Method for biocatalytic synthesis of Sitagliptin and intermediate thereof
11459549 · 2022-10-04
Assignee
Inventors
- Fahao Wu (Nanjing, CN)
- Wen YANG (Beijing, CN)
- Gang Li (Nanjing, CN)
- Hongwei SHI (Beijing, CN)
- Yangzhe Gao (Nanjing, CN)
- Lili Liu (Beijing, CN)
- Zhifa Yuan (Beijing, CN)
Cpc classification
C12P13/02
CHEMISTRY; METALLURGY
C07D233/60
CHEMISTRY; METALLURGY
International classification
Abstract
Provided is a method for biocatalytic synthesis of Sitagliptin and intermediates thereof, in particular, provided are compounds of Formula (I) and Formula (II), or pharmaceutically acceptable salts thereof, a polypeptide capable of catalyzing conversion of a compound of Formula (I) to a compound of Formula (II), a nucleic acid encoding the polypeptide, a vector and a cell comprising the nucleic acid. In addition, also provided are a method for producing a compound of Formula (II) and Sitagliptin by using the polypeptide and the compound of Formula (I), and a method for preparing the polypeptide.
Claims
1. A polypeptide having the activity of catalyzing the conversion of a carbonyl group to an amino group, wherein the polypeptide comprises the amino acid sequence as set forth in SEQ ID NO: 1.
2. An isolated nucleic acid, encoding the polypeptide according to claim 1.
3. A vector comprising the isolated nucleic acid according to claim 2.
4. A cell comprising the isolated nucleic acid according to claim 2 and/or a vector comprising the isolated nucleic acid, wherein, the isolated nucleic acid is heterogenous or exogenous for the cell.
5. A composition, comprising the polypeptide according to claim 1.
6. A method for producing a compound of Formula II or a pharmaceutically acceptable salt thereof, comprising the step of converting a compound of Formula I or a pharmaceutically acceptable salt thereof to the compound of Formula II or a pharmaceutically acceptable salt thereof by using the polypeptide according to claim 1 or a composition comprising the polypeptide, wherein, the chemical structures of Formula I and Formula II are as follows: ##STR00014## wherein, R.sub.1 and R.sub.2 are independently selected from the group consisting of hydrogen, C.sub.1-6alkyl, 3-8-membered cycloalkyl, 3-8-membered heterocyclic alkyl, 6-10-membered aryl and 5-10-membered heteroaryl; or, R.sub.1 and R.sub.2 together with the N atom to which they are linked form a 4-7 membered heterocycle.
7. A method for synthesizing Sitagliptin or a salt thereof, comprising the following steps: ##STR00015## the first step: the compound of Formula II is prepared according to the method of claim 6; the second step: the compound of Formula II is hydrolyzed in the presence of a base to produce a compound of Formula III; the third step: the amino group in the compound of Formula III is protected to produce a compound of Formula IV; and the fourth step: the compound of Formula IV and a compound of Formula V are subjected to condensation reaction, and the amino-protecting group of the product is removed, to produce Sitagliptin or a salt thereof; wherein, -Pg represents an amino-protecting group; R.sub.1 and R.sub.2 have the same meanings as defined in claim 6.
8. The method according to claim 7, characterized by one or more of the following items: (1) the compound of Formula I is produced by the following method: ##STR00016## a) Compound 1 is reacted with Compound 2 in an aprotic solvent (e.g. EtOAc, DCM, DMF, DMA or DMSO; preferably DMA), in the presence of an organic base (e.g. methylamine, triethylamine, n-butyl amine or tert-butyl amine; preferably triethylamine), at room temperature or under heating (e.g. 20-50° C.; preferably 35° C.), to produce Compound 3; preferably, the molar ratio of Compound 1 and Compound 2 is 1: (1-5); preferably, after the reaction at room temperature or under heating, the step of adding an acidification agent is further comprised; more preferably, the acidification agent is selected from the group consisting of hydrochloric acid, thionyl chloride and pivaloyl chloride, and hydrochloric acid is preferred; preferably, the acidification agent is added in such an amount that the reaction system has an acidic pH; b) Compound 3 and NHR.sub.1R.sub.2 in a non-alcohol solvent (e.g. benzene, toluene or tetrahydronaphthalene), are catalyzed by an inorganic base (e.g. sodium hydroxide or potassium hydroxide), under heating (e.g. 40-120° C.; e.g. 100-105° C.), to produce a compound of Formula I; preferably, the molar ratio of Compound 3 and NHR1R.sub.2 is 1: (2-4); (3) in the second step, the base is an inorganic base, e.g. sodium hydroxide or potassium hydroxide; (4) in the third step, Boc anhydride reacts with a compound of Formula III in the presence of a base to protect the amino group; preferably, the molar ratio of the compound of Formula III, the Boc anhydride and the base is 1: (1.5-3): (2-4); preferably, the base is selected from the group consisting of sodium hydroxide, potassium hydroxide and triethylamine; (5) in the fourth step, an active intermediate of the compound of Formula IV, and a compound of Formula V are subjected to condensation reaction; preferably, the active intermediate of the compound of Formula IV is an acyl chloride, an anhydride or an amide thereof; preferably, the molar ratio of the compound of Formula IV and the compound of Formula V is 1: (1-1.2); preferably, the condensation reaction is carried out in a non-alcohol solvent (e.g. ethyl acetate, dichloromethane or chloroform); preferably, the condensation reaction is carried out at room temperature (e.g. 25° C.); preferably, the condensation reaction is carried out in the presence of a base, more preferably, the base is triethylamine.
9. A method for preparing the polypeptide according to claim 1, comprising (a) culturing a host cell comprising and expressing a nucleic acid encoding the polypeptide, and (b) collecting the polypeptide expressed in the cell.
