CN105274027A - Pseudomonas pseudoalcaligenes and application of pseudomonas pseudoalcaligenes to preparation of sitagliptin intermediate - Google Patents
Pseudomonas pseudoalcaligenes and application of pseudomonas pseudoalcaligenes to preparation of sitagliptin intermediate Download PDFInfo
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Abstract
The invention discloses pseudomonas pseudoalcaligenes and application of the pseudomonas pseudoalcaligenes to preparation of a sitagliptin intermediate. A colony of the pseudomonas pseudoalcaligenes XW-40 is flat and round in surface morphology, smooth and neat in edge, moist, milk white and opaque and is about 1.9mm in diameter. The pseudomonas pseudoalcaligenes is capable of effectively catalyzing 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7(8H)-yl]-1-(2,4,5)-trifluorophenyl)-butan-2-one to be reduced into a sitagliptin chiral intermediate; in optimal reaction conditions, the pseudomonas pseudoalcaligenes can be tolerant to 10 g/L of high substrate concentration while keeps high selectivity, and is weak in substrate inhibition effect, so that high-substrate-concentration biological catalysis can be achieved; the pseudomonas pseudoalcaligenes is recyclable and removes inhibition effect of a substrate and a product on cells as compared with fed-batch, so that catalytic activity of the pseudomonas pseudoalcaligenes is used maximally.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of pseudomonas pseudoalcaligenes strain and preparing the application in sitagliptin intermediate.
Background technology
Sitagliptin phosphate (sitagliptinphosphate) is dipeptidyl peptidase-4 (DPP-4) inhibitor being used for the treatment of diabetes B of MSD Corp. first development, this medicine can effectively for the dysfunction of the insulin resistant in diabetes B and pancreatic islet alpha, β cell, by the degraded suppressing DPP-4 to slow down incretin GLP-1, thus play blood sugar reducing function.Increase with easily causing hypoglycemia, body weight and compare with the conventional oral ofhypoglycemic medicine of the side effects such as vomitting that causes nausea, this inhibitor has obvious advantage and good market outlook.The chiral intermediate of sitagliptin phosphate key mainly contains two, and one is chiral beta-amino acids, and one is its corresponding chiral alcohol intermediate, and the synthetic work emphasis at present about sitagliptin mainly launches around these two chiral intermediates.
The synthetic method of current this chiral beta-amino acids of bibliographical information mainly contains following several:
(1) chiral source is adopted to induce the a-amino acid of chirality, and after through diazotization reaction produce beta-amino acids, thus structure chiral centre, this route is costly raw materials used, reaction conditions is quite harsh, as cold condition such as needs-78 DEG C and-30 DEG C, and some reaction required time longer and operation comparatively loaded down with trivial details, the refining of intermediate product needs through column chromatography for separation;
(2) adopt chiral phosphorus ruthenium catalyst to carry out asymmetric catalytic hydrogenation to ketone ester, build chiral secondary alcohol, be then secondary amine by chiral secondary alcohol chiral inversion, in this route, chiral catalyst is expensive, and scale effect is obvious;
(3) asymmetric hydrogenation of β-enamino acid intermediate, asymmetric hydrogenation carries out or uses the metal ruthenium catalyst of costliness under the metal catalyst of costliness is as the existence of the rhodium combined with chiral phosphine/diphosphine ligand;
(4) take Isopropylamine as ammonia donor, with sitagliptin precursor ketone for ammonia acceptor, transaminase biocatalysis is utilized to prepare sitagliptin, the method has environmental friendliness, the advantages such as reaction conditions is gentle, but the high characteristic (separation scheme etc.) of not easily acquisition and the price of specific enzymes is very important;
(5) split with trifluro benzaldehyde and acetylaminoacetic acid through condensation, reduction, biological enzyme, be hydrolyzed, protect amino, diazotization, Arndt-Fistert rearrangement reaction, be hydrolyzed to obtain R-beta-aminobutyric acid; but described synthetic route is long; the yield that chiral separation obtains chiral amino acid is no more than 50%; the recycle of half enantiomer is a great problem in addition; and reaction process uses diazomethane; it is unstable toxic gas under diazomethane room temperature; there is explosivity, operational hazards.
