CN103555683A - Synthesis method of saxagliptin chiral intermediate - Google Patents

Synthesis method of saxagliptin chiral intermediate Download PDF

Info

Publication number
CN103555683A
CN103555683A CN201310586363.3A CN201310586363A CN103555683A CN 103555683 A CN103555683 A CN 103555683A CN 201310586363 A CN201310586363 A CN 201310586363A CN 103555683 A CN103555683 A CN 103555683A
Authority
CN
China
Prior art keywords
pdh
mutant
aminoacid sequence
amino acids
hag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201310586363.3A
Other languages
Chinese (zh)
Other versions
CN103555683B (en
Inventor
罗煜
丁时诚
瞿旭东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yikelai (Taizhou) Pharmaceutical Co.,Ltd.
Original Assignee
NANJING ABIOCHEM BIOLOGICAL PHARMACEUTICAL TECHNOLOGY Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by NANJING ABIOCHEM BIOLOGICAL PHARMACEUTICAL TECHNOLOGY Co Ltd filed Critical NANJING ABIOCHEM BIOLOGICAL PHARMACEUTICAL TECHNOLOGY Co Ltd
Priority to CN201310586363.3A priority Critical patent/CN103555683B/en
Publication of CN103555683A publication Critical patent/CN103555683A/en
Application granted granted Critical
Publication of CN103555683B publication Critical patent/CN103555683B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • C12N9/0018—Phenylalanine dehydrogenase (1.4.1.20)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/0102—Phenylalanine dehydrogenase (1.4.1.20)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides a phenylalanine dehydrogenase (PDH) mutant derived from geobacillus, which is high in enzyme activity and thermal stability compared to wild PDH. Furthermore, the invention provides a method for catalytic synthesis of a saxagliptin chiral intermediate, namely (S)-N-t-butyloxycarbonyl-3-hydroxy-1-adamantyl-D-glycine (Boc-HAG) through the PDH mutant. According to the method provided by the invention, Boc-HAG can be directly prepared through a reaction in two steps, and e.e. (enantiomeric excess) value exceeds 99.9%; generation of side products can be reduced, yield of 95% can be achieved within 12 hours, catalysis time is greatly shortened, energy consumption is reduced and a post-treatment process is simplified.