10. The method according to claim 6, comprising (a) reacting the compound of Formula I with an amino donor in the presence of the polypeptide or the composition and an amino transmitter; and (b) collecting the compound of Formula II produced in the step (a).
11. The method according to claim 10, in the step (a), V(ml).sub.the composition: m(g).sub.the compound of Formula I=(2-5): 1, the polypeptide is used in an amount of 10 wt. %-80 wt. % of the compound of Formula I.
12. The method according to claim 10, in the step (a), the amino donor is selected from C.sub.1-6alkylamine, ammonium formate, ammonium chloride and ammonium sulfate, the molar ratio of the compound of Formula I to the amino donor is 1: (1-3).
13. The method according to claim 10, in the step (a), the amino donor is isopropyl amine, and the molar ratio of the compound of Formula I to the amino donor is 1: (1.2-3).
14. The method according to claim 10, in the step (a), the amino transmitter is selected from pyridoxal phosphate and pyridoxamine phosphate.
15. The method according to claim 10, in the step (a), the reaction is carried out in an aqueous phase.
16. The method according to claim 10, in the step (a), the compound of Formula I is dissolved in an alcohol solvent (e.g. methanol, ethanol or isopropanol) before being added to a reaction system (e.g. the compound of Formula I is dissolved in an alcohol solvent to form a 1-5 Kg/L solution, e.g. 1-4 Kg/L, e.g. 3-4 Kg/L), the concentration of the compound of Formula I is 100 g/L-250 g/L in the reaction system.
17. The method according to claim 10, in the step (a), the reaction is carried out at 30-50° C. (preferably 45° C.); the reaction system has a pH of 7.0-9.0 (preferably 8.0-9.0), and an organic amine (e.g. isopropyl amine, butyl amine or pentyl amine) is used to adjust pH of the reaction system.
18. The method according to claim 10, in the step (a), the reaction system is in contact with air.
19. The method according to claim 10, in the step (b), the compound of Formula II is collected by the following method: the product obtained in the step (a) is extracted with an organic solvent and concentrated, the organic solvent is selected from the group consisting of dichloromethane, ethyl acetate and isopropyl acetate.
Description
DESCRIPTION OF THE DRAWINGS
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SEQUENCE INFORMATION
(11) Information of the sequences involved in the present invention is provided in the following Table 1.
(12) TABLE-US-00002 TABLE 1 Description of sequences SEQ ID NO: Description 1 the amino acid sequence of Arthrobacter-derived transaminase mutant 1 2 the gene sequence of Arthrobacter-derived transaminase mutant 1 3 the amino acid sequence of Arthrobacter-derived transaminase mutant 2 4 the gene sequence of Arthrobacter-derived transaminase mutant 2 5 the amino acid sequence of Arthrobacter-derived transaminase mutant 3 6 the gene sequence of Arthrobacter-derived transaminase mutant 3 7 the amino acid sequence of Arthrobacter-derived transaminase mutant 4 8 the gene sequence of Arthrobacter-derived transaminase mutant 4
