CN105274027B - Pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared - Google Patents
Pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared Download PDFInfo
- Publication number
- CN105274027B CN105274027B CN201510753925.8A CN201510753925A CN105274027B CN 105274027 B CN105274027 B CN 105274027B CN 201510753925 A CN201510753925 A CN 201510753925A CN 105274027 B CN105274027 B CN 105274027B
- Authority
- CN
- China
- Prior art keywords
- pseudomonas pseudoalcaligenes
- substrate
- prepared
- thalline
- sitagliptin
- 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.)
- Active
Links
Abstract
The invention discloses a kind of pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared, 40 bacterium colony configurations of surface of pseudomonas pseudoalcaligenes XW are flat, circular, the smooth of the edge is neat, and in moist shape, color is creamy white, opaque, colony diameter is about 1.9mm.The bacterial strain can effectively be catalyzed 4 oxygen, 4 [3 (trifluoromethyls) 5,6 dihydros [1,2,4] triazol [4,3 a] pyrazine 7 (8H) base] 1 (2,4,5) trifluorophenyl) fourth 2 ketone be reduced to sitagliptin chiral intermediate, the high concentration of substrate of 10g/L is resistant under optimum reaction conditions, and is still kept highly selective, substrate inhibition is weaker, it can be achieved that the living things catalysis of high concentration of substrate.The thalline can also recycle, and compared with batch feeding, while eliminate the inhibitory action of substrate and products upon cell, reach the catalytic activity of thalline and utilize to greatest extent.
Description
Technical field
The invention belongs to biopharmaceutical technologies, and in particular to a kind of pseudomonas pseudoalcaligenes strain and its prepare west he
Arrange the application in the intermediate of spit of fland.
Background technology
Sitagliptin phosphate (sitagliptin phosphate) is first development of MSD Corp. for treatment 2
Dipeptidyl peptidase-4 (DPP-4) inhibitor of patients with type Ⅰ DM, the medicine can effectively for the insulin resistance in diabetes B and
The dysfunction of pancreatic islet alpha, β cells slows down the degradation of incretin GLP-1 by inhibiting DPP-4, makees so as to play hypoglycemic
With.Compared with easily leading to hypoglycemia, weight gain and causing the conventional oral hypoglycemic medicine of the side effects such as nausea and vomiting, the inhibition
Agent has apparent advantage and good market prospects.There are two the chiral intermediate of sitagliptin phosphate key is main, one
It is chiral beta-amino acids, one is its corresponding chiral alcohol intermediate, and the synthetic work emphasis on sitagliptin is main at present
It is unfolded around the two chiral intermediates.
The synthetic method of this chiral beta-amino acids of document report is mainly the following at present：
(1) induce chiral a-amino acid using chiral source, and after through diazo-reaction generate beta-amino acids, so as to
Chiral centre is built, the route is raw materials used costly, and reaction condition is quite harsh, and low temperature are waited if desired for -78 DEG C and -30 DEG C
Condition, and longer the time required to some reactions and operation is relatively complicated, the refined of intermediate product is needed by column chromatography for separation；
(2) asymmetric catalytic hydrogenation is carried out to ketone ester using chiral phosphorus ruthenium catalyst, builds chiral secondary alcohol, then will
Chiral secondary alcohol chiral inversion is secondary amine, and chiral catalyst is expensive in the route, and enlarge-effect is apparent；
(3) asymmetric hydrogenation of β-enamino acid intermediate, asymmetric hydrogenation expensive metallic catalyst such as with
It is carried out in the presence of the combined rhodium of chiral phosphine/diphosphine ligand or using expensive metal ruthenium catalyst；
(4) using isopropylamine as ammonia donor, using sitagliptin precursor ketone as ammonia receptor, prepared using transaminase living things catalysis
Sitagliptin, this method have environmental-friendly a, advantages such as reaction condition is mild, but specific enzymes be not easy to obtain and price it is high
Characteristic (separation scheme etc.) is very important；
(5) with trifluro benzaldehyde and N- acetoglycocolls through being condensed, reducing, biological enzyme is split, hydrolysis, protection amino, again
Nitridation, Arndt-Fistert rearrangement reactions hydrolyze to obtain R- beta-aminobutyric acids, but the synthetic route is long, and chiral resolution obtains
The yield for obtaining chiral amino acid is no more than 50%, and in addition recycling for half enantiomter is a great problem, and is reacted
Journey uses diazomethane, and diazomethane is unstable toxic gas at room temperature, has explosivity, operational hazards.