And the synthetic method of bibliographical information corresponding chiral alcohol intermediate is mainly as follows at present:
(1) WO09/045507 utilizes suitable carbonyl reductase to carry out asymmetric reduction to β-carbonyl moiety and obtains beta-hydroxy intermediate, obtain sitagliptin with azetidinone intermediate reaction again, the shortcoming of the method is: under high pressure react, use very expensive metal chiral catalyst (Rh or Ru), low stereoselectivity and rhodium pollution products and the finalization compound that causes thus to be difficult to purifying;
(2) WO12/046254 openly prepares the method for the intermediate being prepared sitagliptin by Enzymatic transformation, provide the method for two kinds of biocatalysis synthesis of chiral alcohol intermediates, sitagliptin precursor ketone body powder, as biological catalyst, is the alcohol of corresponding (S) configuration by the crude extract being respectively the carbonyl reductase that the full cell of utilizing works bacterium and engineering bacteria produce; Its method has environmental friendliness, reaction conditions is gentle, the high advantage of ee value, but on the other hand, engineering bacteria be built with very large difficulty and both expensive, using and slightly carrying enzyme as biological catalyst Problems existing is that free thick enzyme is unstable in reaction solution, the catalytic capability of enzyme is lower, the easy inactivation of enzyme, not easily realizes recycling of enzyme, and the lock out operation in later stage is complicated; And using intact cells as catalyzer, the problem of carrying out existing for biphasic catalysis reaction in toluene that is dissolved in by substrate is, substrate solubleness in toluene is little, and the concentration of substrate that reaction transforms is lower, is not suitable for large-scale industrial production.
All there is its weak point in the method for existing chemical method and biological catalysis synthesis sitagliptin and intermediate, therefore, the method preparing the intermediate of sitagliptin developing more clean environment close friend is significantly.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of pseudomonas pseudoalcaligenes strain, and provide the purposes of this bacterial strain, can economically obtain sitagliptin intermediate by the biocatalysis of this bacterial strain thalline.
The present invention is with 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a]-pyrazine-7 (8H)-Ji]-1-(2,4,5)-trifluorophenyl) fourth-2-ketone (II) is raw material, utilize the carbonyl reductase in microorganism, regioselectivity and enantioselective catalyses are reduced to (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazine-7 (8H)-Ji]-1-(2,4,5-trifluorophenyl) fourth-1-ketone (I).
The technology of the present invention route is as follows:
The technical scheme that the present invention takes is as follows:
1, pseudomonas pseudoalcaligenes (Pseudomonaspseudoalcaligenes) XW-40, by China typical culture collection center preservation, preserving number is CCTCCNO:M2015521.
2, above-mentioned pseudomonas pseudoalcaligenes XW-40 is preparing the application in sitagliptin intermediate.
Preferably, using pseudomonas pseudoalcaligenes XW-40 thalline as catalyzer, by raw material 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a]-pyrazine-7 (8H)-Ji]-1-(2,4,5)-trifluorophenyl) fourth-2-ketone catalytic reduction is (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazine-7 (8H)-Ji]-1-(2,4,5-trifluorophenyl) fourth-1-ketone.
Preferably, regenerating coenzyme substrate and organic cosolvent is also comprised in described reaction system.
Preferably, described catalyzer pseudomonas pseudoalcaligenes XW-40 thalline through again collecting, for the preparation of next batch sitagliptin intermediate after washing.
Preferably, described pseudomonas pseudoalcaligenes XW-40 cell concentration is 90g (dry weight)/L, and material concentration is 10g/L, and regenerating coenzyme substrate is the glucose of massfraction 5%, organic cosolvent is the dimethyl sulfoxide (DMSO) of volume fraction 10%, and the reaction times is 28 hours.
