminase, amino donor and pyridoxal phosphate, carrying out a transamination reaction on the compound B to obtain a compound C;
the aminotransferase has an amino acid sequence shown as SEQ ID NO. 2 or SEQ ID NO. 3;
R 1 is H;
R 2 h, C of a shape of H, C 1 -C 3 Alkyl or CN;
R 3 is H, halogen or C 1 -C 3 Alkoxy or CN;
R 4 h, C of a shape of H, C 1 -C 3 Alkyl or CN;
R 5 h.
2. A process for the preparation of compound C as claimed in claim 1,
in the halogen, the halogen is fluorine, chlorine, bromine or iodine;
and/or, at C 1 -C 3 In the alkyl group, the C 1 -C 3 Alkyl is methyl, ethyl, n-propyl or isopropyl;
and/or, at C 1 -C 3 In the alkoxy group, the C 1 -C 3 Alkoxy is methoxy, ethoxy, n-propoxy or isopropoxy.
3. A process for the preparation of compound C as claimed in claim 2,
among the halogens, the halogen is fluorine or chlorine;
and/or, at C 1 -C 3 In the alkyl group, the C 1 -C 3 Alkyl is methyl;
and/or, at C 1 -C 3 In the alkoxy group, the C 1 -C 3 Alkoxy is methoxy.
4. A process for the preparation of compound C as claimed in claim 1,
R 1 is H;
and/or R 2 Is H, methyl or CN;
and/or R 3 Is H, fluorine, chlorine, methoxy or CN;
and/or R 4 Is H, methyl or CN;
and/or R 5 H.
5. A process for preparing compound C as claimed in claim 4,
R 2 is H;
and/or R 3 Is CN;
and/or R 4 H.
6. The method for preparing compound C according to claim 1, wherein the compound B is any one of the following compounds:
7. the process for preparing compound C according to any one of claims 1 to 6, wherein in the deammonification reaction, the ph=8.0;
and/or, in the transamination reaction, the buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, tris-HCl buffer solution or HEPES buffer solution;
and/or, the deammonification reaction further comprises the step of adding acid to maintain the pH of the reaction system;
and/or, in the transamination reaction, the amino donor is NH 2 C 1 -C 6 An alkyl group;
and/or, the tranamination reaction further comprises a cosolvent;
and/or in the transamination reaction, the mass ratio of the pyridoxal phosphate to the compound B is 0.01:1-0.1:1;
and/or in the transamination reaction, the mass ratio of the transaminase to the compound B is 0.1:1-1.0:1;
and/or, in the transamination reaction, the reaction temperature is 30-45 ℃;
and/or in the transamination reaction, the reaction time is 2-80 h;
and/or in the deammonification reaction, the mass-volume ratio of the compound B to the water in the buffer solution is 1.0 g/L-80 g/L;
and/or, in the deammonification reaction, the deammonification reaction comprises the following steps: adding the amino donor and acid into a buffer solution to adjust the pH to 7.5-9.0, dissolving the compound B into a cosolvent, and then adding the transaminase and the pyridoxal phosphate for the transamination reaction;
and/or, after the reammonification reaction is finished, the method further comprises post-treatment, wherein the post-treatment comprises the following steps: adding alkali, regulating pH to 11-13, layering, extracting water phase with methyltetrahydrofuran, separating organic phase, concentrating to dryness to obtain the compound C.
8. The process for preparing compound C according to claim 7, wherein the deammonification reaction further comprises the step of maintaining the pH of the reaction system by adding an acid selected from the group consisting of hydrochloric acid, phosphoric acid and sulfuric acid;
and/or, in the transamination reaction, the amino donor is NH 2 C 1 -C 6 Alkyl, said C 1 -C 6 Alkyl is methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl;
and/or, the transfer ammoniation reaction further comprises a cosolvent, wherein the cosolvent is a sulfone solvent; the mass volume ratio of the compound B to the cosolvent is 0.1 g/mL-1.0 g/mL;
and/or, in the transamination reaction, the mass ratio of the pyridoxal phosphate to the compound B is 0.01:1 or 0.02:1;
and/or in the transamination reaction, the mass ratio of the transaminase to the compound B is 0.1:1, 0.2:1 or 0.5:1;
and/or, in the deammonification reaction, the reaction temperature is 40 ℃ or 45 ℃;
and/or, in the transamination reaction, the reaction time is 4-39.5 h;
and/or in the deammonification reaction, the mass-volume ratio of the compound B to the water in the buffer solution is 1.1 g/L-54 g/L.
9. The process for preparing compound C according to claim 8, wherein in the deammonification reaction, the amino donor is NH 2 C 1 -C 6 Alkyl, said C 1 -C 6 Alkyl is isopropyl;
and/or, the transfer ammoniation reaction further comprises a cosolvent, wherein the cosolvent is dimethyl sulfoxide; the mass volume ratio of the compound B to the cosolvent is 0.5g/mL.
10. The process for producing compound C according to any one of claims 1 to 6, wherein when compound B isWhen (1):
the compound B is obtained by carrying out the cyanation reaction shown in the following way on the compound A;
11. the process for the preparation of compound C according to claim 10, wherein the cyanation reaction comprises the steps of:
in [ Pd (C) 3 H 5 )Cl] 2 And carrying out the cyanation reaction on the compound A in the presence of triphenylphosphine, a solvent and a cyanation reagent to obtain the compound B.