And the synthetic method of document report chiral alcohol intermediate accordingly is mainly as follows at present：
(1) WO 09/045507 β-carbonyl moiety is carried out using appropriate carbonyl reductase asymmetric reduction obtain β-
Hydroxy intermediate, then sitagliptin is obtained with aza cyclo-butanone intermediate reaction, the disadvantages of this method is：It is anti-under high pressure
Should, it cause using very expensive metal chiral catalyst (Rh or Ru), low stereoselectivity and rhodium pollution products and thus
Final compound be difficult purifying；
(2) WO12/046254 discloses the method for preparing the intermediate that sitagliptin is prepared by enzymatic conversion, provides
The method of two kinds of living things catalysis synthesis of chiral alcohol intermediates, the respectively full cell of utilizing works bacterium and the produced carbonyl of engineering bacteria are also
Sitagliptin precursor ketone is reduced to the alcohol of corresponding (S) configuration as biocatalyst by the crude extract of protoenzyme；Its method has
Environmental-friendly, reaction condition is mild, the high advantage of ee values, but on the other hand, engineering bacteria be built with very big difficulty and
Both expensive, using slightly carry enzyme as biocatalyst there are the problem of be that free thick enzyme is unstable in reaction solution, enzyme
Catalytic capability is relatively low, and enzyme easily inactivates, and is not easy to realize the recycling of enzyme, and the lock out operation in later stage is complicated；And it uses whole thin
For born of the same parents as catalyst, it is that substrate dissolves in toluene that substrate is dissolved in the problems of progress biphasic catalysis reaction in toluene
Spend small, the converted concentration of substrate of reaction is relatively low, is not suitable for large-scale industrial production.
Existing chemical method and the method for biological catalysis synthesis sitagliptin and intermediate all there are its shortcoming,
Therefore, the method for developing the intermediate for preparing sitagliptin of cleaner environment close friend is significantly.
The content of the invention
In view of this, it is an object of the invention to provide a kind of pseudomonas pseudoalcaligenes strain, and the use of the bacterial strain is provided
On the way, sitagliptin intermediate can economically be obtained by the living things catalysis of the bacterial strain thalline.
The present invention with 4- oxygen -4- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-a]-pyrazine -7 (8H) -
Base] -1- (2,4,5)-trifluorophenyl) butyl- 2- ketone (II) be raw material, utilize the carbonyl reductase in microorganism, regioselectivity
(S) -3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazols [4,3-a] are reduced to enantioselectivity catalysis
Pyrazine -7 (8H)-yl] -1- (2,4,5- trifluorophenyls) butyl- 1- ketone (I).
The technology of the present invention route is as follows：
The technical solution that the present invention takes is as follows：
1st, pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) XW-40, by Chinese Typical Representative culture
Collection preservation, preserving number are CCTCC NO:M2015521.
2nd, applications of the above-mentioned pseudomonas pseudoalcaligenes XW-40 in sitagliptin intermediate is prepared.
Preferably, using pseudomonas pseudoalcaligenes XW-40 thalline as catalyst, by raw material 4- oxygen -4- [3- (fluoroforms
Base) -5,6- dihydros [1,2,4] triazol [4,3-a]-pyrazine -7 (8H)-yl] -1- (2,4,5)-trifluorophenyl) butyl- 2- ketone urges
Change be reduced to (S) -3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-a] pyrazine -7 (8H) -
Base] -1- (2,4,5- trifluorophenyls) butyl- 1- ketone.
Preferably, regenerating coenzyme substrate and organic cosolvent are further included in the reaction system.
Preferably, the catalyst pseudomonas pseudoalcaligenes XW-40 thalline are used for next group by collecting again after washing
The preparation of secondary sitagliptin intermediate.
Preferably, the pseudomonas pseudoalcaligenes XW-40 cell concentrations be 90g (dry weight)/L, material concentration 10g/L,
Regenerating coenzyme substrate is the glucose of mass fraction 5%, and organic cosolvent is the dimethyl sulfoxide (DMSO) of volume fraction 10%, during reaction
Between for 28 it is small when.