(13) TABLE-US-00003 Sequence 1 (SEQ ID NO: 1) SVLHRGQQRRRFHIQFPVTTDNALGNRTRHTVRNGITIDRNERPDATAGGATQNFVSIVQFCRRDIGQNRFVAQRFRDFQNG LARNTRQSCTTRATNHTIFDDNHIKARTFSQQVVTIQQQRQFETAIMGFLDCTNQVAPLKVLHLRINGTTRGATDALCNHQV HTVADTIERNNPLVRARTHIHLRAMFGDITFKRRRAIAAGNRHGDHGFTQFGLGHQFQRDFFDFILRQRRDQANRFCIAEQAF NVVAQTESITVPNMKRGVGCVRRIETLIKDGNTGFTRRHKRTLNPCCTASQRVCRVQFVVAVGNVVQARIVGVNNFRRVCG ECHRILAVVMMVMAA Sequence 2 (SEQ ID NO: 2) ATGGCCTCTATGGACAAAGTCTTTTCGGGATATTATGCGCGCCAGAAGCTGCTTGAACGGAGCGACAATCCTTTCTCTAAG GGCATTGCTTATGTGGAAGGAAAGCTCGTCTTTCCTAGTGATGCTAGAATACCGCTACTCGACGAAGGTTTCATGCACAGT GACCTAACCTATGATGTTATATCGGTTTGGGATGGTCGCTTCTTTCGATTGGACGATCATTTGCAACGGATTTTGGAAAGC TGCGATAAGATGCGGCTCAAGTTCCCACTTGCACTGAGCACCGTGGAAAATATTCTGGCTGAGATGGTCGCCAAGAGTGG TATCCGGGATGCGTTTGTGGAAGTTATTGTGACACGTGGTCTGACAGGTGTACGTGGTTCGAAGCCTGAGGATCTGTATA ATAACAACATATACCTGCTTGTTCTTCCATACATTTGGGTTATGGCGCCTGAGAACCAGCTCCATGGTGGCGAGGCTATCA TTACAAGGACAGTGCGACGAACACCCCCAGGTGCATTTGATCCTACTATCAAAAATCTACAGTGGGGTGATTTAACAAAG GGACTTTTTGAGGCAATGGACCGTGGCGCCACATACCCATTTCTCACTGATGGAGACACCAACCTTACTGAAGGATCTGGT TTCAACATTGTTTTGGTGAAGAACGGTATTATCTATACCCCTGATCGAGGTGTCTTGCGAGGGATCACACGTAAAAGTGTG ATTGACGTTGCCCGAGCCAACAGCATCGACATCCGCCTTGAGGTCGTACCAGTGGAGCAGGCTTATCACTCTGATGAGAT CTTCATGTGCACAACTGCCGGCGGCATTATGCCTATAACATTGCTTGATGGTCAACCTGTTAATGACGGCCAGGTTGGCCC AATCACAAAGAAGATATGGGATGGCTATTGGGAGATGCACTACAATCCGGCGTATAGTTTTCCTGTTGACTATGGCAGTG GCTAA Sequence 3 (SEQ ID NO: 3) AAAITIITTARIRYCTGVSREEDSTFSSQGRRMIDWVTGPGTPSEIGLPSTETNGQTPPPVVQPRTSSASSSSARVMSARIASGP RDSAISRTVLRVIPGRAAASRPSPSSSSGASKPRSWVSGTARIRSPHWKFLTCGSIEERGVRRTDGATIAGTPSRMRSNGTIHW YGTAYMGTCGRCLVMSRSPGVEEGPRVIETETTASRSSVLATSSRAISLTSSWVSGGMIRIDSALENRRSMWSSRRKALPFQT WNPVGVTSECRGPWSKIEIRASDGGTKAPSIQAAPPASGLAGSSSGSEGVIGSRPVSWVGTISEVSAEKA Sequence 4 (SEQ ID NO: 4) GCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGGTACTGTACCGGGGTCAGCAGAGAAGAAGATTCAACGTTCAG TTCCCAGTAACGACGGATGATAGACTGGGTAACCGGACCCGGAACACCGTCAGAGATCGGGTTACCGTCAACAGAAACG AACGGCCAAACACCACCACCGGTAGTGCAACCCAGAACTTCGTCAGCGTCCAGCAGTTCAGCCAGGGTGATGTCAGCCAG GATAGCTTCGTGACCCAGAGATTCAGCGATTTCCAGAACGGTTTTACGGGTGATACCCGGCAGAGCAGCAGCCAGCAGAC CGTCACCGTCCAGCAGCAGCGGAGCTTCGAAACCACGGTCGTGGGTTTCCTGAACTGCACGGATCAGGTCACCCCACTGG AAGTTTTTAACCTGCGGGTCGATAGAAGAACGCGGAGTACGACGAACAGACTGAGCAACCATAGCGTGAACACCGTCAC GGATGCGGTCAAACGGTACGATCCACTGGTACGGAACAGCGTACATGTAAACCTGCGGACGATGTTTGGTGATGTCACGT TCACCTGGGGTAGAAGAGTAACCACGGGTGATAGAAACAGAAACGACTGCTTCACGCAGTTCGGTTTTAGCAACCAGTTC CAGAGCGATTTCTTTAACTTCGTCCTGGGTCAGCGGCGGGATGATACGCATAGATTCAGCGTTAGAGAACAGACGTTCGA TGTGGTCGTCCAGACGGAAAGCGTTACCGTTCCAAACGTGGAACCCGGTGTAGGTAACGTCAGAGTGCAGGTAACCCTG GTCGAAGATAGAGATACGAGCTTCAGACGGCGGAACGAAAGCACCTTCGATCCAAGCAGCACCACCAGCCAGCGGGTTA GCCGGGTCCAGTTCGTAGTCAGAGTAGGTGATATAGTCCAGACCGGTGTCGTGGGTGTAAACGATTTCAGAGGTGTCAG CAGAGAAAGCCAT Sequence 5 (SEQ ID NO: 5) AAAITIITTARIRYCTGVSREEDSTFSSQGRRMIDWVTGPGTPSEIGLPSTETNGQTPPPVEQPRTSSASSSSARVMSARIASGP RDSAISRTVLRVIPGRAARPGERTTPSLITTTLKPGPSASRPSPSSSSGSSKPRSWVSGIARIRSPHWKFLTCGSIEERGVRRTDGA TIAGTPSRMRSNGTIHWYGTAYMGTCGRCLVMSRIYGVEEGPRVIETETIASRSSVLATSSRAISLTSSWVSGGMIRIDSALENR RSMWSSRRKALPFQTWNPVGVASEVRGPWSKIEIRASDGGTKAPSIQAAPPASGLAGSSSWSEGVIGSRPVSWVGTISEVSA EKA Sequence 6 (SEQ ID NO: 6) GCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGGTACTGTACCGGGGTCAGCAGAGAAGAAGATTCAACGTTCAG