Beneficial effect of the present invention is: the product carbonyl reductase strain Pseudomonas pseudoalcaligenesXW-40 that (1) screens has regioselectivity and the enantioselectivity of height, can effective catalysis 4-oxygen-4-[3-(trifluoromethyl)-5, 6-dihydro [1, 2, 4] triazolo [4, 3-a]-pyrazine-7 (8H)-Ji]-1-(2, 4, 5)-trifluorophenyl) fourth-2-ketone (II) is reduced to sitagliptin chiral intermediate (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5, 6-dihydro [1, 2, 4] triazolo [4, 3-a] pyrazine-7 (8H)-Ji]-1-(2, 4, 5-trifluorophenyl) fourth-1-ketone (I), no coupling product produces, simplify sepn process, (2) for the screening of bacterial strain in soil, select to be the screening that unique carbon source carries out bacterial strain with the methyl phenyl ketone of substrate (II) structural similitude, can comform rapidly in multiple soil and filter out suitable doubtful primary dcreening operation bacterial strain fast, use it for the reduction of (II) again, select best bacterial strain fast, (3) the strain Pseudomonas pseudoalcaligenesXW-40 screened can tolerate the high concentration of substrate of 10g/L under the suitableeest reaction conditions, and still keep highly selective, substrate inhibition is more weak, thus the biocatalysis of high concentration of substrate can be realized, reported to utilize wildtype bacterium to carry out biological reducing relatively rare for current for this point, (4) PseudomonaspseudoalcaligenesXW-40 thalline can recycle, after a batch reaction terminates, thalline is centrifugal, can again drop in the catalyzed reaction of next batch, compared with batch feeding, eliminate the restraining effect of substrate and products upon cell simultaneously, the catalytic activity of thalline is reached and utilizes to greatest extent, can be recycled 5 times with a collection of thalline, yield and optical purity high, process is green, with low cost.
Culture presevation
In the present invention, pseudomonas pseudoalcaligenes (Pseudomonaspseudoalcaligenes) XW-40 is separated to obtain from soil, send China typical culture collection center (CCTCC) preservation, deposit number is CCTCCNO:M2015521, address is positioned at Wuhan City, Hubei Province Wuhan University, preservation date is on September 10th, 2015, and Classification And Nomenclature is pseudomonas pseudoalcaligenes Pseudomonaspseudoalcaligenes.Bacterium colony is that configuration of surface is flat in the form of solid medium, and circular, edge section is neatly smooth, and whole bacterium colony is moist shape, and color is creamy white, opaque, and colony diameter is about 1.9mm, and physiological characteristics is in table 2.Be pseudomonas pseudoalcaligenes through 16SrDNA molecular biology identification, homology reaches 100%.
Embodiment
Below the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
The primary dcreening operation of embodiment 1 soil bacterial strain
Take about 1g pedotheque in 10ml sterilized water, centrifugal after vibration mixing, it is in the minimum salt culture medium of sole carbon source with methyl phenyl ketone that Aspirate supernatant 1ml joins 100ml, cultivates 5-7 days for 30 DEG C.Then the bacteria suspension of cultivation is applied to methyl phenyl ketone be sole carbon source minimum salt nutrient agar in, the bacterium colony separation and purification that flat board grows is inoculated in (substratum composition: peptone 5g/L on agar slant, yeast extract paste 1.5g/L, glucose 10g/L, extractum carnis 1.5g/L, NaCl5g/L, pH7.0), cultivate 48h for 30 DEG C, as the doubtful bacterial strain of screening further, numbering is preserved.
Embodiment 2 produces the screening of carbonyl reductase bacterial strain
Strain inoculation embodiment 1 separation and purification obtained is in consisting of peptone 5g/L, yeast extract paste 1.5g/L, glucose 10g/L, extractum carnis 1.5g/L, in the substratum of NaCl5g/L, pH7.0, cultivate 48h for 30 DEG C, the centrifugal 10min of 7000rpm/min at 4 DEG C, collect thalline, wash twice with cold saline, obtain cell wet thallus.