The beneficial effects of the present invention are：(1) the production carbonyl reductase strain Pseudomonas of screening
Pseudoalcaligenes XW-40 have the regioselectivity and enantioselectivity of height, can effectively be catalyzed 4- oxygen -4-
[3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-a]-pyrazine -7 (8H)-yl] -1- (2,4,5)-trifluorophenyl)
Butyl- 2- ketone (II) is reduced to sitagliptin chiral intermediate (S) -3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4]
Triazol [4,3-a] pyrazine -7 (8H)-yl] -1- (2,4,5- trifluorophenyl) butyl- 1- ketone (I), no coupling product generate, simplify
Separation process；(2) for the screening of bacterial strain in soil, select the acetophenone similar to substrate (II) structure for unique carbon source into
The screening of row bacterial strain quickly filters out suitable doubtful primary dcreening operation bacterial strain in a variety of soil that can rapidly comform, then is used
In the reduction of (II), optimal bacterial strain is quickly selected；(3) strain Pseudomonas screened
Pseudoalcaligenes XW-40 are resistant to the high concentration of substrate of 10g/L under most suitable reaction condition, and still keep high
Selectivity, substrate inhibition is weaker, and so as to can realize the living things catalysis of high concentration of substrate, this point for being reported at present
It is carried out using wildtype bacterium relatively rare for biological reducing；(4) Pseudomonas pseudoalcaligenes XW-40 bacterium
Body can recycle, and treat that a batch after reaction centrifuges thalline, can put into again in the catalytic reaction of next batch, with dividing
It criticizes feed supplement to compare, while eliminates the inhibitory action of substrate and products upon cell, the catalytic activity of thalline is made to reach to greatest extent
Utilization, can be recycled 5 times with a batch thalline, yield and optical purity are high, process green, of low cost.
Culture presevation
Pseudomonas pseudoalcaligenes (Pseudomonas pseudoalcaligenes) XW-40 is from soil in the present invention
Separation obtains, and send China typical culture collection center (CCTCC) preservation, and deposit number is CCTCC NO:M2015521, address
Positioned at Wuhan City, Hubei Province Wuhan University, preservation date is September in 2015 10 days, and Classification And Nomenclature is pseudomonas pseudoalcaligenes
Pseudomonas pseudoalcaligenes.Bacterium colony is flat for configuration of surface in the form of solid medium, circular, edge
Portion smooth is neat, and entire bacterium colony is in moist shape, and color is creamy white, opaque, and colony diameter is about 1.9mm, physiology
Matter is shown in Table 2.It is pseudomonas pseudoalcaligenes through 16S rDNA molecular biology identifications, homology is up to 100%.
Specific embodiment
The preferred embodiment of the present invention is described in detail below.The experiment side of actual conditions is not specified in embodiment
Method, usually according to normal condition or according to the condition proposed by manufacturer.
The primary dcreening operation of 1 soil bacterial strain of embodiment
About 1g pedotheques are weighed in 10ml sterile waters, are centrifuged after vibrating mixing, Aspirate supernatant 1ml is added to
100ml is using acetophenone as in the minimum salt culture medium of sole carbon source, 30 DEG C are cultivated 5-7 days.Then the bacteria suspension of culture is coated with
To using acetophenone in the minimum salt agar medium of sole carbon source, the bacterium colony to grow on tablet to be isolated and purified and is inoculated in fine jade
(culture medium forms on fat inclined-plane：Peptone 5g/L, yeast extract 1.5g/L, glucose 10g/L, beef extract 1.5g/L, NaCl 5g/
L, pH7.0), 30 DEG C of culture 48h, as the doubtful bacterial strain further screened, number preserves.
Embodiment 2 produces the screening of carbonyl reductase bacterial strain
The strain that embodiment 1 isolates and purifies is inoculated in composition as peptone 5g/L, yeast extract 1.5g/L, glucose
In the culture medium of 10g/L, beef extract 1.5g/L, NaCl 5g/L, pH7.0,30 DEG C are cultivated 48h, and 7000rpm/min is centrifuged at 4 DEG C
10min collects thalline, is washed twice with cold saline, obtain cell wet thallus.