TTCCCAGTAACGACGGATGATAGACTGGGTAACCGGACCCGGAACACCGTCAGAGATCGGGTTACCGTCAACAGAAACG AACGGCCAAACACCACCACCGGTTGAGCAACCCAGAACTTCGTCAGCGTCCAGCAGTTCAGCCAGGGTGATGTCAGCCAG GATAGCTTCGTGACCCAGAGATTCAGCGATTTCCAGAACGGTTTTACGGGTGATACCCGGCAGAGCAGCACGACCCGGAG AACGAACAACACCGTCTTTGATAACAACAACGTTGAAACCCGGACCTTCAGCCAGCAGACCGTCACCGTCCAGCAGCAGC GGCAGCTCGAAACCACGGTCGTGGGTTTCCTGAATTGCACGGATCAGGTCACCCCACTGGAAGTTTTTAACCTGCGGGTC GATAGAAGAACGCGGAGTACGACGAACAGACTGAGCAACCATAGCGTGAACACCGTCACGGATGCGGTCAAACGGTAC GATCCACTGGTACGGAACAGCGTACATGTAAACCTGCGGACGATGTTTGGTGATGTCACGAATATATGGGGTAGAAGAG TAACCACGGGTGATAGAAACAGAAACGATTGCTTCACGCAGTTCGGTTTTAGCAACCAGTTCCAGAGCGATTTCTTTAACT TCGTCCTGGGTCAGCGGCGGGATGATACGCATAGATTCAGCGTTAGAGAACAGACGTTCGATGTGGTCGTCCAGACGGA AAGCGTTACCGTTCCAAACGTGGAACCCGGTGTAGGTAGCGTCAGAGGTCAGGTAACCCTGGTCGAAGATAGAGATACG AGCTTCAGACGGCGGAACGAAAGCACCTTCGATCCAAGCAGCACCACCAGCCAGCGGGTTAGCCGGGTCCAGTTCGTGG TCAGAGTAGGTGATATAGTCCAGACCGGTGTCGTGGGTGTAAACGATTTCAGAGGTGTCAGCAGAGAAAGCCAT Sequence 7 (SEQ ID NO: 7) AAAITIITTARIRYCTGVSREEDSTFSSQGRRMIDWVTGPGTPSEIGLPSTETNGQTPPPVEQPRTSSASSSSARVMSARIASGP RDSAISRTVLRVIPGRAARPGERTTPSLITTTLKPGPSASRPSQSSSSGSSKPRSWVSGIARIRSPHWKFLTCGSIEERGVRRTDG ATIAGTPSRMRSNGTIHWYGTAYMGTCGRCLVMSRSNGVEEGPRVIETETIASRSSVLATSSRAISLTSSWVSGGMIRIDSALE NRRSMWSSRRKALPFQTWKVVGVASEVGGPWSKIEIRASDGGTKAPSIQAAPPASGLAGSSSGSEGVIGSRPVSWVGTISEV SAEKA Sequence 8 (SEQ ID NO: 8) GCAGCAGCCATCACCATCATCACCACAGCCAGGATCCGGTACTGTACCGGGGTCAGCAGAGAAGAAGATTCAACGTTCAG TTCCCAGTAACGACGGATGATAGACTGGGTAACCGGACCCGGAACACCGTCAGAGATCGGGTTACCGTCAACAGAAACG AACGGCCAAACACCACCACCGGTTGAGCAACCCAGAACTTCGTCAGCGTCCAGCAGTTCAGCCAGGGTGATGTCAGCCAG GATAGCTTCGTGACCCAGAGATTCAGCGATTTCCAGAACGGTTTTACGGGTGATACCCGGCAGAGCAGCACGACCCGGAG AACGAACAACACCGTCTTTGATAACAACAACGTTGAAACCCGGACCTTCAGCCAGCAGACCGTCGCAGTCCAGCAGCAGC GGCAGCTCGAAACCACGGTCGTGGGTTTCCTGAATTGCACGGATCAGGTCACCCCACTGGAAGTTTTTAACCTGCGGGTC GATAGAAGAACGCGGAGTACGACGAACAGACTGAGCAACCATAGCGTGAACACCGTCACGGATGCGGTCAAACGGTAC GATCCACTGGTACGGAACAGCGTACATGTAAACCTGCGGACGATGTTTGGTGATGTCACGCTCGAATGGGGTAGAAGAG TAACCACGGGTGATAGAAACAGAAACGATTGCTTCACGCAGTTCGGTTTTAGCAACCAGTTCCAGAGCGATCTCTTTAACT TCGTCCTGGGTCAGCGGCGGGATGATACGGATAGATTCCGCGTTAGAGAACAGACGTTCGATGTGGTCGTCCAGACGGA AAGCGTTACCGTTCCAAACGTGGAAGGTGGTGTAGGTAGCGTCAGAAGTATAGTAACCCTGGTCGAAGATAGAGATACG AGCTTCAGACGGCGGAACGAAAGCACCTTCGATCCAAGCAGCACCACCAGCCAGCGGGTTAGCCGGGTCCAGTTCGTAGT CAGAGTAGGTGATATAGTCCAGACCGGTGTCGTGGGTGTAAACGATTTCAGAGGTGTCAGCTGAGAAAGCCAT
SPECIFIC MODES FOR CARRYING OUT THE INVENTION
(14) The invention is illustrated by reference to the following examples which are intended to exemplify the present invention, rather than limiting the protection scope of the present invention.
(15) Unless indicated otherwise, the molecular biological experimental methods used in the present invention are carried out substantially in accordance with the methods as described in Sambrook J et al., Molecular Cloning: A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3.sup.rd Edition, John Wiley & Sons, Inc., 1995. The assays used here are the conventional assays in the art, and are carried out according to the steps described in the relevant documents or according to the steps recommended by the manufacturers of the instruments used.
