CN103555683B - A kind of synthetic method of saxagliptin chiral intermediate - Google Patents
A kind of synthetic method of saxagliptin chiral intermediate Download PDFInfo
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- CN103555683B CN103555683B CN201310586363.3A CN201310586363A CN103555683B CN 103555683 B CN103555683 B CN 103555683B CN 201310586363 A CN201310586363 A CN 201310586363A CN 103555683 B CN103555683 B CN 103555683B
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- 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)
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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- 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)
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Abstract
The invention provides a kind of Phenylalanine dehydrogenase (PDH) mutant deriving from ground bacillus, it has enzyme activity and the thermostability of raising compared with wild-type PDH.Additionally providing one utilizes PDH mutant of the present invention to catalyze and synthesize the method for saxagliptin chiral intermediate (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG).According to method of the present invention, two-step reaction directly obtains Boc-HAG, and e.e. value more than 99.9%, and reduces the generation of by product, within 12 hours, can reach the productive rate of 95%, significantly shorten catalysis time, reduce energy consumption, simplify last handling process.
Description
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, selectivity, competitive dipeptidyl peptidase-IV (DPP-IV) inhibitor efficient by the one executing your treasured and AstraZeneca joint development, in 2009 by European drug administration (EMA) and FDA Food and Drug Administration (FDA) approval listing, commodity are called Onglyza, and its chemical structure is as follows:
BMS-477118 (Onglyza) is first DPP-IV inhibitor obtaining 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 is convenient to features such as taking, and be Fast Growth after listing, within 2009, global marketing volume has exceeded 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 factor in diabetes medicament market.Estimate that the share in global diabetic market in 2017 will reach 30,000,000,000 dollars, wherein DDP-IV will account for the share of 25%.In May, 2011, SFDA official approval BMS-477118 is in Discussion on Chinese Listed, and be used for the treatment of Adult type II diabetes, commodity are called An Lize.Show extremely strong growth potential after BMS-477118 goes on the market at home, although domestic 16 key cities in 2011 sample hospital Xi Gelieting medication only reaches more than 7,000,000 yuan, increase 200% than the previous year.The time that this medicine gets the Green Light is shorter, and the domestic report imitated to it still belongs 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 document (WO2011117393 about its synthesis; WO2010032129; US20060035954; US2005090539; J.Med.Chem, 2005,48,5025-5037; Org.Process.Res.Dev; 2009; 13; after 1169-1176) derivative amidation coughed up by main employing adamantine amino acid derivative and carbamyl tetrahydrochysene arsenic; primary amide groups forms cyano group through trifluoroacetic anhydride dehydration; finally sloughing tertbutyloxycarbonyl (Boc) protecting group in acid condition obtains target compound, and synthetic route is as follows:
Therefore, synthesis BMS-477118 needs two crucial intermediate (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 synthesis 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) and enzyme catalysis ammonification reduction (WO2010032129; US2005090539; AdvancedSynthesis & Catalysis, 2007,349,1369-1378; Bioorganic.Med.Chem, 2011,19 (3), 1136-1154; US2010291642).Concrete chemical synthesis route is as follows:
Routes one: DavidAugeri etc. set out with adamantane acid and to reduce through LAH; Swern oxidation and asymmetric Strecker reaction construct 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.
Route two: US2005090539 reports that adamantane acid is after bromo; react with nitration mixture and hydroxylation occurs; introduce amino and obtain raceme; again Boc protection is carried out to amino; means finally by chemical resolution obtain A, and route is longer, overall yield about 30%; after splitting, enantiomeric excess value (e.e. value) is still not high, only has 70%-80%.
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.Compared with pure chemistry synthetic route, enzyme catalysis biotransformation method can carry out mutually at pure water, and reduce the catalyzer using and people and environment are had to harm, reduce the generation of refuse, environment friendly is good; More importantly, enzyme has excellent stereoselectivity, and intermediate product does not need to split, and therefore enzymatic bio-transformation effectively can improve the optical purity of productive rate and product, has fabulous industrialization potential.The people such as RonaldL.Hanson report under the existence of DPNH (NADH), obtain the amino acid (S) of S-type-(3-hydroxyl-1-adamantyl)-D-glycine (HAG) with the reduction amination of Phenylalanine dehydrogenase (PDH) catalysis keto acid substrate 2-(3-hydroxyl-1-the adamantyl)-2-oxoethanoic acid in actinomycetes source; Under ammonium formiate exists, the hydrogenlyase (FDH) in pichia source makes coenzyme NAD H regenerate.Amino acid is introduced Boc protecting group and namely obtain Boc-HAG, route is as follows:
The Phenylalanine dehydrogenase (PDH) used in reduction amination process is through N end and the transformation of C end, its enzyme to keto acid substrate is lived and is raised, but thermostability declines, the overlong time of reacting is carried out at the temperature of 40 DEG C, reach and transform needs more than 38 hours completely, energy consumption is too high, and production capacity is difficult to improve.
Summary of the invention
The present invention is by improve enzyme activity and the stability of the Phenylalanine dehydrogenase in ground bacillus source to the orthogenesis of DNA sequence dna, this enzyme may be used for the synthesis of key intermediate (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG) in BMS-477118 synthesis.
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 group deamination is formed 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
3when concentration is higher, Phenylalanine dehydrogenase can the reversed reaction of the above-mentioned reaction of catalysis, and phenyl-pyruvic acid reduction amination is generated L-Phe, and this reaction needed consumes NADH.
Contriver transforms to improve the enzyme activity of its catalysis onglyza intermediate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid reduction amination by the Phenylalanine dehydrogenase (PDH) that the method for random mutation is originated to ground bacillus.In order to make NADH regenerate, contriver clones and obtains hydrogenlyase (FDH) from pichia spp, and NAD can be made to be reduced to NADH, thus promotes the carrying out of reduction amination.Above-mentioned two kinds of enzymes can utilize E. coli recombinant stain to carry out expressing and being obtained by high density fermentation; under their catalysis; keto 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 is more than 99.9%.The recombinase obtained by random mutation has higher enzyme activity and good thermostability, thus concentration of substrate is improved, the reaction times is shortened, and energy consumption is reduced, can thoroughly transform in 20 hours, 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 deriving from ground bacillus, it is characterized in that, compared with wild-type PDH, having at the 85th and 294 amino acids residues of aminoacid sequence and replace sudden change.This PDH mutant compares wild-type PDH, has enzyme activity and/or the thermostability of raising.In present patent application file, the numbering of PDH aminoacid sequence is to derive from the aminoacid sequence (SEQIDNO:1) of the wild-type PDH of ground bacillus (Geobacillussp.) Y412MC61 for benchmark.Preferably, the 93rd and 288 amino acids residues of described PDH variant amino acid sequence sport leucine (L) and α-amino-isovaleric acid (V) respectively; More preferably, the replacement sudden change of the 93rd and 288 amino acids residues of described PDH variant amino acid sequence is respectively I93L and L288V.
In a specific embodiment, PDH mutant of the present invention, compared with wild-type PDH, only has at the 93rd and 288 amino acids residues of aminoacid sequence and replaces sudden change.Preferably, the 93rd and 288 amino acids residues of described PDH variant amino acid sequence sport leucine (L) and α-amino-isovaleric acid (V) respectively; More preferably, the replacement sudden change of the 93rd and 288 amino acids residues of its aminoacid sequence is respectively I93L and L288V.
Compared with wild-type PDH, PDH mutant of the present invention can also have sudden change in other sites, and preferably, the described replacement that other sport the 75th, 184 and 301 amino acids residues of its aminoacid sequence suddenlys change; More preferably, the 75th, 184 and 301 amino acids residues of its aminoacid sequence sport L-Ala (A), L-glutamic acid (E) and Serine (S) respectively; Most preferably, the replacement sudden change of the 75th, 184 and 297 amino acids residues of its aminoacid sequence is respectively M75A, K184E and V301S.
In another specific embodiment, PDH mutant of the present invention, compared with wild-type PDH, only has at the 75th, 93,184,288 and 301 amino acids residues of aminoacid sequence and replaces sudden change.Preferably, the 75th, 93,184,288 and 301 amino acids residues of described PDH variant amino acid sequence sport L-Ala (A), leucine (L), L-glutamic acid (E), α-amino-isovaleric acid (V) and Serine (S) respectively; More preferably, the replacement sudden change of the 75th, 93,184,294 and 297 amino acids residues of its aminoacid sequence is respectively M75A, I93L, K184E, L288V and V301S.
PDH mutant of the present invention derives from ground bacillus (Geobacillussp.), preferred Geobacillussp.Y412MC61; Most preferably, the aminoacid sequence of described wild-type PDH is for deriving from the aminoacid sequence (SEQIDNO:1) of the wild-type PDH of ground bacillus (Geobacillussp.) Y412MC61.
In a most preferred embodiment, the aminoacid sequence of PDH mutant of the present invention is as shown in SEQIDNO:5 or 6.
Second aspect present invention provides the nucleic acid molecule of PDH mutant of the present invention of encoding.Described nucleic acid molecule can be DNA or RNA, double-strand or strand, as long as contain the genetic information of PDH mutant of the present invention of encoding.
A third aspect of the present invention provides the carrier comprising above-mentioned nucleic acid molecule.Described carrier can be cloning vector or expression vector, can be various types of carrier, 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 comprising 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 synthesis (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG).Further, described PDH mutant or described host cell can also for the synthesis of BMS-477118s.
A sixth aspect of the present invention provides a kind of method of synthesis saxagliptin 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), amino acid (S)-(3-hydroxyl-1-adamantyl)-D-glycine (HAG) of the amino acid S-type of S-type is obtained with PDH mutant catalysis keto acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid reduction amination of the present invention;
(2) under ammonium formiate exists, hydrogenlyase (FDH) makes NADH regenerate;
(3) HAG introduces tertbutyloxycarbonyl (Boc) protecting group and obtain Boc-HAG.
Wherein, described FDH preferably derives from pichia (Pichiapastoris); Most preferably, described FDH is for deriving from the aminoacid sequence (SEQIDNo.7) of the wild-type FDH of pichia (Pichiapastoris) GS115.
A seventh aspect of the present invention provides a kind of method of synthesizing BMS-477118, comprises and uses aforesaid method synthesis saxagliptin chiral intermediate (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG).
Accompanying drawing explanation
Fig. 1 clones the gel electrophoresis spectrum of the pcr amplification product of the pdh gene of ground bacillus (Geobacillussp.) 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 for induce contrast, and swimming lane B is PDH mutant 2, and swimming lane C is FDH.
The amino acid alignment result of Fig. 3 wild-type PDH, PDH mutant 1, PDH mutant 2.SEQNO1, SEQNO5, SEQNO6 are respectively the aminoacid sequence of wild-type PDH, PDH mutant 1, PDH mutant 2.Compared with wild-type PDH, 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 DEG C, and reaction system contains 0.4mMNADH, and 50mM ketone acid A1(is dissolved in the NaOH of 1 equivalent), 0.75M ammoniacal liquor (HCl adjusts pH8.75).Add the change of the ultraviolet absorption value of detection reaction system under 340nm after Phenylalanine dehydrogenase.
The enzyme activity determination of FDH carries out at 30 DEG C, and reaction system contains 1mMNAD, 100mM ammonium formiate, 100mM potassiumphosphate (pH8.0) damping fluid, detects the change of the ultraviolet absorption value in 30 minutes under 340nm after adding hydrogenlyase.
Enzyme is lived and is defined:
Under 340nm, ultraviolet absorption value often changes 0.1, and the concentration corresponding to NADH changes 0.016mM.Therefore enzyme activity calculation formula is obtained:
A: absorbancy reduced value
N: extension rate
T: reaction times (min)
Embodiment 2 substrate and product analysis method
High performance liquid chromatography (HPLC) method is adopted to detect product: with water and acetonitrile (45:55) for moving phase, 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 keto 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 uses AD-H chiral column (DiacelChemical) to analyze on Agilent1260 series HPLC, and the product retention time of R-configuration is the product retention time of 3.5min, S-configuration is 5.1min.Moving phase is ethanol: normal hexane=98:2.
TLC developping agent is butanols: water: acetic acid=4:1:1, and 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 (Geobacillussp.) Y412MC61 is introduced E.coli.For the pdh gene (SEQIDNo.2) of amplification coding PDH enzyme (SEQIDNo.1), respectively using primers F 1(SEQIDNo.3) and R1(SEQIDNo.4) as forward and reverse primer.Two primers all contain the site compatible with the pcr amplification deoC gene fragment using GatewayTechnology to be obtained by recombinant clone of fixing a point.The gel electrophoresis spectrum of pcr amplification product as shown in Figure 1.Use Random Mutagenesis Kit, by changing MnSO
4concentration carries out multiple reaction, thus the pdh gene (SEQIDNo.2) of Geobacillussp.Y412MC61 is introduced in 1 to 3 point mutation, and 1-3 amino-acid residue in PDH enzyme amino acid sequence is replaced.
The sequence (SEQIDNo.3) of forward primer F1:
5’-GAGCATATGAATGTCATGCTATCGCC-3’
The sequence (SEQIDNo.4) of reverse primer R1:
5’-AGACTCGAGTTAACGTCGAATATCCCAC-3’
Fallibility pcr amplification uses following temperature program(me): 94 DEG C of 2min, 25 circulations of 94 DEG C of 30s and 68 DEG C 1min, then 68 DEG C of 10min.First fallibility PCR fragment is cloned into pDONR carrier, prepare extensive pENTR plasmid library, initial more than 20,000 bacterium colony.Then take pDEST14 as carrier, plasmid library of being got started by pENTR is configured to expression library.Then expression library is proceeded to Competent E.coilBL21Star(DE3), for expressing the pdh gene (coding PDH enzyme mutant) of sudden change.
The expression of embodiment 4PDH enzyme
The expression of pdh gene in deep hole microtiter plate of sudden change: the mutant bacteria obtained according to embodiment 3, choose sudden change bacterium colony, be seeded in microtiter plate (MTP) 200 μ L2 × YT substratum (penbritins containing 100 μ g/ml), culture condition 37 DEG C, the time is 1 day.Then the above-mentioned inoculation of precultures of 100 μ L is got to expressing in the deep-well plates of culture (2 × YT, containing 100 μ g/ml penbritin and 1mMIPTG) containing 500 μ L, then 25 DEG C of cultivation 24h on shaking table.
Embodiment 5 screening has the PDH mutant improving catalytic capability
PDH mutant deep hole culture 3500rpm embodiment 4 obtained carries out centrifugal 15 minutes, and add 400 μ L lysis buffers (50mM phosphoric acid buffer, pH7.4,1mg/mL N,O-Diacetylmuramidase) and make its resuspension, 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 NADH and keto acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid respectively to 1mM and 10mg/mL final concentration, 30 DEG C hatch 15 minutes, 30 minutes, 45 minutes and 1 hour after detect uv-absorbing A under its 340nm respectively
340.Not add the sample of keto acid substrate as blank group.According to the calculating enzyme activity that underspeeds of NADH under 340nm, screening obtains the PDH mutant 1(SEQIDNo.5 that enzyme activity improves), experimental result is as shown in table 1.
Table 1PDH enzyme activity determination
Embodiment 6 screening has the PDH mutant of thermostability
The PDH mutant 1 that the enzyme activity obtained with embodiment 5 improves is for masterplate, and carry out random mutation by fallibility PCR and build new mutant library, 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) and makes it suspend.50 DEG C temperature bath 30min, then add 1mMNADH and 10mg/mL keto acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid, 30 DEG C hatch 60min after detect the decay intensity of NADH in 340nm uv-absorbing, compare PDH mutant enzyme vigor.Screen in contrast with the sample without 50 DEG C of incubations and obtain heat-staple PDH mutant 2(SEQIDNo.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
Being inoculated in by the high-performance PDH enzyme mutant gene engineering colon bacillus obtained according to embodiment 6 is equipped with in the 1L shaking flask of 200mLLB substratum, and in 37 DEG C, 180-220rpm cultivates 10-16h.Above-mentioned cultured seed to be inoculated on 3L (glucose 4g/L in tank fermention medium (M9) 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 DEG C, 300-800rpm, cultivate under the condition of air flow quantity 2-6L/min.After cultivating 6-10h, add the supplemented medium containing 60% glycerine with the data rate stream of 5-20mL/h, continue to fermentation ends.Flow feeding substratum a few hours are to OD
600when reaching 20-40, add 0.1-1mMIPTG and start induction.After induction 10-20h, put tank, 5000rpm collected by centrifugation thalline.
The clonal expression of embodiment 8FDH enzyme
Hydrogenlyase (FDH) gene of pichia (Pichiapastoris) GS115 is cloned into coli expression carrier pET21a by PCR.For the fdh gene (SEQIDNo.8) of amplification coding FDH enzyme (SEQIDNo.7), respectively using primers F 2(SEQIDNo.9) and R2(SEQIDNo.10) as forward and reverse primer.The gel electrophoresis spectrum of pcr amplification product as shown in Figure 1.
The sequence SEQIDNo.9 of forward primer F2:
5’-GGGCATATGAAAATCGTTCTCGTTTTG-3’
The sequence SEQIDNo.10 of reverse primer R2:
5’-GGGCTCGAGTTATGCGACCTTTTTGTC-3’
Pcr amplification uses following temperature program(me): 94 DEG C of 2min, 1 circulation; 30 circulations of 94 DEG C of 30s, 60 DEG C of 1min and 72 DEG C 2min, then 72 DEG C of 10min.PCR fragment NdeI and XhoI is carried out double digestion, then clone into the linearizing pET21a carrier of same restriction endonuclease, T4 ligase enzyme spends the night 16 DEG C of connections, connect product conversion to bacillus coli DH 5 alpha, picking list bacterium colony also identifies the genotype of plasmid, correct Plastid transformation carries out FDH protein expression to e. coli bl21 (DE3), and the SDS-PAGE electrophoretogram of expression product as shown in Figure 2.
The fermentation of embodiment 9FDH enzyme
Being inoculated in by the FDH enzyme gene engineering colibacillus obtained according to embodiment 8 is equipped with in the 1L shaking flask of 200mLLB substratum, and in 37 DEG C, 180-220rpm cultivates 10-16h.Above-mentioned cultured seed to be inoculated on 3L (glucose 4g/L in tank fermention medium (M9) 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 DEG C, 300-800rpm, cultivate under the condition of air flow quantity 4L/min.After cultivating 10h, add the supplemented medium containing 60% glycerine with the data rate stream of 5-20mL/h, continue to fermentation ends.Flow feeding substratum a few hours are to OD
600when reaching 20, add 1mMIPTG and start induction.After induction 15h, put tank, 5000rpm collected by centrifugation thalline.
Embodiment 10 enzyme catalysis reduction amination and Boc protection
The coli somatic of Example 7 and 9 collection, adds the ammonium phosphate buffer liquid (pH7.5) of 1 ~ 3 times of volume 0.05M respectively.60-600W ultrasonication 30min, crude enzyme liquid is directly used in enzymic catalytic reaction.1.31g keto acid substrate 2-(3-hydroxyl-1-adamantyl)-2-oxoethanoic acid (5.8mmol) and 740mg ammonium formiate (11.7mmol) are dissolved in 5mL ammonium phosphate buffer liquid.Then 16.4mgNAD(24.7 μm of ol is added) and 3.6mgDTT(23.3 μm of ol), mend and add water to cumulative volume 12mL, and regulate pH to 8.0 with ammoniacal liquor.Add 22UPDH and 114UFDH, 30 DEG C, 40rpm carries out catalyzed reaction 20 hours, and TLC monitors reaction process.Question response terminates rear 80 DEG C of water-bath 30min makes protein precipitation, crosses and filters protein, and filtrate rotary evaporation removing excess of ammonia, is settled to 9ml.Add potassiumphosphate 3.09g, add under stirring containing 1.15g (Boc)
2o(5.27mmol) THF solution, reacts 3h under room temperature, TLC detection reaction process.Bottom is abandoned after reaction terminates; add 7ml distilled water and adjust pH to 2.5 ~ 3 with concentrated hydrochloric acid; 3 times are extracted with equal-volume Iso Butyl Acetate; organic phase anhydrous sodium sulfate drying; add normal heptane and obtain suspension; amino acid (BOC-HAG) 1.32g obtaining Boc protection is filtered, two step total recovery 84%, e.e. value >99.9% after stirring 1h.
Claims (25)
1. one kind derives from Phenylalanine dehydrogenase (PDH) mutant of ground bacillus (Geobacillussp.), it is characterized in that, compared with wild-type PDH, have at the 93rd and 288 amino acids residues of aminoacid sequence and replace sudden change, sport leucine (L) and α-amino-isovaleric acid (V) respectively, and there is enzyme activity and/or the thermostability of raising; Wherein, the numbering of PDH aminoacid sequence take SEQIDNO:1 as benchmark.
2. PDH mutant according to claim 1, it is compared with wild-type PDH, only has at the 93rd and 288 amino acids residues of aminoacid sequence and replaces sudden change.
3. the PDH mutant described in claim 1 or 2, the replacement sudden change of the 93rd and 288 amino acids residues of its aminoacid sequence is respectively I93L and L288V.
4. PDH mutant according to claim 1, its also the 75th of aminoacid sequence the, 184 and 301 amino acids residues have and replace sudden change, sport L-Ala (A), L-glutamic acid (E) and Serine (S) respectively.
5. PDH mutant according to claim 4, it is compared with wild-type PDH, only has at the 75th, 93,184,288 and 301 amino acids residues of aminoacid sequence and replaces sudden change.
6. the PDH mutant described in claim 4 or 5, 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.
7. the PDH mutant according to any one of claim 1,2,4 and 5, wherein, described ground bacillus is Geobacillussp.Y412MC61.
8. PDH mutant according to claim 3, wherein, described ground bacillus is Geobacillussp.Y412MC61.
9. PDH mutant according to claim 6, wherein, described ground bacillus is Geobacillussp.Y412MC61.
10. PDH mutant according to claim 7, wherein, the aminoacid sequence of described wild-type PDH is as shown in SEQIDNO:1.
PDH mutant described in 11. claims 8 or 9, wherein, the aminoacid sequence of described wild-type PDH is as shown in SEQIDNO:1.
12. PDH mutant according to claim 1, the aminoacid sequence of described PDH mutant is as shown in SEQIDNO:5 or 6.
The nucleic acid molecule of the PDH mutant described in 13. any one of coding claim 1-12.
14. carriers comprising nucleic acid molecule according to claim 13.
15. carriers according to claim 14, it is pET21a carrier.
16. host cells comprising the carrier described in claims 14 or 15.
The host cell of 17. claims 16, it is intestinal bacteria.
18. host cells according to claim 17, it is e. coli bl21 (DE3).
PDH mutant described in 19. any one of claim 1-12 or the purposes of the host cell described in any one of claim 16-18 in synthesis (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG).
PDH mutant described in 20. any one of claim 1-12 or the purposes of the host cell described in any one of claim 16-18 in synthesis BMS-477118.
The method of 21. 1 kinds of synthesis (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG), comprises the following steps:
(1) under the existence of DPNH (NADH), amino acid (S)-(3-hydroxyl-1-adamantyl)-D-glycine (HAG) of S-type is obtained with PDH mutant catalysis keto acid substrate 2-(3-hydroxyl-1-the adamantyl)-2-oxoethanoic acid reduction amination described in any one of claim 1-12;
(2) under ammonium formiate exists, hydrogenlyase (FDH) makes NADH regenerate;
(3) HAG introduces tertbutyloxycarbonyl (Boc) protecting group and obtain Boc-HAG.
22. methods according to claim 21, wherein, described FDH derives from pichia (Pichiapastoris).
23. methods according to claim 22, wherein, the aminoacid sequence of described FDH is as shown in SEQIDNO:7.
Method described in 24. any one of claim 21-23, wherein, catalyzed reaction, at 30 DEG C, is carried out under 40rpm, and the reaction times is 20 hours.
25. 1 kinds of methods of synthesizing BMS-477118, comprise method synthesis of chiral intermediate (S)-N-tertbutyloxycarbonyl-3-hydroxyl-1-adamantyl-D-glycine (Boc-HAG) used described in any one of claim 21-24.
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| 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 |
| 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 |
| 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 |
| CN109593802B (en) * | 2018-12-24 | 2021-10-01 | 上海健康医学院 | A kind of preparation method of (R)-2-(2,5-difluorophenyl) pyrrolidine or its salt |
| CN110628739B (en) * | 2019-08-14 | 2021-04-09 | 华东理工大学 | An amine dehydrogenase mutant and its application in the synthesis of chiral amines and amino alcohols |
| CN112391363B (en) * | 2021-01-21 | 2021-04-06 | 凯莱英生命科学技术(天津)有限公司 | Amino acid dehydrogenase mutants and their applications |
| CN116253804A (en) * | 2023-02-22 | 2023-06-13 | 天津科技大学 | Self-assembled protein of phenylalanine dehydrogenase and formate dehydrogenase and its application |
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