It is screened using following bacterial strain screening system：1g/L substrates (II), the glucose of mass fraction 5%, 10% (v/
V) absolute ethyl alcohol, 80g/L thalline (dry weight), sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution of 0.1M, pH7.0 are stirred at 30 DEG C
12h, centrifugation are reacted, supernatant is extracted with ethyl acetate 3 times, and organic layer merges, and anhydrous magnesium sulfate is dried overnight, reverse phase C18Liquid phase
Chromatography and Chiral liquid chromatography measure conversion ratio and enantiomeric excess (ee).
Liquid phase chromatogram condition is：Chromatographic column C when detecting conversion ratio18(250 × 4.6mm × 5 μm, Agilent America),
Mobile phase：Water/acetonitrile (v/v)=70/30, flow velocity 1ml/min, Detection wavelength：268nm, temperature：25℃；4- oxygen -4- [3- (three
Methyl fluoride) -5,6- dihydros [1,2,4] triazol [4,3-a]-pyrazine -7 (8H)-yl] -1- (2,4,5)-trifluorophenyl) butyl- 2-
Ketone and 3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-a] pyrazine -7 (8H)-yl] -1- (2,4,
5- trifluorophenyls) retention time of butyl- 1- ketone is respectively 19.7 and 26.8min；Chromatographic column Chiracel AY-H (5 when detecting ee
× 250mm), mobile phase：N-hexane/absolute ethyl alcohol (v/v)=85：15, flow velocity：1.0ml/min, Detection wavelength：268nm, temperature
Degree：25℃；In chemical synthesis racemic alcohol 3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-a]
Pyrazine -7 (8H)-yl] -1- (2,4,5- trifluorophenyl) butyl- 1- ketone analysis in, the retention time of S and R isomers is respectively
11.3min and 17.8min.It determines to prepare the chiral configuration of alcohol product using same procedure, conversion ratio is more than to 5% bacterial strain
Result be expressed in table 1,
Table 1 produces the screening of carbonyl reductase bacterial strain
As shown in Table 1, enantiomeric excess (ee) highest and the best bacterial strain for number 40-1 of conversion ratio, observe
40-1 bacterium colonies are flat for configuration of surface in the form of solid medium, circular, and marginal portion is smooth neat, and entire bacterium colony is in tide
Wet shape, color are creamy white, opaque, and colony diameter is about 1.9mm, and physiological characteristics are shown in Table 2.Through 16S rDNA molecular biosciences
Be accredited as pseudomonas pseudoalcaligenes, and homology is named as Pseudomonas pseudoalcaligenes XW- up to 100%
40, DNA sequence dna is shown in SEQ ID No.1.
Table 140-1 bacterial strain Physiology and biochemistry properties
It is prepared by 3 product of embodiment (I)
(S) -3- hydroxyls -1- [3- (trifluoros are prepared using Pseudomonas pseudoalcaligenes XW-40 bacteriums
Methyl) -5,6- dihydros [1,2,4] triazol [4,3-a] pyrazine -7 (8H)-yl] -1- (2,4,5- trifluorophenyls) butyl- 1- ketone
(I).5ml seed liquors is taken to be forwarded in the fresh culture mediums of 100ml (shaking flask of 250ml capacity) (nutrient media components and culture
Condition is shown in embodiment 2), at 30 DEG C, when 190 revs/min of shake cultures 48 are small, at 4 DEG C, centrifuged under the conditions of 7000 revs/min
10 minutes, precipitation thalline is collected, and is washed twice with cold saline, obtains wet thallus as biocatalyst.Gained is wet
It is 50g (dry weight)/L bacteria suspensions that thalline is made into cell concentration with the phosphate buffer of pH 7.0.5ml reaction systems, substrate
(II) concentration is 1g/L, adds in the glucose of mass fraction 5% as regenerating coenzyme substrate, 10% absolute ethyl alcohol of volume fraction is made
For organic cosolvent, 30 DEG C, 190 revs/min, when conversion 12 is small, reaction mixture is extracted three times with monoploid product ethyl acetate,
Merge organic layer and dried with anhydrous sodium sulfate, vacuum distillation removes ethyl acetate, gained residue 2ml absolute ethyl alcohol (colors
Compose pure) fully dissolving, 10 μ l samples is taken to carry out HPLC detections (method and pillar type are such as embodiment 2), calculating conversion ratio is
57%, ee value ＞ 99%.