Following bacterial strain screening system is adopted to screen: 1g/L substrate (II), the glucose of massfraction 5%, 10% (v/v) dehydrated alcohol, 80g/L thalline (dry weight), SODIUM PHOSPHATE, MONOBASIC-Sodium phosphate dibasic the damping fluid of 0.1M, pH7.0, at 30 DEG C, stirring reaction 12h, centrifugal, supernatant liquor is extracted with ethyl acetate 3 times, organic layer merges, and anhydrous magnesium sulfate drying spends the night, anti-phase C
18liquid chromatography and Chiral liquid chromatography measure transformation efficiency and enantiomeric excess (ee).
Liquid phase chromatogram condition is: chromatographic column C during detection transformation efficiency
18(250 × 4.6mm × 5 μm, AgilentAmerica), moving phase: water/acetonitrile (v/v)=70/30, flow velocity 1ml/min, determined wavelength: 268nm, temperature: 25 DEG C; 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a]-pyrazine-7 (8H)-Ji]-1-(2,4,5)-trifluorophenyl) fourth-2-ketone and 3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazine-7 (8H)-Ji]-1-(2,4,5-trifluorophenyl) retention time of fourth-1-ketone is respectively 19.7 and 26.8min; Chromatographic column ChiracelAY-H (5 × 250mm) during detection ee, moving phase: normal hexane/dehydrated alcohol (v/v)=85:15, flow velocity: 1.0ml/min, determined wavelength: 268nm, temperature: 25 DEG C; At chemosynthesis racemic alcohol 3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazine-7 (8H)-Ji]-1-(2,4,5-trifluorophenyl) fourth-1-ketone analysis in, the retention time of S and R isomer is respectively 11.3min and 17.8min.Adopt same procedure to determine the chiral configuration preparing alcohol product, transformation efficiency be greater than the expression of results of the bacterial strain of 5% in table 1,
Table 1 produces the screening of carbonyl reductase bacterial strain
As shown in Table 1, the bacterial strain for numbering 40-1 that enantiomeric excess (ee) is the highest and transformation efficiency is best, observing 40-1 bacterium colony in the form of solid medium is that configuration of surface is flat, circle, edge section is neatly smooth, and whole bacterium colony is moist shape, color is creamy white, opaque, colony diameter is about 1.9mm, and physiological characteristics is in table 2.Be pseudomonas pseudoalcaligenes through 16SrDNA molecular biology identification, homology reaches 100%, called after PseudomonaspseudoalcaligenesXW-40, and its DNA sequence dna is shown in SEQIDNo.1.
Table 140-1 bacterial strain Physiology and biochemistry character
Prepared by embodiment 3 product (I)
PseudomonaspseudoalcaligenesXW-40 bacterium is utilized to prepare (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazine-7 (8H)-Ji]-1-(2,4,5-trifluorophenyl) fourth-1-ketone (I).Get 5ml seed liquor and be forwarded to (shaking flask of 250ml capacity) (nutrient media components and culture condition are shown in embodiment 2) in the fresh substratum of 100ml, at 30 DEG C, 190 revs/min shake cultivation 48 hours, at 4 DEG C, under 7000 revs/min of conditions centrifugal 10 minutes, collecting precipitation thalline, and wash twice with cold saline, obtain wet thallus as biological catalyst.The phosphate buffered saline buffer of gained wet thallus pH7.0 being made into cell concentration is 50g (dry weight)/L bacteria suspension.5ml reaction system, substrate (II) concentration is 1g/L, add the glucose of massfraction 5% as regenerating coenzyme substrate, volume fraction 10% dehydrated alcohol is as organic cosolvent, 30 DEG C, 190 revs/min, transform 12 hours, reaction mixture monoploid amasss extraction into ethyl acetate three times, merge organic layer and use anhydrous sodium sulfate drying, underpressure distillation removing ethyl acetate, gained resistates 2ml dehydrated alcohol (chromatographically pure) fully dissolves, get 10 μ l samples and carry out HPLC detection (method and pillar type are as embodiment 2), calculating transformation efficiency is 57%, ee value > 99%.
Utilize product (I) prepared by multiple classical tool analysis enzymatic, i.e. fusing point (m.p.), specific optical rotation (SOR), NMR (Nuclear Magnetic Resonance) spectrum (NMR), result is as follows:
m.p.：114-119℃；SOR[α]D25：23.2°(c＝1，CHCl
3)；1HNMR(400MHz，DMSO-D6)：δ2.46-2.48(m，1H)，2.67-2.79(m，3H)，3.90-3.97(m，2H)，4.00-4.09(m，2H)，4.21-4.26(m，1H)，4.84-5.06(overlappingm，3H)，7.40-7.48(m，2H)；13CNMR(100MHz，DMSO-D6)：δ35.4，37.3，38.3，40.1，41.3，42.1，43.0，43.7，67.3，105.1，115.2，117.3，119.5，123.0，142.3，144.4，146.5，148.7，151.0，154.8，156.8，170.2。
The data of embodiment 4 ~ 25 product (I) are identical with embodiment 3.
Prepared by embodiment 4 product (I)
In order to screen optimum organic cosolvent further, to volume fraction 10% dehydrated alcohol be adopted in embodiment 3 during the experiment of bacterial strain primary transformants to be replaced by the different organic solvents listed in table 2 as the solubility promoter in reaction system as organic cosolvent, other conditions be with embodiment 3.
Table 2 organic cosolvent is on the impact of reaction
As shown in Table 2, when selecting dimethyl sulfoxide (DMSO) as organic cosolvent, the transformation efficiency of substrate is the highest, and ee value is > 99%.Therefore, in embodiment 5 ~ 25, organic cosolvent is dimethyl sulfoxide (DMSO).
Prepared by embodiment 5 product (I)
Culture medium prescription and seed liquor preparation method are see embodiment 3.Yeast culture for bio-transformation is centrifugal after 48 hours, washing, gained wet thallus phosphate buffered saline buffer being made into cell concentration is that 30g (dry weight)/L bacteria suspension is as biological catalyst, 5mL reaction system, substrate (II) concentration is 1g/L, add the glucose of massfraction 5% as regenerating coenzyme substrate, volume fraction 10% dimethyl sulfoxide (DMSO) is as organic cosolvent, 30 DEG C, 190 revs/min, transform 12 hours, reaction mixture monoploid amasss extraction into ethyl acetate three times, merge organic layer and use anhydrous sodium sulfate drying, underpressure distillation removing ethyl acetate, gained resistates 2ml dehydrated alcohol (chromatographically pure) fully dissolves, get 10 μ l samples and carry out HPLC detection (method and pillar type detailed in Example 2), calculating transformation efficiency is 56%, ee value > 99%.
Prepared by embodiment 6 product (I)
In reaction system, PseudomonaspseudoalcaligenesXW-40 cell concentration is 40g (dry weight)/L, and all the other are with embodiment 5.It is 78%, ee value > 99% that HPLC detection computations obtains transformation efficiency.
Prepared by embodiment 7 product (I)
PseudomonaspseudoalcaligenesXW-40 cell concentration in reaction system is 50g (dry weight)/L, and all the other conditions are with embodiment 5.It is 89%, ee value > 99% that HPLC detection computations obtains transformation efficiency.
Prepared by embodiment 8 product (I)
PseudomonaspseudoalcaligenesXW-40 cell concentration in reaction system is 60g (dry weight)/L, and all the other experiment conditions are with embodiment 5.HPLC detects and calculates transformation efficiency is 95%, ee value > 99%.
Prepared by embodiment 9 product (I)
PseudomonaspseudoalcaligenesXW-40 cell concentration in reaction system is 70g (dry weight)/L, and all the other experiment conditions are with embodiment 5.HPLC detects and calculates transformation efficiency is 98%, ee value > 99%.
Prepared by embodiment 10 product (I)
PseudomonaspseudoalcaligenesXW-40 cell concentration in reaction system is 80g (dry weight)/L, and all the other experiment conditions are with embodiment 5.HPLC detects and calculates transformation efficiency is 99%, ee value > 99%.
Prepared by embodiment 11 product (I)
PseudomonaspseudoalcaligenesXW-40 cell concentration in reaction system is 90g (dry weight)/L, and all the other experiment conditions are with embodiment 5.HPLC detects and calculates transformation efficiency is > 99%, ee value > 99%.