Description

A kind of synthetic method of BMS-477118 chiral intermediate
Technical field
The invention belongs to field of biological pharmacy, be specifically related to the synthetic method of the crucial chiral intermediate of a kind of BMS-477118.
Background technology
BMS-477118 (saxagliptin) is by a kind of efficient, the selectivity of executing your treasured and AstraZeneca develop jointly, competitive dipeptidyl peptidase-IV (DPP-IV) inhibitor, in 2009, by European drug administration (EMA) and FDA Food and Drug Administration (FDA) approval, gone on the market, commodity are called Onglyza, and its chemical structure is as follows:
Figure BDA0000417623560000011
BMS-477118 (Onglyza) is that first obtains the DPP-IV inhibitor of listing approval in European market, worldwide 90 countries submit new drug registration, and get the Green Light in 56 countries, comprise 30 countries of the U.S., Canada, Chile, India, Brazil and European Union.Because its side effect is little, formulation such as is convenient to take at the feature, is Fast Growth after listing, and within 2009, global marketing volume has surpassed 100,000,000 dollars.To 2012, the global marketing volume of Onglyza rose to nearly 700,000,000 U.S. dollars, and rising trend is swift and violent.In developed country's pharmaceutical market, DPP-IV inhibitor has become the main growth point in diabetes medicament market.The share of estimating global diabetic market in 2017 will reach 30,000,000,000 dollars, and wherein DDP-IV is by the share that accounts for 25%.In May, 2011, SFDA official approval BMS-477118, in Discussion on Chinese Listed, is used for the treatment of adult's type ii diabetes, and commodity are called An Lize.BMS-477118 shows extremely strong growth potential after going on the market at home, although the domestic 16Ge key cities Xi Gelieting of sample hospital in 2011 medication only reaches more than 7,000,000 yuan, increased by 200% than the previous year.The time that this medicine gets the Green Light is shorter, domestic its imitated report is still belonged to blank, does not have enterprise to have bulk drug and the preparation certification of BMS-477118.Therefore, development have autonomous property right and efficiently BMS-477118 chemical-biological method synthesis technique there is great economic benefit and social benefit.
The synthetic method report of BMS-477118 is more, most about its synthetic document (WO2011117393; WO2010032129; US20060035954; US2005090539; J.Med.Chem, 2005,48,5025-5037; Org.Process.Res.Dev; 2009; 13; 1169-1176) mainly adopt adamantine amino acid derivative and carbamyl tetrahydrochysene arsenic to cough up after derivative amidation; primary amide group forms cyano group through trifluoroacetic anhydride dehydration; finally under acidic conditions, slough tertbutyloxycarbonyl (Boc) protecting group and obtain target compound, synthetic route is as follows:
Figure BDA0000417623560000021
Therefore, synthetic BMS-477118 needs two crucial intermediates (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG, intermediate A) and cis-4,5-methyl prolineamide (L-cis-4,5-methanoprolinamide, intermediate B), wherein the synthetic key of intermediate A is the introducing of chiral carbon, and its synthetic method mainly contains chemical resolution method (WO2011117393; US2005090539; J.Med.Chem, 2005,48,5025-5037) with enzyme catalysis ammonification reduction (WO2010032129; US2005090539; Advanced Synthesis & Catalysis, 2007,349,1369-1378; Bioorganic.Med.Chem, 2011,19 (3), 1136-1154; US2010291642).Concrete chemical synthesis route is as follows:
Route one: David Augeri etc. sets out with adamantane acid and reduces through LAH; Swern oxidation and asymmetric Strecker reaction have built chiral intermediate; follow-up again through four-step reactions such as hydrolysis, debenzylation, Boc protection, oxidation introducing hydroxyls; prepare chiral intermediate A; yield 21%, but this route is difficult to amplify.
Figure BDA0000417623560000022
Route two: US2005090539 report adamantane acid is after bromo; react with nitration mixture hydroxylation occurs; introduce amino and obtain raceme; again amino is carried out to Boc protection; the means that finally split by chemistry obtain A, and route is longer, overall yield 30% left and right; after splitting, enantiomeric excess value (e.e. value) is still not high, only has 70%-80%.
Figure BDA0000417623560000031
Two kinds of chemical process routes are above longer, and overall combined coefficient is not high; The use of the chemical reagent such as Li-Al hydrogen causes cost higher, and can produce comparatively serious environmental problem and subsequent disposal problem.Compare with pure chemistry synthetic route, enzyme catalysis biotransformation method can carry out mutually at pure water, and reducing use has the catalyzer of harm to people and environment, reduced the generation of refuse, and environment friendly is good; More importantly, enzyme has excellent stereoselectivity, and intermediate product does not need to split, and therefore enzymatic bio-transformation can effectively improve the optical purity of productive rate and product, has fabulous industrialization potential.The people such as Ronald L.Hanson have reported under the existence of DPNH (NADH), obtain amino acid (S)-(3-hydroxyl-1-adamantyl)-D-glycine (HAG) of S-type with Phenylalanine dehydrogenase (PDH) catalysis ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-reduction amination of 2-oxoethanoic acid in actinomycetes source; Under ammonium formiate exists, the hydrogenlyase (FDH) in pichia source makes coenzyme NAD H regeneration.On amino acid, introduce Boc protecting group and obtain Boc-HAG, route is as follows:
Figure BDA0000417623560000032
The Phenylalanine dehydrogenase using in reduction amination process (PDH) is through N end and the transformation of C end, its enzyme to ketone acid substrate is lived and is raise, but thermostability declines, the overlong time of reacting at the temperature of 40 ℃, reach completely and transform and need more than 38 hours, energy consumption is too high, and production capacity is difficult to improve.
Summary of the invention
The present invention is by having improved enzyme activity and the stability of the Phenylalanine dehydrogenase in ground bacillus source to the orthogenesis of DNA sequence dna, this enzyme can synthesizing for synthetic middle key intermediate (the S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG) of BMS-477118.