The product (I) prepared using a variety of classical tools analysis enzymatic, i.e. fusing point (m.p.), specific rotation (SOR), nuclear-magnetism are common
Vibrational spectrum (NMR), it is as a result as follows：
m.p.：114-119℃；SOR[α]D25：23.2 ° of (c=1, CHCl3)；1H NMR (400MHz, DMSO-D6)：δ
2.46-2.48 (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 (overlapping m, 3H), 7.40-7.48 (m, 2H)；13C NMR (100MHz, DMSO-D6)：δ
35.4,37.3,38.3,40.1,41.3,42.1,43.0,43.7,67.3,105.1,115.2,117.3,119.5,123.0,
142.3,144.4,146.5,148.7,151.0,154.8,156.8,170.2.
The data of 4~25 product of embodiment (I) are same as Example 3.
It is prepared by 4 product of embodiment (I)
In order to further screen optimal organic cosolvent, volume is used when bacterial strain primary transformants in embodiment 3 are tested
10% absolute ethyl alcohol of fraction is changed to the different organic solvents listed in table 2 as organic cosolvent as helping in reaction system
Solvent, other conditions are the same as embodiment 3.
Influence of 2 organic cosolvent of table to reaction
As shown in Table 2, when selecting dimethyl sulfoxide (DMSO) as organic cosolvent, the conversion ratio highest of substrate, ee values are ＞
99%.Therefore, organic cosolvent is dimethyl sulfoxide (DMSO) in embodiment 5~25.
It is prepared by 5 product of embodiment (I)
Culture medium prescription and seed liquid and preparation method thereof are referring to embodiment 3.After when small for the thalline culture 48 of bioconversion
Centrifugation, washing, it is that 30g (dry weight)/L bacteria suspensions are urged as biology that gained wet thallus is made into cell concentration by the use of phosphate buffer
Agent, 5mL reaction systems, substrate (II) concentration are 1g/L, add in the glucose of mass fraction 5% as regenerating coenzyme substrate,
10% dimethyl sulfoxide (DMSO) of volume fraction is as organic cosolvent, and 30 DEG C, 190 revs/min, when conversion 12 is small, reaction mixture is used
The extraction of monoploid product ethyl acetate three times, merges organic layer and is dried with anhydrous sodium sulfate, vacuum distillation removes ethyl acetate, institute
It obtains residue fully to be dissolved with 2ml absolute ethyl alcohols (chromatographically pure), 10 μ l samples is taken to carry out HPLC detections, and (method and pillar type are detailed
See embodiment 2), calculating conversion ratio is 56%, ee values ＞ 99%.
It is prepared by 6 product of embodiment (I)
Pseudomonas pseudoalcaligenes XW-40 cell concentrations are 40g (dry weight)/L in reaction system,
Remaining same embodiment 5.HPLC detection calculate conversion ratio be 78%, ee values ＞ 99%.
It is prepared by 7 product of embodiment (I)
Pseudomonas pseudoalcaligenes XW-40 cell concentrations in reaction system are 50g (dry weight)/L,
Remaining condition is the same as embodiment 5.HPLC detection calculate conversion ratio be 89%, ee values ＞ 99%.
It is prepared by 8 product of embodiment (I)
Pseudomonas pseudoalcaligenes XW-40 cell concentrations in reaction system are 60g (dry weight)/L,
Remaining experiment condition is the same as embodiment 5.HPLC detect and calculate conversion ratio be 95%, ee values ＞ 99%.
It is prepared by 9 product of embodiment (I)
Pseudomonas pseudoalcaligenes XW-40 cell concentrations in reaction system are 70g (dry weight)/L,
Remaining experiment condition is the same as embodiment 5.HPLC detect and calculate conversion ratio be 98%, ee values ＞ 99%.
It is prepared by 10 product of embodiment (I)
Pseudomonas pseudoalcaligenes XW-40 cell concentrations in reaction system are 80g (dry weight)/L,
Remaining experiment condition is the same as embodiment 5.HPLC detect and calculate conversion ratio be 99%, ee values ＞ 99%.