Prepared by embodiment 12 product (I)
Culture medium prescription and seed liquor preparation method are shown in embodiment 2.For the yeast culture 48 hours of bio-transformation, as biological catalyst after the washing of collected by centrifugation thalline, 5ml reaction system, add the glucose of massfraction 5% as regenerating coenzyme substrate, PseudomonaspseudoalcaligenesXW-40 cell concentration 90g (dry weight)/L, concentration of substrate is 1g/L, under 30 DEG C of conditions, 190 revs/min, transform 12 hours, extraction into ethyl acetate is amassed twice with monoploid, collected organic layer anhydrous sodium sulfate drying, underpressure distillation removing ethyl acetate, resistates 5ml dehydrated alcohol (chromatographically pure) fully dissolves, get 2 μ l samples and carry out HPLC detection, calculating transformation efficiency is > 99%, ee value > 99%.
Prepared by embodiment 13 product (I)
Concentration of substrate in reaction system is 3g/L, and all the other experiment conditions are with embodiment 12.Detecting with HPLC and calculating transformation efficiency is 99%, ee value > 99%.
Prepared by embodiment 14 product (I)
Concentration of substrate in reaction system is 6g/L, and all the other experiment conditions are with embodiment 12.Detecting with HPLC and calculating transformation efficiency is 95%, ee value > 99%.
Prepared by embodiment 15 product (I)
Concentration of substrate in reaction system is 8g/L, and all the other experiment conditions are with embodiment 12.Detecting with HPLC and calculating transformation efficiency is 89%, ee value > 99%.
Prepared by embodiment 16 product (I)
Concentration of substrate in reaction system is 10g/L, and all the other experiment conditions are with embodiment 12.Detecting with HPLC and calculating transformation efficiency is 80%, ee value > 99%.
Prepared by embodiment 17 product (I)
Culture medium prescription and seed liquor preparation method are with embodiment 2.For the yeast culture 48 hours of bio-transformation, as biological catalyst after the washing of collected by centrifugation thalline, operation is with embodiment 3, 5ml reaction system, add the glucose of 5% as regenerating coenzyme substrate, volume fraction 10% dimethyl sulfoxide (DMSO) is as solubility promoter, PseudomonaspseudoalcaligenesXW-40 cell concentration 90g (dry weight)/L, concentration of substrate 10g/L, transform 16 hours, extraction into ethyl acetate is amassed three times with monoploid, collected organic layer anhydrous sodium sulfate drying, underpressure distillation removing ethyl acetate, resistates 5ml chromatographically pure level dehydrated alcohol fully dissolves, get 2 μ l sample feedings, detect with HPLC, calculating transformation efficiency is 89%, ee value > 99%.
Prepared by embodiment 18 product (I)
Bioconversion time is 20 hours, and all the other experiment conditions are with embodiment 16.Detecting with HPLC and calculating transformation efficiency is 96%, ee value > 99%.
Prepared by embodiment 19 product (I)
Bioconversion time is 24 hours, and all the other experiment conditions are with embodiment 16.Detecting with HPLC and calculating transformation efficiency is 99%, ee value > 99%.
Prepared by embodiment 20 product (I)
It is 28 hours that biology turns the time, and all the other experiment conditions are with embodiment 16.Detecting with HPLC and calculating transformation efficiency is > 99%, ee value > 99%
Prepared by embodiment 21 product (I)
Reaction system expands 500ml to, and bio-transformation 28 hours, all the other experiment conditions are with embodiment 16.Detecting with HPLC and calculating transformation efficiency is 99%, ee value > 99%
Prepared by embodiment 22 product (I)
Reaction system expands 1000ml to, and bio-transformation 28 hours, all the other experiment conditions are with embodiment 16.After reacting completely, by reaction solution at 4 DEG C, 7000 revs/min centrifugal 10 minutes, collecting precipitation thalline, uses cold saline to wash twice, centrifugal collecting precipitation thalline, Eddy diffusion in pH7.0 phosphate buffered saline buffer as second batch secondary pollutant transform catalyzer, the reaction system of the biocatalytic reaction of second batch is 1000ml, and bio-transformation 24 hours, all the other experiment conditions are with embodiment 16.Detecting with HPLC and calculating transformation efficiency is 99%, ee value > 99%
Prepared by embodiment 23 product (I)
The reaction system of the biocatalytic reaction of the 3rd batch is 1000ml, and bio-transformation 28 hours, all the other experiment conditions are with embodiment 22.Detecting with HPLC and calculating transformation efficiency is 93%, ee value > 99%.
Prepared by embodiment 24 product (I)
The reaction system of the biocatalytic reaction of the 4th batch is 1000ml, and bio-transformation 28 hours, all the other experiment conditions are with embodiment 22.Detecting with HPLC and calculating transformation efficiency is 91%, ee value > 99%.
Prepared by embodiment 25 product (I)
The reaction system of the biocatalytic reaction of the 5th batch is 1000ml, and bio-transformation 28 hours, all the other experiment conditions are with embodiment 22.Detecting with HPLC and calculating transformation efficiency is 90%, ee value > 99%.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (6)
1. pseudomonas pseudoalcaligenes (Pseudomonaspseudoalcaligenes) XW-40, by China typical culture collection center preservation, preserving number is CCTCCNO:M2015521.
2. pseudomonas pseudoalcaligenes XW-40 described in claim 1 is preparing the application in sitagliptin intermediate.
3. application according to claim 2, it is characterized in that, using pseudomonas pseudoalcaligenes XW-40 thalline as catalyzer, by raw material 4-oxygen-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a]-pyrazine-7 (8H)-Ji]-1-(2,4,5)-trifluorophenyl) fourth-2-ketone catalytic reduction is (S)-3-hydroxyl-1-[3-(trifluoromethyl)-5,6-dihydro [1,2,4] triazolo [4,3-a] pyrazine-7 (8H)-Ji]-1-(2,4,5-trifluorophenyl) fourth-1-ketone.
4. application according to claim 3, is characterized in that, also comprises regenerating coenzyme substrate and organic cosolvent in described reaction system.
5. the application according to claim 3 or 4, is characterized in that, described catalyzer pseudomonas pseudoalcaligenes XW-40 thalline through again collecting, for the preparation of next batch sitagliptin intermediate after washing.
6. application according to claim 4, it is characterized in that, described pseudomonas pseudoalcaligenes XW-40 cell concentration is 90g (dry weight)/L, material concentration is 10g/L, regenerating coenzyme substrate is the glucose of massfraction 5%, organic cosolvent is the dimethyl sulfoxide (DMSO) of volume fraction 10%, and the reaction times is 28 hours.
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| CN105925506A (en) * | 2016-05-27 | 2016-09-07 | 浙江工业大学 | Pseudomonas aeruginosa ZJPH1504 and application thereof in preparation of sitagliptin chiral intermediate |
| CN106755152A (en) * | 2017-01-23 | 2017-05-31 | 苏州引航生物科技有限公司 | A kind of method for preparing Ao Gelieting intermediates |
| CN114507699A (en) * | 2022-02-15 | 2022-05-17 | 上海微巨实业有限公司 | Low-cost preparation method of sitagliptin phosphate |
| US11459549B2 (en) | 2018-05-10 | 2022-10-04 | China Fortune Way Company | Method for biocatalytic synthesis of Sitagliptin and intermediate thereof |
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| CN106755152B (en) * | 2017-01-23 | 2020-06-16 | 苏州引航生物科技有限公司 | Method for preparing augustine intermediate |
| US11459549B2 (en) | 2018-05-10 | 2022-10-04 | China Fortune Way Company | Method for biocatalytic synthesis of Sitagliptin and intermediate thereof |
| CN114507699A (en) * | 2022-02-15 | 2022-05-17 | 上海微巨实业有限公司 | Low-cost preparation method of sitagliptin phosphate |
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| CN105274027B (en) | 2018-06-05 |
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