12. A process for the preparation of compound C according to claim 11, characterized in that:
in the cyanation reaction, the cyanation reagent is K 4 [Fe(CN) 6 ]；
And/or, in the cyanation reaction, the solvent is an amide solvent and/or water;
and/or, in the cyanation reaction, the [ Pd (C) 3 H 5 )Cl] 2 The molar ratio of the compound A to the compound A is 0.001:1-0.01:1;
and/or in the cyanation reaction, the molar ratio of the triphenylphosphine to the compound A is 0.01:1-0.1:1;
and/or in the cyanation reaction, the molar ratio of the cyanation reagent to the compound A is 0.4:1-1:1;
and/or, in the cyanation reaction, the concentration of the compound A in the solvent is 0.1mol/L to 1mol/L;
and/or, in the cyanation reaction, the reaction temperature is 60-95 ℃;
and/or in the cyanation reaction, the reaction time is 15-40 h.
13. A process for the preparation of compound C according to claim 12, characterized in that:
in the cyanation reaction, the amide solvent is dimethyl acetyl;
and/or, in the cyanation reaction, the [ Pd (C) 3 H 5 )Cl] 2 And said at least one ofThe mol ratio of the compound A is 0.005:1-0.01:1;
and/or in the cyanation reaction, the molar ratio of the triphenylphosphine to the compound A is 0.02:1-0.03:1;
and/or, in said cyanation reaction, the molar ratio of said cyanation reagent to said compound a is 0.5:1;
and/or, in the cyanation reaction, the concentration of the compound A in the solvent is 0.2mol/L to 0.5mol/L;
and/or, in the cyanation reaction, the reaction temperature is 85-95 ℃;
and/or in the cyanation reaction, the reaction time is 18-25 h.
14. A process for preparing compound D, wherein when compound C isIn the time-course of which the first and second contact surfaces,
carrying out hydrolysis reaction on the compound C as shown below to obtain a compound D;
the preparation method of the compound C is as claimed in any one of claims 1 to 13.
15. A process for the preparation of compound D according to claim 14, wherein:
the hydrolysis reaction comprises the following steps:
in buffer solution with pH=6.5-8.0, in the presence of cyano hydrolase, performing the hydrolysis reaction on the compound C to obtain a compound D;
the cyano hydrolase has an amino acid sequence shown as SEQ ID NO. 4.
16. A process for the preparation of compound D as claimed in claim 15, wherein:
in the hydrolysis reaction, the buffer solution is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, tris-HCl buffer solution or HEPES buffer solution;
and/or in the hydrolysis reaction, the mass ratio of the cyano hydrolase to the compound C is 0.05:1-0.3:1;
and/or, in the hydrolysis reaction, the mass-volume ratio of the compound C to the water in the buffer solution is 0.1 g/L-1.0 g/L;
and/or the hydrolysis reaction further comprises a cosolvent, wherein the cosolvent is a sulfone solvent,
and/or, the hydrolysis reaction further comprises the step of adding acid to maintain the pH of the reaction system;
and/or, in the hydrolysis reaction, the reaction temperature is 10-40 ℃;
and/or, in the hydrolysis reaction, the reaction time is 20-40 hours;
and/or, in the hydrolysis reaction, the hydrolysis reaction comprises the following steps: adding a compound C dissolved in a cosolvent into a buffer solution, adding acid to adjust the pH to 6.5-8.0, and then adding the cyano hydrolase to carry out hydrolysis reaction to obtain the compound D.
17. A process for the preparation of compound D as claimed in claim 16, wherein:
in the hydrolysis reaction, the mass ratio of the cyano hydrolase to the compound C is 0.08:1-0.1:1;
and/or, in the hydrolysis reaction, the mass-volume ratio of the compound C to the water in the buffer solution is 0.6 g/L-1.0 g/L;
and/or the hydrolysis reaction further comprises a cosolvent, wherein the cosolvent is dimethyl sulfoxide; the mass volume ratio of the compound C to the cosolvent is 1.2 g/mL-1.5 g/mL;
and/or the hydrolysis reaction further comprises the step of adding acid to maintain the pH of the reaction system, wherein the acid is hydrochloric acid, phosphoric acid or sulfuric acid;
and/or, in the hydrolysis reaction, the reaction temperature is 25-30 ℃;
and/or, in the hydrolysis reaction, the reaction time is 23-30 hours.
18. A process for the preparation of compound D as claimed in claim 17, wherein:
the hydrolysis reaction further comprises a cosolvent, and the mass volume ratio of the compound C to the cosolvent is 1.2 g/mL-1.5 g/mL;
and/or, in the hydrolysis reaction, the reaction temperature is 30 ℃;
and/or, in the hydrolysis reaction, the reaction time is 23 hours.
19. A transaminase having an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 3.
20. Use of a transaminase having an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 3 for the preparation of compound C and/or compound E;
R 1 、R 2 、R 3 、R 4 and R is 5 Is as defined in any one of claims 1 to 6.
21. Use of a transaminase according to claim 20 for the preparation of compounds C and/or compounds E, wherein compounds C areThe compound E is +.>
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| PCT/CN2021/143627 WO2022143977A1 (en) | 2020-12-31 | 2021-12-31 | Transaminase and method for catalytic preparation using same |
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| WO2015078267A1 (en) * | 2013-11-26 | 2015-06-04 | 凯莱英医药集团（天津）股份有限公司 | Transaminase and use thereof |
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