It is prepared by 11 product of embodiment (I)
Pseudomonas pseudoalcaligenes XW-40 cell concentrations in reaction system are 90g (dry weight)/L,
Remaining experiment condition is the same as embodiment 5.HPLC detect and calculate conversion ratio be ＞ 99%, ee values ＞ 99%.
It is prepared by 12 product of embodiment (I)
Culture medium prescription and seed liquid and preparation method thereof are shown in embodiment 2.When small for the thalline culture 48 of bioconversion, centrifugation
Biocatalyst is used as after collecting thalline washing, 5ml reaction systems add the glucose of mass fraction 5% as regenerating coenzyme
Substrate, Pseudomonas pseudoalcaligenes XW-40 cell concentrations 90g (dry weight)/L, concentration of substrate 1g/L, 30
Under the conditions of DEG C, 190 revs/min, when conversion 12 is small, it is extracted twice with monoploid product ethyl acetate, the anhydrous sulphur of collected organic layer
The drying of sour sodium, vacuum distillation remove ethyl acetate, and residue is fully dissolved with 5ml absolute ethyl alcohols (chromatographically pure), take 2 μ l samples into
Row HPLC detect, calculate conversion ratio be ＞ 99%, ee values ＞ 99%.
It is prepared by 13 product of embodiment (I)
Concentration of substrate in reaction system is 3g/L, remaining experiment condition is the same as embodiment 12.It is detected and calculated with HPLC
Conversion ratio is 99%, ee values ＞ 99%.
It is prepared by 14 product of embodiment (I)
Concentration of substrate in reaction system is 6g/L, remaining experiment condition is the same as embodiment 12.It is detected and calculated with HPLC
Conversion ratio is 95%, ee values ＞ 99%.
It is prepared by 15 product of embodiment (I)
Concentration of substrate in reaction system is 8g/L, remaining experiment condition is the same as embodiment 12.It is detected and calculated with HPLC
Conversion ratio is 89%, ee values ＞ 99%.
It is prepared by 16 product of embodiment (I)
Concentration of substrate in reaction system is 10g/L, remaining experiment condition is the same as embodiment 12.It is detected and calculated with HPLC
Conversion ratio is 80%, ee values ＞ 99%.
It is prepared by 17 product of embodiment (I)
Culture medium prescription and seed liquid and preparation method thereof are the same as embodiment 2.When small for the thalline culture 48 of bioconversion, centrifugation
It collects after thalline washing as biocatalyst, operates same embodiment 3,5ml reaction systems add 5% glucose as auxiliary
Enzyme regenerates substrate, and 10% dimethyl sulfoxide (DMSO) of volume fraction is as cosolvent, Pseudomonas pseudoalcaligenes XW-
40 cell concentration 90g (dry weight)/L, concentration of substrate 10g/L when conversion 16 is small, with the extraction of monoploid product ethyl acetate three times, is received
Collection organic layer is dried with anhydrous sodium sulfate, and vacuum distillation removes ethyl acetate, and residue is abundant with 5ml chromatographically pure grade absolute ethyl alcohols
Dissolving, take 2 μ l sample feedings, detected with HPLC, calculate conversion ratio be 89%, ee values ＞ 99%.
It is prepared by 18 product of embodiment (I)
When bioconversion time is 20 small, remaining experiment condition is the same as embodiment 16.Conversion ratio is detected and calculated and to obtain with HPLC
For 96%, ee values ＞ 99%.
It is prepared by 19 product of embodiment (I)
When bioconversion time is 24 small, remaining experiment condition is the same as embodiment 16.Conversion ratio is detected and calculated and to obtain with HPLC
For 99%, ee values ＞ 99%.
It is prepared by 20 product of embodiment (I)
Biology turn the time for 28 it is small when, remaining experiment condition is the same as embodiment 16.Detected and calculated with HPLC conversion ratio is
＞ 99%, ee value ＞ 99%
It is prepared by 21 product of embodiment (I)
Reaction system is expanded to 500ml, and when bioconversion 28 is small, remaining experiment condition is the same as embodiment 16.It is detected with HPLC
And calculate conversion ratio be 99%, ee values ＞ 99%
It is prepared by 22 product of embodiment (I)
Reaction system is expanded to 1000ml, and when bioconversion 28 is small, remaining experiment condition is the same as embodiment 16.The reaction was complete
Afterwards, by reaction solution at 4 DEG C, 7000 revs/min centrifuge 10 minutes, collect precipitation thalline, are washed twice using cold saline,
Precipitation thalline is collected by centrifugation, is resuspended in the catalyst as second lot bioconversion in pH7.0 phosphate buffers, the
The reaction system of the biocatalytic reaction of two batches is 1000ml, and when bioconversion 24 is small, remaining experiment condition is the same as embodiment 16.