Amino acid dehydrogenase is the important albumen in amino acid metabolism, at cofactor NAD +effect under the oxidative deamination of catalytic amino acid, amino acid whose amino oxidative deamination is formed to corresponding ketone acid.As the member of this family, Phenylalanine dehydrogenase (PDH) can catalysis L-Phe to the conversion of phenyl-pyruvic acid; But at NH 3in the higher situation of concentration, the reversed reaction that Phenylalanine dehydrogenase can the above-mentioned reaction of catalysis, generates L-Phe by phenyl-pyruvic acid reduction amination, and this reaction needed consumes NADH.
Contriver transforms to improve the enzyme activity of its catalysis BMS-477118 intermediate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid reduction amination by the method for random mutation to the Phenylalanine dehydrogenase (PDH) in ground bacillus source.In order to make NADH regeneration, contriver clones and has obtained hydrogenlyase (FDH) from pichia spp, can make NAD be reduced to NADH, thereby promotes the carrying out of reduction amination.Above-mentioned two kinds of enzymes can utilize intestinal bacteria recombinant bacterial strain express and obtain by high density fermentation; under their catalysis; ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid is through reduction amination and Boc protection; two-step reaction directly obtains the chiral intermediate Boc-HAG of BMS-477118, and e.e. value surpasses 99.9%.The recombinase obtaining by random mutation has higher enzyme activity and good thermostability, thereby concentration of substrate is improved, and the reaction times is shortened, and energy consumption is reduced, in 20 hours, can thoroughly transform, therefore this technique have extraordinary industrialization prospect.
On the basis of above-mentioned achievement in research, a first aspect of the present invention provides a kind of Phenylalanine dehydrogenase (PDH) mutant that derives from ground bacillus, it is characterized in that, PDH compares with wild-type, at the 85th and 294 amino acids residues of aminoacid sequence, has the sudden change of replacement.This PDH mutant is compared wild-type PDH, has enzyme activity and/or the thermostability of raising.In present patent application file, it is benchmark that the numbering of PDH aminoacid sequence be take the aminoacid sequence (SEQ ID NO:1) of the wild-type PDH that derives from ground bacillus (Geobacillus sp.) Y412MC61.Preferably, the 93rd of described PDH mutant aminoacid sequence the and 288 amino acids residues sport respectively leucine (L) and α-amino-isovaleric acid (V); More preferably, the replacement of the 93rd of described PDH mutant aminoacid sequence the and 288 amino acids residues sudden change is respectively I93L and L288V.
In a specific embodiment, PDH mutant of the present invention is compared with wild-type PDH, and only the 93rd and 288 amino acids residues at aminoacid sequence have the sudden change of replacement.Preferably, the 93rd of described PDH mutant aminoacid sequence the and 288 amino acids residues sport respectively leucine (L) and α-amino-isovaleric acid (V); More preferably, the replacement of the 93rd of its aminoacid sequence the and 288 amino acids residues sudden change is respectively I93L and L288V.
PDH compares with wild-type, and PDH mutant of the present invention can also have sudden change in other sites, and preferably, described other sport the replacement sudden change of the 75th, 184 and 301 amino acids residues of its aminoacid sequence; More preferably, the 75th, 184 of its aminoacid sequence and 301 amino acids residues sport respectively L-Ala (A), L-glutamic acid (E) and Serine (S); Most preferably, the replacement of the 75th, 184 of its aminoacid sequence and 297 amino acids residues sudden change is respectively M75A, K184E and V301S.
In another specific embodiment, PDH mutant of the present invention is compared with wild-type PDH, and only the 75th, 93,184, the 288 and 301 amino acids residues at aminoacid sequence have the sudden change of replacement.Preferably, the 75th, 93,184,288 of described PDH mutant aminoacid sequence and 301 amino acids residues sport respectively L-Ala (A), leucine (L), L-glutamic acid (E), α-amino-isovaleric acid (V) and Serine (S); More preferably, the replacement of the 75th, 93,184,294 of its aminoacid sequence and 297 amino acids residues sudden change is respectively M75A, I93L, K184E, L288V and V301S.
PDH mutant of the present invention derives from ground bacillus (Geobacillus sp.), preferably Geobacillus sp.Y412MC61; Most preferably, the aminoacid sequence of described wild-type PDH is for deriving from the aminoacid sequence (SEQ ID NO:1) of the wild-type PDH of ground bacillus (Geobacillus sp.) Y412MC61.
In a most preferred embodiment, the aminoacid sequence of PDH mutant of the present invention is as shown in SEQ ID NO:5 or 6.
Second aspect present invention provides the nucleic acid molecule of the PDH mutant of the present invention of encoding.Described nucleic acid molecule can be DNA or RNA, and two strands or strand, as long as the genetic information that has comprised the PDH mutant of the present invention of encoding.
A third aspect of the present invention provides the carrier that comprises above-mentioned nucleic acid molecule.Described carrier can be cloning vector or expression vector, can be various types of carriers, includes but not limited to plasmid vector, virus vector, cosmid vector, YAC, BAC etc.Preferably, described carrier is pET21a carrier.
A fourth aspect of the present invention provides the host cell that comprises above-mentioned carrier, and it can be used for expressing PDH mutant of the present invention.Described host cell can be protokaryon or eukaryotic host cell, preferred intestinal bacteria, and most preferably, it is e. coli bl21 (DE3).
A fifth aspect of the present invention provides described PDH mutant or the purposes of described host cell in synthetic (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG).Further, described PDH mutant or described host cell can also be for the synthesis of BMS-477118s.
A sixth aspect of the present invention provides the method for a kind of synthetic BMS-477118 chiral intermediate (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG), and the method comprises the following steps:
(1), under the existence of DPNH (NADH), with PDH mutant catalysis ketone acid substrate 2-of the present invention (3-hydroxyl-1-adamantyl)-2-oxoethanoic acid reduction amination, obtain amino acid (S)-(3-hydroxyl-1-adamantyl)-D-glycine (HAG) of the amino acid S-type of S-type;
(2), under ammonium formiate exists, hydrogenlyase (FDH) makes NADH regeneration;
(3) the upper introducing of HAG tertbutyloxycarbonyl (Boc) protecting group obtains Boc-HAG.
Wherein, described FDH preferably derives from pichia (Pichia pastoris); Most preferably, described FDH is for deriving from the aminoacid sequence (SEQ ID No.7) of the wild-type FDH of pichia (Pichia pastoris) GS115.
A seventh aspect of the present invention provides a kind of method of synthetic BMS-477118, comprises and uses synthetic BMS-477118 chiral intermediate (the S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG) of aforesaid method.
Accompanying drawing explanation
Fig. 1 clones the gel electrophoresis spectrum of the pcr amplification product of the pdh gene of ground bacillus (Geobacillus sp.) Y412MC61 and the fdh gene of pichia GS115.M is molecular weight marker, and swimming lane 1 is pdh gene, and swimming lane 2 is fdh gene.
The SDS-PAGE electrophoretogram of Fig. 2 PDH mutant 2 and FDH protein expressioning product.M is molecular weight marker, and swimming lane A is not induction contrast, and swimming lane B is PDH mutant 2, and swimming lane C is FDH.
The aminoacid sequence comparison result of Fig. 3 wild-type PDH, PDH mutant 1, PDH mutant 2.SEQ NO1, SEQ NO5, SEQ NO6 are respectively the aminoacid sequence of wild-type PDH, PDH mutant 1, PDH mutant 2.PDH compares with wild-type, and PDH mutant 1 has I93L+L288V sudden change, and PDH mutant 2 has M75A+I93L+K184E+L288V+V301S sudden change.
Embodiment
The enzyme activity determination of embodiment 1PDH and FDH
The enzyme activity determination of PDH carries out at 30 ℃, and reaction system contains 0.4mM NADH, and 50mM ketone acid A1(is dissolved in the NaOH of 1 equivalent), 0.75M ammoniacal liquor (HCl adjusts pH8.75).Add the variation of the ultraviolet absorption value of detection reaction system under 340nm after Phenylalanine dehydrogenase.
The enzyme activity determination of FDH carries out at 30 ℃, and reaction system contains 1mM NAD, 100mM ammonium formiate, and 100mM potassiumphosphate (pH8.0) damping fluid, adds the variation that detects the ultraviolet absorption value under 340nm in 30 minutes after hydrogenlyase.
Enzyme is lived and is defined:
The every variation 0.1 of ultraviolet absorption value under 340nm, corresponding to the concentration change 0.016mM of NADH.Therefore obtain enzyme activity calculation formula:
U = A · N · 0.016 t
A: absorbancy minimizing value
N: extension rate
T: reaction times (min)
Embodiment 2 substrates and product analysis method
Adopt high performance liquid chromatography (HPLC) method to detect product: take water and acetonitrile (45:55) is moving phase, and chromatographic column is ODS-18 reversed-phase column, and Shimadzu LC-15C high performance liquid chromatography detects uv-absorbing under 210nm; Reaction system water and acetonitrile (45:55) dilute; centrifugal and with nylon membrane, filter after sample detection; the retention time of ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid is 4.19 minutes; amino acid product peak retention time is 2.6 minutes, and the amino acid whose retention time of Boc protection was at 2.1 minutes.
Optical purity e.e. value is used AD-H chiral column (Diacel Chemical) to analyze on Agilent1260 series HPLC, and the product retention time of R-configuration is 3.5min, and the product retention time of S-configuration is 5.1min.Moving phase is ethanol: normal hexane=98:2.
TLC developping agent is butanols: water: acetic acid=4:1:1, with triketohydrindene hydrate, to amino acid and the colour developing of Boc derivative thereof, phospho-molybdic acid is to raw material and amino acid colour developing.
The clone of embodiment 3PDH also builds E.coli mutant library
The pdh gene of ground bacillus (Geobacillus sp.) Y412MC61 is introduced to E.coli.For the pdh gene (SEQ ID No.2) of amplification coding PDH enzyme (SEQ ID No.1), using respectively primers F 1(SEQ ID No.3) and R1(SEQ ID No.4) as forward and reverse primer.Two primers all contain the site compatible with the pcr amplification deoC gene fragment of using Gateway Technology to obtain by fixed point recombinant clone.The gel electrophoresis spectrum of pcr amplification product as shown in Figure 1.Use random mutagenesis test kit, by changing MnSO 4concentration is carried out a plurality of reactions, thereby 1 to 3 point mutation is introduced to the pdh gene (SEQ ID No.2) of Geobacillus sp.Y412MC61, and 1-3 amino-acid residue in PDH enzyme amino acid sequence is replaced.
The sequence of forward primer F1 (SEQ ID No.3):
5’-GAGCATATGAATGTCATGCTATCGCC-3’
The sequence of reverse primer R1 (SEQ ID No.4):
5’-AGACTCGAGTTAACGTCGAATATCCCAC-3’
Fallibility pcr amplification is used following temperature program(me): 94 ℃ of 2min, 25 circulations of 94 ℃ of 30s and 68 ℃ of 1min, then 68 ℃ of 10min.First fallibility PCR fragment is cloned to the carrier into pDONR, prepare extensive pENTR plasmid library, initially surpass 20,000 bacterium colonies.Then take pDEST14 as carrier, the pENTR plasmid library of geting started is configured to expression library.Then expression library is proceeded to chemoreception state E.coil BL21Star(DE3), for expressing the pdh gene (coding PDH enzyme mutant) of sudden change.
The expression of embodiment 4PDH enzyme
The expression of the pdh gene of sudden change in deep hole microtiter plate: the mutant bacteria obtaining according to embodiment 3, choose sudden change bacterium colony, be seeded in microtiter plate (MTP) 200 μ L2 * YT substratum (penbritin that contains 100 μ g/ml), 37 ℃ of culture condition, the time is 1 day.Then get the above-mentioned preculture thing of 100 μ L and be seeded in the deep-well plates containing 500 μ L expression cultures (2 * YT, containing 100 μ g/ml penbritins and 1mM IPTG), then 25 ℃ of cultivation 24h on shaking table.
Embodiment 5 screenings have the PDH mutant that improves catalytic capability
The PDH mutant deep hole culture 3500rpm that embodiment 4 is obtained carries out centrifugal 15 minutes, adds 400 μ L lysis buffers (50mM phosphoric acid buffer, pH7.4,1mg/mL N,O-Diacetylmuramidase) to make its resuspension, and multigelation makes cytoclasis.Centrifugal 4000rpm removes cell debris, takes out the not celliferous lysate of 210 μ L to quartz micropores titer plate in each hole.Add respectively NADH and ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid to 1mM and 10mg/mL final concentration, hatch for 30 ℃ and after 15 minutes, 30 minutes, 45 minutes and 1 hour, detect respectively uv-absorbing A under its 340nm 340.Using do not add ketone acid substrate sample as blank group.According to the calculating enzyme activity that underspeeds of NADH under 340nm, screening obtains the PDH mutant 1(SEQ ID No.5 that enzyme activity improves), experimental result is as shown in table 1.