Example 1 Expression of Mutant Proteins of Transaminase from Arthrobacter
(16) The wild-type gene sequence of transaminase from Arthrobacter was designed artificially, and the designed gene sequences were set forth in SEQ ID 2, 4, 6 and 8. These sequences were obtained by total gene synthesis, and were cloned into plasmid PET24a at two restriction enzyme recognition sites XhoI and NcoI. The plasmid constructed above was transfected into E. coli DH5α competent cells. The positive transformants were selected and identified by sequencing to afford the recombinant expression vector, which was named as 00-8 plasmid (see
(17) The genetically engineered bacteria obtained above were subjected to streak culture on kanamycin-resistant LB solid medium in a 37° C. biochemical incubator overnight. Large colonies were seeded in kanamycin-resistant LB liquid medium, and incubated in a shaker at 37° C., 220 rpm for 6˜8 h, or incubated at 30° C., 200 rpm overnight, thereby obtaining a primary seed culture. The primary seed culture was seeded into kanamycin-resistant TB liquid medium at a seeding amount of 1%, and incubated in a shaker at 37° C., 220 rpm for 4˜6 h. When the bacterial solution was observed to be turbid, a secondary seed culture was obtained. The secondary seed culture was seeded to a fermenter at a seeding amount of 1% (the secondary seed culture was seeded into three fermenters, respectively, and the formulations of the media contained in the fermenters were shown in Table 2). Initial control: incubated at 37° C. under a constant pressure with air introduced. When the dissolved oxygen level reduced, it was enhanced by gradually increasing ventilatory capacity, rotational speed, and the pressure of fermenter, wherein the pressure of fermenter was not higher than 0.08 MPa. During fermentation, a supplementary medium was added (the formulation of the supplementary medium was shown in Table 3), so as to control the dissolved oxygen level between 20-40%. When OD600 reached about 25, the temperature was reduced to 28° C., 30° C. and 25° C., respectively, and IPTG was added at a concentration of 0.15 mM, 0.2 mM and 0.3 mM, respectively. The fermentation broth was subjected to centrifugation or membrane filtration to collect bacteria, and the collected bacteria were washed with a phosphate buffer, and then subjected to cell disruption in an ultrasonic disrupter or a high pressure homogenizer. The cell disrupting solution was subjected to centrifugation or membrane filtration, thereby obtaining a crude Mutant 1, which was dissolved in a pH 8.0 KH.sub.2PO.sub.4 buffer at a mass concentration of 10-20% for further use.
(18) TABLE-US-00004 TABLE 2 Formulations of media in fermenters (exemplified as a 30 L fermentation liquid, unit: g) No. Ingredient Group I Group II Group III 1 dipotassium hydrogen 194 294 294 phosphate 2 potassium dihydrogen 30 33 30 phosphate 3 ammonium sulfate 25 25 25 4 anhydrous magnesium 17.2 17.2 17.2 sulfate 5 citric acid 160 160 160 6 glycerol 50 50 150 7 fish peptone 140 440 540 8 yeast extract powder 360 160 360 9 sodium chloride 15 15 15 10 manganese chloride 0.6 2 1.6 tetrahydrate 11 ferric chloride 0.6 0.6 1.6 12 defoamer 15 15 15 Note: the ingredients were weighed and added to the fermenter, water was balanced to a suitable volume, the mixture was stirred for better disolution, and then caustic soda flake was used to adjust pH to 7.
(19) TABLE-US-00005 TABLE 3 Formulation of the supplementary medium (exemplified as a 6 L supplementary medium for a 30 L fermentation liquid, unit: g) No. Ingredient Group I Group II Group III 1 glycerol 7400 2400 3400 2 fish peptone 140 140 240 3 yeast extract powder 1080 1080 180 4 magnesium sulfate 15.6 14.6 25.6 5 defoamer 3 3 3
(20) According to the method above, the Mutants 2-4 were obtained.
Example 2 Assay on Enzyme Activity of the Mutants
(21) Method for Determining Enzyme Activity:
(22) (1) Preparation of 4M isopropyl amine hydrochloride (100 mL): 100% isopropyl amine (23.64 g) was weighed and added with about 40 mL water, followed by a slow addition of HCl (about 30 mL, fuming) at a low temperature in a fume cupboard until pH reached 8.5, then volumed to 100 mL with water for further use.
(23) (2) Preparation of an isopropyl amine aqueous solution (40% by mass).
(24) (3) Preparation of a substrate solution: the substrate solution was prepared by dissolving the substrate 3-oxo-4-(2,4,5-trifluorophenyl)butyrylpiperazine in ethanol, at a ratio of per 100 g substrate dissolved with 200 mL ethanol, and contained in a feeding bottle at 45° C. for further use.
(25) (4) Water (600 mL) (the total volume of water and the mutant solution prepared in example 1 was 1000 mL), 4M isopropyl amine hydrochloride (1.2 eq, 99.4 mL), TEA (3 g) and coenzyme pyridoxal phosphate (0.7 g) were added, and heated to 45° C. 40% isopropyl amine was used to adjust pH to 8.5. The mutant solution (400 mL) was added, and a suitable amount of air was introduced (whilst controlling bubbling). The substrate solution (about 270 mL in total) was fed to a reaction system at a relatively fixed rate in 5 h-7 h. The reaction was carried out at 45° C. for 12 h. During the reaction, pH was controlled at 8.5 by using 40% isopropyl amine. If the liquid level reduced, a suitable amount of pure water was added. The product was extracted with ethyl acetate, and the ethyl acetate phase was collected and concentrated. The concentrated residue was qualitatively and quantitatively determined by HPLC. The substrate conversion rate was recorded as enzyme activity index, which was used to evaluate the catalytic activity of the Mutants 1-4. The result was shown in the following table.
(26) TABLE-US-00006 Comparative table of enzyme activity No. SEQ ID NO. Enzyme activity index 1 1 100 2 3 30 3 5 35 4 7 40
Example 3 Synthesis of Sitagliptin
(27) In the present application, 2,4,5-trifluorophenylacetic acid, Meldrum's acid, NHR.sub.1R.sub.2 and 3-trifluoromethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride were used as raw materials; a transamination substrate β-carbonyl amide Compound I was prepared, and then the mutant of transaminase from Arthrobacter obtained in the present application was used to catalyze the carbonyl-to-amino conversion, thereby obtaining β-amino amide Compound II in a high optical purity; the Compound II was then subjected to hydrolysis, amino group protection, condensation with 3-trifluoromethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride, and de-protection, thereby affording Sitagliptin in a high yield. The particular synthetic scheme was as followed:
(28) ##STR00009##
(29) 1. Synthesis of Compound 3:
(30) Condition 1:
(31) To diethylacetamide (800 ml), 2,4,5-trifluorophenylacetic acid (190 g) and Meldrum's acid (210 g) were added. The resultant mixture was heated to 35° C. followed by an addition of triethylamine (50 g) and reacted at this temperature for 5 h. The resultant solution was cooled to room temperature, and added with water (2000 ml). The resultant solution was acidified with hydrochloric acid to pH=2, crystallized for 2 h, and filtrated. The crystal was dried, providing the dry product of Compound 3 (303 g), with a purity of 99.2%, and a yield of 96%.
(32) Condition 2;
(33) To dimethylformamide (782 ml), 2,4,5-trifluorophenylacetic acid (220 g) and Meldrum's acid (260 g) were added. The resultant mixture was heated to 35° C. followed by an addition of triethylamine (61 g) and reacted at this temperature for 4-7 h. The resultant solution was cooled to room temperature, and added with water (2500 ml) and pivaloyl chloride (89 g). The resultant solution was crystallized for 2 h, and filtrated. The crystal was dried, providing the dry product of Compound 3 (348 g), with a purity of 99.1%, and a yield of 95%.
(34) 2. Synthesis of Compound I:
(35) Condition 1, wherein in Compound I, R.sub.1 and R.sub.2 formed an imidazole ring, and the compound of Formula I here had a structure as shown below:
(36) ##STR00010##
(37) To toluene (300 ml), Compound 3 (30 g) was added, followed by the addition of imidazole (12 g) and sodium hydroxide (0.3 g). The resultant mixture was heated to 105° C. and reacted for 10 min. The resultant solution was cooled to room temperature and crystallized to obtain a crude product as a solid. The solid was washed with water and dried, providing 3-oxo-4-(2,4,5-trifluorophenyl)butyrylimidazole (25.3 g), with a purity of 99.5%, and a yield of 94%.
(38) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 8.26 (1H), 7.69 (1H), 7.25 (1H), 6.73 (1H), 6.54 (1H), 3.71 (2H), 3.56 (2H).
(39) Condition 2, wherein in Compound I, R.sub.1 and R.sub.2 formed a piperazine ring, and the compound of Formula I here had a structure as shown below:
(40) ##STR00011##
(41) To tetrahydronaphthalene (400 ml), Compound 3 (35 g) was added, followed by the addition of piperazine (17 g). The resultant mixture was heated to 100° C., and sodium hydroxide (0.25 g) was added. After being reacted for 20 min, the resultant mixture was cooled to room temperature to obtain a crude product as a solid. The solid was washed with water and dried, providing 3-oxo-4-(2,4,5-trifluorophenyl)butyrylpiperazine (31.7 g), with a purity of 99.4%, and a yield of 95%.
(42) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.73 (1H), 6.54 (1H), 3.71 (2H), 3.34 (2H), 3.32 (4H), 2.81 (4H), 2.0 (1H).
(43) Condition 3, wherein in Compound I, R.sub.1 and R.sub.2 formed an isoxazolidine ring, and the compound of Formula I here had a structure as shown below:
(44) ##STR00012##
(45) To toluene (500 ml), Compound 3 (32 g) was added, followed by the addition of 4-hydroisoxazolehydrochloride (25 g). The resultant mixture was heated to 101° C., and sodium hydroxide (40 g) was added. After being reacted for 30 min, the resultant mixture was cooled to room temperature, and filtrated. The solid was washed with water and 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole (28 g) was obtained, with a purity of 99.4%, and a yield of 96%.
(46) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.73 (1H), 6.54 (1H), 3.71 (2H), 3.53 (2H), 3.34 (2H), 3.20 (2H), 1.74 (2H).
(47) Condition 4, wherein in Compound I, R.sub.1 and R.sub.2 formed an morpholine ring, and the compound of Formula I here had a structure as shown below:
(48) ##STR00013##
(49) To toluene (90 ml) and tetralin (200 ml), Compound 3 (30 g) was added, followed by an addition of morpholine (14 g) and sodium hydroxide (0.25 g). The resultant mixture was heated to 102° C. and reacted for 30 min, then was cooled to room temperature to precipitate a crude crystal. The crystal was washed with water and 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-morpholine (27.1 g) was obtained, with a purity of 99.4%, and a yield of 95%. .sup.1H NMR was shown in
(50) 3. Synthesis of Compound II:
(51) Condition 1:
(52) 4M isopropyl amine hydrochloride (100 ml), TEA (2 g), and pyridoxal phosphate (0.1 g) were added to water (1000 ml). The resultant mixture was heated to 45° C., and 40% isopropyl amine aqueous solution was used to adjust pH to 8.5. The mutant 1 solution prepared in Example 1 (600 ml) was added, and a suitable amount of air was introduced (whilst controlling bubbling). A solution of the compound 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole (i.e. 150 g 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole was dissolved in 40 ml methanol) was added to the reaction system at a relatively fixed flow rate in 5 h-7 h. During the addition, 40% isopropyl amine was used to control the pH. The reaction was carried out at 45° C. for 12 h, and HPLC showed a conversion rate of above 99.9%. The resultant solution was extracted with dichloromethane (200 ml), and dichloromethane was recovered and R-3-amino-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole (142.5 g) was obtained, with a purity of 99.7%, and a yield of 95.0%.
(53) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.79 (1H), 6.61 (1H), 3.32 (4H), 3.23 (1H), 2.81 (4H), 2.77 (2H), 2.40 (2H), 2.0 (1H), 2.0 (2H).
(54) By reference to the reaction conditions above, the polypeptide with a sequence of SEQ ID NO: 86 as disclosed in the U.S. Pat. No. 8,293,507 of Codeixs Company, was used as the catalyst to catalyze the reaction above. HPLC showed a conversion rate of 22.1%.
(55) Condition 2:
(56) 4M isopropyl amine hydrochloride (90 ml), TEA (1 g), and pyridoxal phosphate (0.5 g) were added to water (1000 ml). The resultant mixture was heated to 40° C., and 40% isopropyl amine aqueous solution was used to adjust pH to 8.0. The mutant 1 solution prepared in Example 1 (1100 ml) was added, and a suitable amount of air was introduced (whilst controlling bubbling). A solution of the compound 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-piperazine (i.e. 250 g 3-oxo-4-(2,4,5-trifluorophenzyl)butyryl-piperazine was dissolved in 80 ml ethanol) was added to the reaction system at a relatively fixed flow rate in 5 h-7 h. During the addition, 40% isopropyl amine was used to control the pH. The reaction was carried out at 40° C. for 12 h, and HPLC showed a conversion rate of above 99.9%. The resultant solution was extracted with ethyl acetate (350 ml), and ethyl acetate was recovered and R-3-amino-4-(2,4,5-trifluorophenyl)butyrylpiperazine (240 g) was obtained, with a purity of 99.8%, and a yield of 96.0%.
(57) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.79 (1H), 6.61 (1H), 3.32 (2H), 3.23 (1H), 3.34 (2H), 3.20 (2H), 1.74 (2H).
(58) By reference to the reaction conditions above, the polypeptide with a sequence of SEQ ID NO: 86 as disclosed in the U.S. Pat. No. 8,293,507 of Codeixs Company, was used as the catalyst to catalyze the reaction above. HPLC showed a conversion rate of 12%.
(59) Condition 3:
(60) 4M isopropyl amine hydrochloride (95 ml), TEA (1.9 g), and pyridoxal phosphate (0.2 g) were added to water (325 ml). The resultant mixture was heated to 45° C., and 40% isopropyl amine aqueous solution was used to adjust pH to 8.5. The mutant 1 solution prepared in Example 1 (625 ml) was added, and a suitable amount of air was introduced (whilst controlling bubbling). A solution of the compound 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-morpholine (i.e. 160 g 3-oxo-4-(2,4,5-trifluorophenzyl)butyryl-4-morpholine was dissolved in 45 ml methanol) was added to the reaction system at a relatively fixed flow rate in 5 h-7 h. During the addition, 40% isopropyl amine was used to control the pH. The reaction was carried out at 45° C. for 12 h, and HPLC showed a conversion rate of above 99.9%. The resultant solution was extracted with dichloromethane (200 ml), and dichloromethane was recovered, and R-3-amino-4-(2,4,5-trifluorophenyl)butyryl-4-morpholine (154 g) was obtained, with a purity of 99.4%, and a yield of 96.3%.
(61) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.79 (1H), 6.61 (1H), 3.67 (4H), 3.47 (4H), 3.23 (1H), 2.77 (2H), 2.40 (2H) 2.0 (2H).
(62) Condition 4:
(63) 4M isopropyl amine hydrochloride (100 ml), TEA (2 g), and pyridoxal phosphate (0.1 g) were added to water (400 ml). The resultant mixture was heated to 45° C., and 40% isopropyl amine aqueous solution was used to adjust pH to 8.5. The mutant 1 solution (600 ml) was added, and a suitable amount of air was introduced (whilst controlling bubbling). A solution of the compound 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole (i.e. 150 g 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole was dissolved in 40 ml methanol) was added to the reaction system at a relatively fixed flow rate in 5 h-7 h. During the addition, 40% isopropyl amine was used to control the pH. The reaction was carried out at 45° C. for 12 h, and HPLC showed a conversion rate of above 99.9%. The resultant solution was extracted with dichloromethane (200 ml), and dichloromethane was recovered and R-3-amino-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole (144.5 g) was obtained, with a purity of 99.8%, and a yield of 96.3%.
(64) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.79 (1H), 6.61 (1H), 3.32 (4H), 3.23 (1H), 2.81 (4H), 2.77 (2H), 2.40 (2H), 2.0 (1H), 2.0 (2H).
(65) By reference to the reaction conditions above, the polypeptide with a sequence of SEQ ID NO: 86 as disclosed in the U.S. Pat. No. 8,293,507 of Codeixs Company, was used as the catalyst to catalyze the reaction above. HPLC showed a conversion rate of 18%.
(66) Condition 5:
(67) 4M isopropyl amine hydrochloride (90 ml), TEA (1 g), and pyridoxal phosphate (0.5 g) were added to water (300 ml). The resultant mixture was heated to 40° C., and 40% isopropyl amine aqueous solution was used to adjust pH to 8.0. The mutant 1 solution prepared in Example 1 (750 ml) was added, and a suitable amount of air was introduced (whilst controlling bubbling). A solution of the compound 3-oxo-4-(2,4,5-trifluorophenyl)butyryl-piperazine (i.e. 250 g 3-oxo-4-(2,4,5-trifluorophenzyl)butyryl-piperazine was dissolved in 80 ml ethanol) was added to the reaction system at a relatively fixed flow rate in 5 h-7 h. During the addition, 40% isopropyl amine was used to control the pH. The reaction was carried out at 40° C. for 12 h, and HPLC showed a conversion rate of above 99.9%. The resultant solution was extracted with ethyl acetate (350 ml), and ethyl acetate was recovered and R-3-amino-4-(2,4,5-trifluorophenyl)butyrylpiperazine (242 g) was obtained, with a purity of 99.6%, and a yield of 96.8%.