Detected and calculated with HPLC conversion ratio be 99%, ee values ＞ 99%
It is prepared by 23 product of embodiment (I)
The reaction system of the biocatalytic reaction of 3rd batch is 1000ml, when bioconversion 28 is small, remaining experiment condition
With embodiment 22.Detected and calculated with HPLC conversion ratio be 93%, ee values ＞ 99%.
It is prepared by 24 product of embodiment (I)
The reaction system of the biocatalytic reaction of 4th batch is 1000ml, when bioconversion 28 is small, remaining experiment condition
With embodiment 22.Detected and calculated with HPLC conversion ratio be 91%, ee values ＞ 99%.
It is prepared by 25 product of embodiment (I)
The reaction system of the biocatalytic reaction of 5th batch is 1000ml, when bioconversion 28 is small, remaining experiment condition
With embodiment 22.Detected and calculated with HPLC conversion ratio be 90%, ee values ＞ 99%.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (6)
1. pseudomonas pseudoalcaligenes（Pseudomonas pseudoalcaligenes）XW-40, it is characterised in that：By Chinese allusion quotation
Type culture collection preservation, preserving number are CCTCC NO:M2015521.
2. applications of the pseudomonas pseudoalcaligenes XW-40 in sitagliptin intermediate is prepared, feature exist described in claim 1
In：The sitagliptin intermediate is (S) -3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-
A] pyrazine -7 (8H)-yl] -1- (2,4,5- trifluorophenyls) butyl- 1- ketone.
3. application according to claim 2, which is characterized in that using pseudomonas pseudoalcaligenes XW-40 thalline as catalyst,
By raw material 4- oxygen -4- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazol [4,3-a]-pyrazine -7 (8H)-yl] -1- (2,
4,5)-trifluorophenyl) butyl- 2- ketone catalysis be reduced to (S) -3- hydroxyls -1- [3- (trifluoromethyl) -5,6- dihydros [1,2,4] triazoles
And [4,3-a] pyrazine -7 (8H)-yl] -1- (2,4,5- trifluorophenyls) butyl- 1- ketone.
4. application according to claim 3, which is characterized in that regenerating coenzyme substrate is further included in catalystic converter system and is had
Co-solvent content.
5. the application according to claim 3 or 4, which is characterized in that the catalyst pseudomonas pseudoalcaligenes XW-40 thalline
By collecting again, the preparation of next batch sitagliptin intermediate is used for after washing.
6. application according to claim 4, which is characterized in that the pseudomonas pseudoalcaligenes XW-40 cell concentrations are
90g/L, in terms of dry cell weight, material concentration 10g/L, regenerating coenzyme substrate be mass fraction 5% glucose, organic co-solvent
Agent is the dimethyl sulfoxide (DMSO) of volume fraction 10%, when the reaction time is 28 small.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510753925.8A CN105274027B (en) | 2015-11-05 | 2015-11-05 | Pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510753925.8A CN105274027B (en) | 2015-11-05 | 2015-11-05 | Pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105274027A CN105274027A (en) | 2016-01-27 |
| CN105274027B true CN105274027B (en) | 2018-06-05 |
Family
ID=55143867
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510753925.8A Active CN105274027B (en) | 2015-11-05 | 2015-11-05 | Pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105274027B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN108586346B (en) | 2018-05-10 | 2019-10-01 | 北京富盛嘉华医药科技有限公司 | A kind of method that biocatalysis synthesizes sitagliptin and its intermediate |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103228658A (en) * | 2010-10-08 | 2013-07-31 | 卡迪拉保健有限公司 | Process for preparing an intermediate of sitagliptin via enzymatic conversion |
| WO2015071861A2 (en) * | 2013-11-14 | 2015-05-21 | Biocon Limited | Fungus mediated stereo-selective bioreduction of keto intermediates of pharmaceutically active compounds to their corresponding hydroxy compounds |