Table 1PDH enzyme activity determination
Figure BDA0000417623560000081
Embodiment 6 screenings have the PDH mutant of thermostability
The PDH mutant 1 that the enzyme activity that the embodiment 5 of take obtains improves is masterplate, carries out random mutation build new mutant library by fallibility PCR, and condition is as embodiment 3.Mutant deep hole culture carries out centrifugal 15 minutes at 3500rpm, adds 400 μ L lysis buffers (50mM phosphoric acid buffer, pH7.4,1mg/mL N,O-Diacetylmuramidase) to make its suspension.50 ℃ of temperature, bathe 30min, then add 1mM NADH and 10mg/mL ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid, 30 ℃ are detected NADH at the decay intensity of 340nm uv-absorbing after hatching 60min, relatively PDH mutant enzyme vigor.With the sample without 50 ℃ of incubations, screen in contrast and obtain heat-staple PDH mutant 2(SEQ ID No.6), experimental result is as shown in table 2.
The thermal stability determination of table 2PDH enzyme
PDH mutant 2 is expressed according to the method for embodiment 4, and the SDS-PAGE electrophoretogram of expression product as shown in Figure 2.
The fermentation of embodiment 7PDH enzyme
The high-performance PDH enzyme mutant gene engineering colibacillus obtaining according to embodiment 6 is inoculated in the 1L shaking flask that 200mL LB substratum is housed, and in 37 ℃, 180-220rpm cultivates 10-16h.Above-mentioned cultured seed is inoculated in to (glucose 4g/L in the upper tank fermention medium (M9) of 3L in the ratio of 10% (v/v), Sodium phosphate dibasic 12.8g/L, potassium primary phosphate 3g/L, ammonium chloride 1g/L, sodium sulfate 0.5g/L, calcium chloride 0.0152g/L, magnesium chloride hexahydrate 0.41g/L), at 20-30 ℃, 300-800rpm, cultivates under the condition of air flow quantity 2-6L/min.Cultivate after 6-10h, with the data rate stream of 5-20mL/h, add the supplemented medium containing 60% glycerine, continue to fermentation ends.Flow feeding substratum a few hours are to OD 600while reaching 20-40, add 0.1-1mM IPTG and start induction.After induction 10-20h, put tank, the centrifugal collection thalline of 5000rpm.
The clonal expression of embodiment 8FDH enzyme
Hydrogenlyase (FDH) gene of pichia (Pichia pastoris) GS115 is cloned into coli expression carrier pET21a by PCR.For the fdh gene (SEQ ID No.8) of amplification coding FDH enzyme (SEQ ID No.7), using respectively primers F 2(SEQ ID No.9) and R2(SEQ ID No.10) as forward and reverse primer.The gel electrophoresis spectrum of pcr amplification product as shown in Figure 1.
The sequence SEQ ID No.9 of forward primer F2:
5’-GGGCATATGAAAATCGTTCTCGTTTTG-3’
The sequence SEQ ID No.10 of reverse primer R2:
5’-GGGCTCGAGTTATGCGACCTTTTTGTC-3’
Pcr amplification is used following temperature program(me): 94 ℃ of 2min, 1 circulation; 94 ℃ of 30s, 30 circulations of 60 ℃ of 1min and 72 ℃ of 2min, then 72 ℃ of 10min.PCR fragment is carried out to double digestion with NdeI and XhoI, then clone into the linearizing pET21a carrier of same restriction endonuclease, T4 ligase enzyme spends the night 16 ℃ of connections, connect product and be converted into bacillus coli DH 5 alpha, picking list bacterium colony is also identified the genotype of plasmid, correct plasmid is converted into e. coli bl21 (DE3) and carries out FDH protein expression, and the SDS-PAGE electrophoretogram of expression product as shown in Figure 2.
The fermentation of embodiment 9FDH enzyme
The FDH enzyme gene engineering colibacillus obtaining according to embodiment 8 is inoculated in the 1L shaking flask that 200mL LB substratum is housed, and in 37 ℃, 180-220rpm cultivates 10-16h.Above-mentioned cultured seed is inoculated in to (glucose 4g/L in the upper tank fermention medium (M9) of 3L in the ratio of 10% (v/v), Sodium phosphate dibasic 12.8g/L, potassium primary phosphate 3g/L, ammonium chloride 1g/L, sodium sulfate 0.5g/L, calcium chloride 0.0152g/L, magnesium chloride hexahydrate 0.41g/L), at 25-35 ℃, 300-800rpm, cultivates under the condition of air flow quantity 4L/min.Cultivate after 10h, with the data rate stream of 5-20mL/h, add the supplemented medium containing 60% glycerine, continue to fermentation ends.Flow feeding substratum a few hours are to OD 600reach at 20 o'clock, add 1mM IPTG and start induction.After induction 15h, put tank, the centrifugal collection thalline of 5000rpm.
Embodiment 10 enzyme catalysis reduction aminations and Boc protection
Get respectively embodiment 7 and 9 coli somatics of collecting, add the ammonium phosphate damping fluid (pH7.5) of 1～3 times of volume 0.05M.60-600W ultrasonication 30min, crude enzyme liquid is directly used in enzymic catalytic reaction.1.31g ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid (5.8mmol) and 740mg ammonium formiate (11.7mmol) are dissolved in 5mL ammonium phosphate damping fluid.Then add 16.4mg NAD(24.7 μ mol) and 3.6mg DTT(23.3 μ mol), mend and add water to cumulative volume 12mL, and regulate pH to 8.0 with ammoniacal liquor.Add 22U PDH and 114U FDH, 30 ℃, 40rpm carries out catalyzed reaction 20 hours, and TLC monitors reaction process.Question response finishes rear 80 ℃ of water-bath 30min makes protein precipitation, removes by filter protein, and filtrate rotary evaporation is removed excess of ammonia, is settled to 9ml.Add potassiumphosphate 3.09g, under stirring, add containing 1.15g (Boc) 2o(5.27mmol) THF solution, reacts 3h under room temperature, TLC detection reaction process.After finishing, reaction abandons bottom; add 7ml distilled water and adjust pH to 2.5～3 with concentrated hydrochloric acid; with equal-volume Iso Butyl Acetate extraction 3 times; organic phase anhydrous sodium sulfate drying; add normal heptane to obtain suspension; after stirring 1h, filter amino acid (BOC-HAG) 1.32g that obtains Boc protection, two step total recoverys 84%, e.e. value >99.9%.
Figure IDA0000417623650000011
Figure IDA0000417623650000031
Figure IDA0000417623650000041
Figure IDA0000417623650000051
Figure IDA0000417623650000061
Figure IDA0000417623650000071
Figure IDA0000417623650000081
Figure IDA0000417623650000091
Figure IDA0000417623650000101
Figure IDA0000417623650000111
Figure IDA0000417623650000121
Figure IDA0000417623650000161
Figure IDA0000417623650000171
Figure IDA0000417623650000181
Figure IDA0000417623650000191
Figure IDA0000417623650000201
Figure IDA0000417623650000221

Claims (24)

1. Phenylalanine dehydrogenase (PDH) mutant that derives from ground bacillus (Geobacillus sp.), it is characterized in that, PDH compares with wild-type, at the 93rd and 288 amino acids residues of aminoacid sequence, has the sudden change of replacement, and has enzyme activity and/or the thermostability of raising; Wherein, to take SEQ ID NO:1 be benchmark to the numbering of PDH aminoacid sequence.
2. PDH mutant claimed in claim 1, it is compared with wild-type PDH, and only the 93rd and 288 amino acids residues at aminoacid sequence have the sudden change of replacement.
3. the PDH mutant described in claim 1 or 2, the 93rd and 288 amino acids residues of its aminoacid sequence sport respectively leucine (L) and α-amino-isovaleric acid (V).
4. PDH mutant claimed in claim 3, the replacement sudden change of the 93rd and 288 amino acids residues of its aminoacid sequence is respectively I93L and L288V.
5. PDH mutant claimed in claim 1, it also has at the 75th, 184 and 301 amino acids residues of aminoacid sequence the sudden change of replacement.
6. PDH mutant claimed in claim 5, it is compared with wild-type PDH, and only the 75th, 93,184, the 288 and 301 amino acids residues at aminoacid sequence have the sudden change of replacement.
7. the PDH mutant described in claim 5 or 6, the 75th, 93,184,288 and 301 amino acids residues of its aminoacid sequence sport respectively L-Ala (A), leucine (L), L-glutamic acid (E), α-amino-isovaleric acid (V) and Serine (S).
8. PDH mutant claimed in claim 7, the replacement sudden change of the 75th, 93,184,288 and 301 amino acids residues of its aminoacid sequence is respectively M75A, I93L, K184E, L288V and V301S.
9. the PDH mutant described in claim 1-8 any one, wherein, described ground bacillus is Geobacillus sp.Y412MC61.
10. PDH mutant claimed in claim 9, wherein, the aminoacid sequence of described wild-type PDH is as shown in SEQ ID NO:1.
11. PDH mutant claimed in claim 10, its aminoacid sequence is as shown in SEQ ID NO:5 or 6.
The nucleic acid molecule of the PDH mutant described in 12. coding claim 1-11 any one.
13. carriers that comprise the nucleic acid molecule described in claim 12.
Carrier described in 14. claims 13, it is pET21a carrier.
15. host cells that comprise the carrier described in claim 13 or 14.
The host cell of 16. claims 15, it is intestinal bacteria.
Host cell described in 17. claims 16, it is e. coli bl21 (DE3).
PDH mutant described in 18. claim 1-11 any one or the host cell described in the claim 15-17 any one purposes in synthetic (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG).
PDH mutant described in 19. claim 1-11 any one or the host cell described in the claim 15-17 any one purposes in synthetic BMS-477118.
The method of 20. 1 kinds of synthetic (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG), comprises the following steps:
(1), under the existence of DPNH (NADH), with PDH mutant catalysis ketone acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid reduction amination described in claim 1-11 any one, obtain amino acid (S)-(3-hydroxyl-1-adamantyl)-D-glycine (HAG) of S-type;
(2), under ammonium formiate exists, hydrogenlyase (FDH) makes NADH regeneration;
(3) the upper introducing of HAG tertbutyloxycarbonyl (Boc) protecting group obtains Boc-HAG.
Method described in 21. claims 20, wherein, described FDH derives from pichia (Pichia pastoris).
Method described in 22. claims 21, wherein, the aminoacid sequence of described FDH is as shown in SEQ ID NO:7.
Method described in 23. claim 20-22 any one, wherein, catalyzed reaction, at 30 ℃, is carried out under 40rpm, and the reaction times is 20 hours.
The method of 24. 1 kinds of synthetic BMS-477118s, comprises that right to use requires method synthesis of chiral intermediate (the S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG) described in 20-23 any one.
CN201310586363.3A 2013-11-19 2013-11-19 A kind of synthetic method of saxagliptin chiral intermediate Active CN103555683B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310586363.3A CN103555683B (en) 2013-11-19 2013-11-19 A kind of synthetic method of saxagliptin chiral intermediate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310586363.3A CN103555683B (en) 2013-11-19 2013-11-19 A kind of synthetic method of saxagliptin chiral intermediate

Publications (2)

Publication Number Publication Date
CN103555683A true CN103555683A (en) 2014-02-05
CN103555683B CN103555683B (en) 2015-11-18

Family

ID=50010038

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310586363.3A Active CN103555683B (en) 2013-11-19 2013-11-19 A kind of synthetic method of saxagliptin chiral intermediate

Country Status (1)

Country Link
CN (1) CN103555683B (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951588A (en) * 2014-04-30 2014-07-30 淮海工学院 Method of synthesizing saxagliptin intermediate N-t-butyloxycarboryl-3-hydroxyl-1-adamantyl-D-glycine
CN106191154A (en) * 2016-07-21 2016-12-07 江苏福瑞生物医药有限公司 A kind of biological preparation method of onglyza intermediate (S) N tertbutyloxycarbonyl 3 hydroxyl 1 adamantyl D glycine
WO2017015154A1 (en) * 2015-07-17 2017-01-26 Children's National Medical Center Methods and compositions for treating phenyltonuria
CN106497843A (en) * 2016-11-30 2017-03-15 浙江工业大学 Pseudomonas putida ZJPH1412 and its application of preparation (S) 3 hydroxyadamantane glycine
CN109593802A (en) * 2018-12-24 2019-04-09 上海健康医学院 The preparation method of one kind (R) -2- (2,5- difluorophenyl) pyrrolidines or its salt
CN110628739A (en) * 2019-08-14 2019-12-31 华东理工大学 Amine dehydrogenase mutant and application thereof in synthesis of chiral amine and amino alcohol

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006015885A1 (en) * 2004-08-13 2006-02-16 University College Dublin, National University Of Ireland, Dublin Amino acid dehydrogenase-derived biocatalysts
JP2006271379A (en) * 2005-03-03 2006-10-12 Toyama Prefecture Function-modified phenylalanine dehydrogenase and method for analyzing amino acid in biological sample using the same
CN102459610A (en) * 2009-04-08 2012-05-16 百时美施贵宝公司 A genetically stable plasmid expressing pdh and fdh enzymes
CN103215321A (en) * 2004-04-14 2013-07-24 布里斯托尔-迈尔斯.斯奎布公司 Process for preparing dipeptidyl iv inhibitors and intermediates therefor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103215321A (en) * 2004-04-14 2013-07-24 布里斯托尔-迈尔斯.斯奎布公司 Process for preparing dipeptidyl iv inhibitors and intermediates therefor
WO2006015885A1 (en) * 2004-08-13 2006-02-16 University College Dublin, National University Of Ireland, Dublin Amino acid dehydrogenase-derived biocatalysts
JP2006271379A (en) * 2005-03-03 2006-10-12 Toyama Prefecture Function-modified phenylalanine dehydrogenase and method for analyzing amino acid in biological sample using the same
CN102459610A (en) * 2009-04-08 2012-05-16 百时美施贵宝公司 A genetically stable plasmid expressing pdh and fdh enzymes

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FRANCESCA PARADISI等: "Phenylalanine dehydrogenase mutants: Efficient biocatalysts for synthesis of non-natural phenylalanine derivatives", 《JOURNAL OF BIOTECHNOLOGY》 *
LUCAS,S.等: "NCBI Reference Sequence: YP_003253915.1", 《GENBANK》 *
STEPHEN Y. K. SEAH等: "Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling", 《EUROPEAN JOURNAL OF BIOCHEMISTRY》 *
无: "NCBI Reference Sequence: WP_014196077", 《GENBANK》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103951588A (en) * 2014-04-30 2014-07-30 淮海工学院 Method of synthesizing saxagliptin intermediate N-t-butyloxycarboryl-3-hydroxyl-1-adamantyl-D-glycine
CN103951588B (en) * 2014-04-30 2016-10-05 淮海工学院 A kind of method synthesizing onglyza intermediate N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine
WO2017015154A1 (en) * 2015-07-17 2017-01-26 Children's National Medical Center Methods and compositions for treating phenyltonuria
US10792339B2 (en) 2015-07-17 2020-10-06 Children's National Medical Center Methods and compositions for treating phenylketonuria
CN106191154A (en) * 2016-07-21 2016-12-07 江苏福瑞生物医药有限公司 A kind of biological preparation method of onglyza intermediate (S) N tertbutyloxycarbonyl 3 hydroxyl 1 adamantyl D glycine
CN106497843A (en) * 2016-11-30 2017-03-15 浙江工业大学 Pseudomonas putida ZJPH1412 and its application of preparation (S) 3 hydroxyadamantane glycine
CN106497843B (en) * 2016-11-30 2019-05-31 浙江工业大学 Pseudomonas putida ZJPH1412 and its application for preparing (S) -3- hydroxyadamantane glycine
CN109593802A (en) * 2018-12-24 2019-04-09 上海健康医学院 The preparation method of one kind (R) -2- (2,5- difluorophenyl) pyrrolidines or its salt
CN109593802B (en) * 2018-12-24 2021-10-01 上海健康医学院 Preparation method of (R) -2- (2, 5-difluorophenyl) pyrrolidine or salt thereof
CN110628739A (en) * 2019-08-14 2019-12-31 华东理工大学 Amine dehydrogenase mutant and application thereof in synthesis of chiral amine and amino alcohol

Also Published As

Publication number Publication date
CN103555683B (en) 2015-11-18

Similar Documents

Publication Publication Date Title
CN103555683B (en) A kind of synthetic method of saxagliptin chiral intermediate
RU2459871C2 (en) Method for enzymatic production of 2-hydroxy-2-methyl carboxylic acids
CN109609582B (en) Method for preparing L-glufosinate-ammonium by microbial catalysis racemization removal
CN105441403B (en) Transaminase for producing C4H9NO2
CN106995808B (en) A kind of recombination transaminase and its application
Tenhaef et al. Production of D-xylonic acid using a non-recombinant Corynebacterium glutamicum strain
CN103898072A (en) Ketoreductase mutant and application thereof
CN110724675A (en) Transaminase catalyst and method for synthesizing (R) -1-tert-butoxycarbonyl-3-aminopiperidine by enzyme method
CN111139270A (en) Enzyme combination for producing L-glufosinate-ammonium and L-glufosinate-ammonium production method
CN114807265A (en) Synthetic method of S-nicotine
CN111019982B (en) Method for preparing L-glufosinate-ammonium by using hydroxy acid dehydrogenase
CN109852593A (en) A kind of recombination ketoreductase and preparing the application in R-3- hydroxybutyric acid and its salt
CN103834631A (en) Penicillin G acylase mutant, and coding gene and application thereof
CN103966275A (en) Method for preparing highly pure L-tertiary leucine through biological process
CN111411128B (en) Whole cell biocatalysis method for producing alpha, omega-dicarboxylic acid and application thereof
CN108410831A (en) Ketone acid reductase, gene, engineering bacteria and the application in synthesis of chiral fragrance 2- hydroxy acids
CN108374017B (en) Novel styrene epoxidase and function thereof
CN110791536B (en) Biosynthesis method of levodopa
CN104846025B (en) A kind of method for preparing (2S, 3R) -2- benzoyl aminomethyls -3-hydroxybutyrate methyl esters
US20100261251A1 (en) Microbial kinetic resolution of ethyl-3,4-epoxybutyrate
CN103409402B (en) Aldolase mutant
EP1290209B1 (en) Method for preparing an r- or s-form of alpha-substituted heterocyclic carboxylic acid and a counter enantiomeric form of alpha-substituted heterocyclic carboxylic acid ester thereto using enzyme
JP2000217590A (en) Production of optically active cyanohydrin
EP1290208B1 (en) Method for optically resolving a racemic alpha-substituted heterocyclic carboxylic acid using enzyme
Yamada Screening of novel enzymes for the production of useful compounds

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20170425

Address after: 200120 China (Shanghai) free trade zone, Pudong South Road, No. 2, building C110, room

Patentee after: Shanghai Yi Ke Lai biological medicine science and Technology Co Ltd

Address before: Pu Si Road in Pukou District of Nanjing City, Jiangsu province 210032 No. 18 Taishan street, science and Technology Innovation Park 1 building 408 room

Patentee before: Nanjing Abiochem Biological Pharmaceutical Technology Co., Ltd.

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20191223

Address after: 317016 No.25, Donghai Fifth Avenue, Linhai medical park, Linhai chemical API base, Taizhou City, Zhejiang Province

Patentee after: Taizhou Bao Ling Pharmaceutical Co., Ltd.

Address before: Room C110, 1st floor, Building 2, 2250 Pudong Southeast Road, China (Shanghai) Free Trade Pilot Area

Patentee before: Shanghai Yi Ke Lai biological medicine science and Technology Co Ltd

CP03 Change of name, title or address
CP03 Change of name, title or address

Address after: No.25, Donghai Fifth Avenue, Linhai medical and chemical park, Zhejiang chemical raw material drug base, Linhai City, Taizhou City, Zhejiang Province

Patentee after: Yikelai (Taizhou) Pharmaceutical Co.,Ltd.

Address before: No.25, Donghai Fifth Avenue, Linhai medical and chemical park, Linhai chemical raw material drug base, Taizhou City, Zhejiang Province, 317016

Patentee before: TAIZHOU POLLENIN PHARMACEUTICAL Co.,Ltd.