(68) .sup.1H NMR (d.sub.6-DMSO) δ(ppm) 6.79 (1H), 6.61 (1H), 3.32 (2H), 3.23 (1H), 3.34 (2H), 3.20 (2H), 1.74 (2H).
(69) By reference to the reaction conditions above, the polypeptide with a sequence of SEQ ID NO: 86 as disclosed in the U.S. Pat. No. 8,293,507 of Codeixs Company, was used as the catalyst to catalyze the reaction above. HPLC showed a conversion rate of 13%.
(70) 4. Synthesis of Compound IV:
(71) Condition 1:
(72) To water (500 ml), R-3-amino-4-(2,4,5-trifluorophenyl)butyryl-4-hydroisoxazole (80 g) and sodium hydroxide (27 g) were added respectively. The resultant mixture was heated to 50° C. and reacted for 2 h. After cooling to room temperature, Boc anhydride (91 g) was added. The reaction was carried out at room temperature for 4-5 h. Then the resultant solution was acidified with hydrochloric acid to pH=1.5, crystallized and filtrated. The crystal was washed with water, and dried, affording the product (88.8 g), with a yield of 96%, and a chemical purity of 99.5%. The .sup.1H NMR spectrum of the product was shown in
(73) Condition 2:
(74) To water (350 ml), R-3-amino-4-(2,4,5-trifluorophenyl)butyrylpiperazine (30 g) and sodium hydroxide (10 g) were added respectively. The resultant mixture was heated to 60° C. and reacted for 3 h. After cooling to room temperature, Boc anhydride (43 g) was added. The reaction was carried out at room temperature for 6 h. Then the resultant solution was acidified with hydrochloric acid to pH=1.7, crystallized and filtrated. The crystal was washed with water, and dried, affording the product (31.5 g), with a yield of 95%, and a chemical purity of 99.4%. The .sup.1H NMR spectrum of the product was shown in
(75) Condition 3:
(76) To water (350 ml), R-3-amino-4-(2,4,5-trifluorophenyl)butyryl-4-morpholine (30 g) and sodium hydroxide (8 g) were added respectively. The resultant mixture was heated to 58° C. and reacted for 2.5 h. After cooling to room temperature, Boc anhydride (46 g) was added. The reaction was carried out at room temperature for 5 h. Then the resultant solution was acidified with hydrochloric acid to pH=1.7, crystallized and filtrated. The crystal was washed with water, and dried, affording the product (31.8 g), with a yield of 96%, and a chemical purity of 99.5%. The .sup.1H NMR spectrum of the product was shown in
(77) 5. Synthesis of Sitagliptin:
(78) Condition 1:
(79) To dichloromethane (300 ml), Compound IV (30 g) was added. The resultant mixture was cooled to 15° C. followed by an addition of thionyl chloride (26.8 g). The resultant mixture was heated to 25° C., and reacted for 2 h. Triethylamine (20 g) and 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride (22.7 g) were added respectively. The resultant mixture was reacted at 25° C. for 5-6 h. Then water (100 ml) was added. The organic phase was separated, concentrated and crystallized, affording the dry product of Sitagliptin (34.8 g), with a purity of 99.8%, a chiral purity 99.9%, and a yield of 95%. The chemical purity and chiral purity of the product were determined by HPLC (as shown in
(80) Condition 2:
(81) To ethyl acetate (250 ml), Compound V (30 g) was added. The resultant mixture was cooled to 15° C. followed by an addition of thionyl chloride (28 g). The resultant mixture was heated to 26° C., and reacted for 2 h. Triethylamine (23 g) and 3-(trifluoromethyl)-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine hydrochloride (223 g) were added respectively. The resultant mixture was reacted at 25° C. for 5-6 h. Then water (200 ml) was added. The organic phase was separated, concentrated and crystallized, affording the dry product of Sitagliptin (35.2 g), with a purity of 99.9%, a chiral purity of 100%, and a yield of 96%. The chemical purity and chiral purity of the product were determined by HPLC (as shown in
(82) Method for Determining Chiral Purity:
(83) Chromatographic column: CHIRALPAK AD-H 4.6 mm×250 mm, 5 um;
(84) Wavelength: 210 nm;
(85) Column temperature: 40° C.;
(86) Flow rate: 1.0 mL/min;
(87) Sample volume: 20 uL;
(88) Mobile phase: n-hexane:ethanol:isopropanol:diethylamine=400:500:100:3;
(89) Diluent:methanol:mobile phase=50:50 (diluent was used to treat a sample and as a blank);
(90) Method for Determining Chemical Purity:
(91) Chromatographic column: Kromasil 100-5-C18 4.6 mm×250 mm, 5 um;
(92) Flow rate: 1.0 ml/min;
(93) Wavelength: 210 nm;
(94) Sample volume: 20 ul;
(95) Column temperature: 30° C.;
(96) Preparation of Buffer:
(97) 9.6 g citric acid was dissolved in 1 L water, and 5% sodium hydroxide was used to adjust pH to 4, for further use.
(98) Preparation of Mobile phase A: buffer:methanol=800:200, mixed, filtrated, and ultrasonically degassed for further use.
(99) Preparation of Mobile phase B: methanol:tetrahydrofuran=900:100, mixed, filtrated, and ultrasonically degassed for further use.
(100) Preparation of a diluent: methanol:water=50:50.
(101) Program:
(102) TABLE-US-00007 Time (min) A % B % 0 90 10 15 60 40 20 90 10 50 90 10
(103) Although the embodiments of the present invention have been described in detail, according to all the disclosed teachings, a person skilled in the art would understand that a variety of modifications and replacements may be performed to the details of the technical solutions of the present invention. These changes all fall into the protection scope of the invention. The whole scope of the present invention is defined by the attached claims and any equivalent thereof.