| CN104893989A (en) * | 2015-05-29 | 2015-09-09 | 浙江工业大学 | Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate |
-
2015
- 2015-11-05 CN CN201510753925.8A patent/CN105274027B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103228658A (en) * | 2010-10-08 | 2013-07-31 | 卡迪拉保健有限公司 | Process for preparing an intermediate of sitagliptin via enzymatic conversion |
| WO2015071861A2 (en) * | 2013-11-14 | 2015-05-21 | Biocon Limited | Fungus mediated stereo-selective bioreduction of keto intermediates of pharmaceutically active compounds to their corresponding hydroxy compounds |
| CN104893989A (en) * | 2015-05-29 | 2015-09-09 | 浙江工业大学 | Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate |
Non-Patent Citations (2)
| Title |
|---|
| 抗2型糖尿病药西他列汀关键手性中间体的合成研究;柳英帅 等;《中国药物化学杂志》;20121031;第22卷(第5期);360-363 * |
| 西他列汀的化学-酶法合成;徐中琦 等;《沈阳药科大学学报》;20141130;第31卷(第11期);867-870 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105274027A (en) | 2016-01-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105274027B (en) | Pseudomonas pseudoalcaligenes strain and its application in sitagliptin intermediate is prepared | |
| Espinoza-Moraga et al. | Bioreduction of β-carboline imines to amines employing Saccharomyces bayanus | |
| CN106497996A (en) | The enzyme catalysiss preparation method of chiral alcohol | |
| JP4394647B2 (en) | Method for producing optically active tetrahydrothiophene derivative and method for crystallizing optically active tetrahydrothiophene-3-ol | |
| KR20080036098A (en) | Method for producing optically active 2-(n-substituted aminomethyl)-3-hydroxybutyric acid ester | |
| CN107119081A (en) | It is prepared by one kind（R）The method of the hexene acid esters of 3 hydroxyl 5 | |
| CN102382780B (en) | Microbacterium oxydans and method for preparing chiral bis(trifluoromethyl) phenyl ethanol by using same | |
| CN107586793A (en) | The method for preparing the alcoholic compound with multiple chiral centers | |
| US20100261251A1 (en) | Microbial kinetic resolution of ethyl-3,4-epoxybutyrate | |
| CN103804384B (en) | The preparation method of benzodiazepine compounds | |
| CN103209949A (en) | Halogenated indenones and method for producing optically active indanones or optically active indanols by using same | |
| CN102947461A (en) | Preparation of (3as,7ar)-hexahydroisobenzofuran-1(3h)-one by catalyzed biological resolution of dimethyl cyclohexane-1,2-dicarboxylate | |
| CN102465159B (en) | Synthesis process for preparing eslicarbazepine with microbial method | |
| WO1996040975A1 (en) | Optical resolution of alkyl chroman-2-carboxylates | |
| Singh et al. | Enantioselective transesterification of (RS)-1-chloro-3-(3, 4-difluorophenoxy)-2-propanol using Pseudomonas aeruginosa lipases | |
| Guieysse et al. | Resolution of 2-bromo-o-tolyl-carboxylic acid by transesterification using lipases from Rhizomucor miehei and Pseudomonas cepacia | |
| CN104651243B (en) | Pichia membranaefaciens and its application in chiral synthesis (R) 1,3 butanediol | |
| JP2710834B2 (en) | FO-608A substance and method for producing the same | |
| CN103122319B (en) | Yeast with stereoselectivity and lipase activity and method for preparing S-type rivastigmine by biological splitting of yeast | |
| JPWO2003097851A1 (en) | Method for producing optically active alkylcarboxylic acid derivative | |
| JP3580875B2 (en) | FO-4259 substance and method for producing the same | |
| JP2872311B2 (en) | FO-608B, C substance and method for producing the same | |
| CN116286380A (en) | Cyclopentapeptide compounds and application thereof in alpha-glycosidase inhibitor hypoglycemic drugs or preventive health products | |
| CN117844876A (en) | Method for synthesizing 3-cyanopropionic acid derivative based on dynamic kinetic resolution, 3-cyanopropionic acid derivative and gamma-aminobutyric acid derivative | |
| CN111808893A (en) | Novel biological preparation method of amino alcohol drug intermediate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |