CN106834376A - A kind of method of Enzyme catalyzed synthesis Xi Gelieting - Google Patents
A kind of method of Enzyme catalyzed synthesis Xi Gelieting Download PDFInfo
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Abstract
The invention belongs to pharmaceutical technology field, it is related to the method and device of a kind of Enzyme catalyzed synthesis Xi Gelieting, comprises the following steps：The transaminase somatic cells of S1, cultivation with catalysis activity, and prepare crude enzyme liquid；The calcium chloride solution of S2, preparation containing spheroidizing immobilized enzyme；S3, to diatomite is added in the calcium chloride solution containing spheroidizing immobilized enzyme, obtain mixed system；S4, preparation reaction cushioning liquid；S5, mixed system are placed in isothermal reaction container, reaction cushioning liquid is filled with isothermal reaction container in the way of circulating and is reacted with mixed system, obtain Xi Gelieting；Its simple to operate, transformation efficiency is high, environmentally friendly and with low cost.
Description
Technical field
The invention belongs to pharmaceutical technology field, and in particular to a kind of method of Enzyme catalyzed synthesis Xi Gelieting.
Background technology
Xi Gelieting is a kind of first DPP-IV inhibitor class medicine developed by United States Merck company, and it is II type for the treatment of
Diabetic, its medicinal forms are phosphoric acid Xi Gelieting monohydrates, and it has following structure：
At present, what synthesis Xi Gelieting in international market was commonly used is full chemistry synthetic method, need to be in height in production process
Precious metal catalytic hydrogenation is used under warm condition of high voltage, catalyst is not easily recycled, operational hazards coefficient is high, its crucial chiral structure
Synthesis difficulty is big, is not readily separated, low yield, causes relatively costly.Common patent synthetic method is as follows：
Xi Gelieting second generation synthetic methods：It is not right using dinaphthalene diphenylphosphine-chlorination catalyzed by ruthenium complexes beta-ketoester is carried out
Claim hydrogenation, further reaction obtains Chiral Amine to obtain chiral alcohol.The method all needs to use metal catalytic twice, costly.Close
It is into technique：
Merck ＆ Co., Inc. latest generation patent (W02005020920) synthetic method：With 2,4,5, trifluoro benzene acetic acid is raw material,
Substrate (1) is obtained through multistep reaction, substrate ammonium acetate acts on obtaining enamine, and enamine is in dimerization chloro (1,5- cyclo-octadiene) rhodium
[(COD)RhCI]2(R)-(-) -1- [(S) -2- [two (4- trifluoromethyls) phosphines] ferrocenyl ethyl -2- tert-butyl group phosphines
There is asymmetric hydrogenation in the presence of (R, S-t-BuJosiphos).Due to [(COD) RhCI]2(R, S-t-
BuJosiphos) expensive, operating condition is harsh, therefore industrialization difficulty is big.
The content of the invention
The present invention provides the method and device of a kind of enzymatic clarification Xi Gelieting, and its simple to operate, transformation efficiency is high, ring
Protect and with low cost.
To realize above-mentioned technical purpose, the specific technical scheme that the present invention takes is, a kind of Enzyme catalyzed synthesis Xi Gelie
The method in spit of fland, S1, cultivate with catalysis activity transaminase somatic cells, transaminase somatic cells are crushed, aqueous suspension,
Centrifugation obtains crude enzyme liquid；S2, to be configured to the buffering containing homeo-osmosis agent using homeo-osmosis agent and sodium chloride solution molten
Agent, to adding sodium alginate to obtain sodium alginate soln in buffer solvent, takes the crude enzyme liquid isometric with sodium alginate soln, will
The crude enzyme liquid for being taken is well mixed with sodium alginate soln and obtains mixed liquor, and mixed liquor is added dropwise in calcium chloride solution, together
When also to adding phosphopyridoxal pyridoxal phosphate to obtain blend solution in calcium chloride solution, blended under agitation solution 3-48h obtains solid containing spheroidizing
Determine the calcium chloride solution of enzyme；S3, to adding the PH of diatomite, control system to be in the calcium chloride solution containing spheroidizing immobilized enzyme
7.5-9.0, obtains mixed system, extracts the moisture in mixed system, and the moisture in control mixed system is not more than 40%；
S4, substrate is dissolved in the aqueous solution of dimethyl sulfoxide (DMSO), and adds isopropylamine hydrochloride to obtain reaction cushioning liquid；It is S5, mixed
Zoarium system is placed in isothermal reaction container, and reaction cushioning liquid is filled with isothermal reaction container simultaneously in the way of circulating
With mixed system reaction, Xi Gelieting is obtained；
Wherein, the structural formula of substrate is：。
Used as improved technical scheme of the invention, Enzyme catalyzed synthesis Xi Gelieting is used using Enzyme catalyzed synthesis Xi Gelieting
Device is carried out, the Enzyme catalyzed synthesis Xi Gelieting with device include constent temperature heater, reaction vessel, peristaltic pump, surge flask with
And magnetic stirring apparatus；Mixed system is equipped with reaction vessel, one end of reaction vessel is provided with import, and the other end is provided with outlet；Institute
Surge flask is stated on magnetic stirring apparatus, surge flask is built with reaction cushioning liquid；The import of reaction vessel uses peristaltic pump
Surge flask is communicated in built with buffer portion, the outlet of reaction vessel is communicated in surge flask on cushioning liquid by valve
The air part at end；Reaction vessel, peristaltic pump and surge flask form the circulatory system of closing；The reaction vessel is built in perseverance
In warm heater；Open peristaltic pump and realize that reaction cushioning liquid is circulated in the circulatory system.
Used as improved technical scheme of the invention, also including core filter screen, the core filter screen faces in reaction vessel
The position of nearly outlet.
Used as improved technical scheme of the invention, reaction vessel is column structure, and the footpath of the reaction vessel of column structure is high
Than being 1：3.5-5.0.
Used as improved technical scheme of the invention, the concentration of enzyme is 13.5-18g/L in the crude enzyme liquid.
As improved technical scheme of the invention, a diameter of 0.1-1.0mm of spheroidizing immobilized enzyme.
Used as improved technical scheme of the invention, the mass concentration of calcium chloride solution is 1%-15%.
As improved technical scheme of the invention, during to adding diatomite in the calcium chloride solution containing spheroidizing immobilized enzyme,
Diatomite is 1 with the mass ratio of spheroidizing immobilized enzyme：（0.15-1.8）.
Used as improved technical scheme of the invention, the mass content of dimethyl sulfoxide (DMSO) is in the aqueous solution of dimethyl sulfoxide (DMSO)
10%-70%。
As improved technical scheme of the invention, isopropylamine hydrochloride be diisopropylamine hydrochloride, isopropanolamine hydrochloride,
Triethylamine hydrochloride or triethanolamine hydrochloride.
The application synthesis Xi Gelieting reaction principle be：
Beneficial effect：
The application transaminase crude enzyme liquid, by the use of diatomite as reaction carriers, by immobilized enzyme scattered adsorption on carrier, substrate is molten
Liquid iterative cycles flow through reactor and are repeatedly fully contacted with enzyme.The reaction time is shortened, reactions steps are reduced, and can be after
Continuous supplement substrate cycle reaction, the recyclable activation cycle of carrier is used.So as to ensure that transformation efficiency higher, production has been saved
Cost, it is easy to industrialize.
Brief description of the drawings
Fig. 1 enzymic catalytic reactions of the invention synthesize the device of Xi Gelieting；
Fig. 2 substrate liquid chromatographic detection figures of the invention；
Xi Gelieting liquid chromatographic detection figures prepared by Fig. 3 present invention；
In figure：1st, reaction vessel；2nd, constent temperature heater；3rd, core filter screen；4 conduits；5th, peristaltic pump；6th, surge flask；7th, magnetic force is stirred
Mix device.
Specific embodiment
To make the purpose of the embodiment of the present invention and technical scheme clearer, below in conjunction with the embodiment of the present invention, to this
The technical scheme of invention is clearly and completely described.Obviously, described embodiment is a part of embodiment of the invention,
Rather than whole embodiments.Based on described embodiments of the invention, those of ordinary skill in the art are without creative
The every other embodiment obtained on the premise of work, belongs to the scope of protection of the invention.
Those skilled in the art of the present technique are appreciated that unless otherwise defined, all terms used herein（Including technology art
Language and scientific terminology）With with art of the present invention in those of ordinary skill general understanding identical meaning.Should also
Understand, those terms defined in such as general dictionary should be understood that the meaning having with the context of prior art
The consistent meaning of justice, and unless defined as here, will not be with idealizing or excessively formal implication be explained.
Embodiment：
The synthesis of the Xi Gelieting of the application is carried out using following device, the Enzyme catalyzed synthesis Xi Gelieting device bags
Include constent temperature heater 2, reaction vessel 1, peristaltic pump 5, surge flask 6 and magnetic stirring apparatus 7；Mixture is equipped with reaction vessel 1
System, one end of reaction vessel 1 is provided with import, and the other end is provided with outlet；The surge flask 6 is located on magnetic stirring apparatus 7, surge flask
6 built with reaction cushioning liquid；The import of reaction vessel 1 is communicated in surge flask 6 built with cushioning liquid portion using peristaltic pump 5
Point, the outlet of reaction vessel 1 is communicated in the air part of cushioning liquid upper end in surge flask 6 by valve；It is reaction vessel 1, compacted
Dynamic pump 5 and surge flask 6 form the circulatory system of closing；The reaction vessel 1 is built in constent temperature heater 2；Open and wriggle
Pump 5 realizes that reaction cushioning liquid is circulated in the circulatory system.More specifically there is the position being close to the exit in reaction vessel 1
It is additionally provided with core filter screen 3；Reaction is fully contacted in order to optimize material in cushioning liquid and reaction vessel 1, reaction vessel 1 is post
Shape structure, the blade diameter length ratio of the reaction vessel 1 of column structure is 1：3.5-5.0.
The synthesis route of Xi Gelieting is comprised the following steps：
A kind of method of Enzyme catalyzed synthesis Xi Gelieting, the transaminase somatic cells of S1, cultivation with catalysis activity, to transaminase
Somatic cells are crushed, aqueous suspension, centrifugation obtain crude enzyme liquid；S2, it is configured to using homeo-osmosis agent and sodium chloride solution
Buffer solvent containing homeo-osmosis agent, to adding sodium alginate to obtain sodium alginate soln in buffer solvent, takes and marine alga
The isometric crude enzyme liquid of acid sodium solution, the crude enzyme liquid that will be taken is well mixed with sodium alginate soln and obtains mixed liquor, will mix
Liquid is added dropwise in calcium chloride solution, while also to adding phosphopyridoxal pyridoxal phosphate to obtain blend solution in calcium chloride solution, stirring is altogether
Miscible fluid 3-48h, obtains the calcium chloride solution containing spheroidizing immobilized enzyme；S3, in the calcium chloride solution containing spheroidizing immobilized enzyme
Diatomite is added, the PH of control system is 7.5-9.0, obtains mixed system, extracts the moisture in mixed system, controls mixture
Moisture in system is not more than 40%；S4, substrate is dissolved in the aqueous solution of dimethyl sulfoxide (DMSO), and adds isopropylamine hydrochloride
Obtain reaction cushioning liquid；S5, mixed system are placed in isothermal reaction container 1, by reaction cushioning liquid circulating
Mode be filled with isothermal reaction container 1 and with mixed system reaction, obtain Xi Gelieting；Surge flask 6 is taken out after completion of the reaction
Middle material adjusts the steps such as pH value, extraction, washing and obtains Xi Gelieting, and yield is up to 72.3%.Reaction can also be to slow after terminating
Rush and new substrate solution is added in bottle 6, continue cycling through reaction, until enzymatic activity disappears；Diatomite is with dilute in taking out reaction vessel 1
Acid steeps, is washed to neutral, drying for recycling.
This method have the advantage that making full use of the stability and diatomaceous adsorptivity of immobilized enzyme, it is ensured that high efficiency is urged
Change so that enzymatic activity time lengthening.
Wherein, the structural formula of substrate is：。
Wherein, the concentration of enzyme is 13.5-18g/L in the crude enzyme liquid.
Wherein, a diameter of 0.1-1.0mm of spheroidizing immobilized enzyme.
Wherein, the mass concentration of calcium chloride solution is 1%-15%.
When wherein, to diatomite is added in the calcium chloride solution containing spheroidizing immobilized enzyme, diatomite and spheroidizing immobilized enzyme
Mass ratio be 1：（0.15-1.8）.
Wherein, the mass content of dimethyl sulfoxide (DMSO) is 10%-70% in the aqueous solution of dimethyl sulfoxide (DMSO).
Wherein, isopropylamine hydrochloride is diisopropylamine hydrochloride, isopropanolamine hydrochloride, triethylamine hydrochloride or three second
Alcohol amine hydrochlorate.
It is specifically：
Prepare catalyst（Transaminase）：By one kind self-control X5 strains（Zhejiang University Wang Lu is in master's thesis of 2014
" high efficiency recombinant expressed and catalysis of the transaminase ATA117 in Escherichia coli prepares Xi Gelieting " is disclosed）It is placed in after activated
13L zymotic fluids containing transaminase are obtained after culture 48h, and induction are carried out in 20L fermentation tanks, zymotic fluid detection is taken, 5 times of dilution is measured
OD600 >=4.5.Zymotic fluid centrifugation is obtained into mycelium 205g, broken, 400ml purifying aqueous suspension, that centrifugation obtains supernatant is thick
Enzyme liquid（Enzyme concentration about 13.5-18g/L）.
Enzyme immobilizatio：To 2g inoses alcohol and 5g sodium chloride, 100g sodium alginates is added in 200ml deionized waters, stir
Uniformly, then by 300ml crude enzyme liquids mixed stirring half an hour, by the calcium chloride of drop mixed above to 80g/300ml,
The phosphopyridoxal pyridoxal phosphate of 0.58g is added, is slowly stirred 20-34 hours.
The filling of immobilised enzymes：To 800g diatomite is added in the system after enzyme immobilization, reaction is added after being well mixed
In container 1, vavuum pump is used（Vacuum≤0.03）Post lower port is connected to, extraction section moisture is no more than filler moisture
40% 。
The preparation of cushioning liquid：Take the DMSO that 100g substrates are dissolved in 600mL（Dimethyl sulfoxide (DMSO)）In, 75g isopropylamine hydrochloric acid
Salt is dissolved in 300ml water, merge add surge flask 6 in and stir；
The fixation and reaction of device：Populated reaction vessel 1 is vertically put into constent temperature heater 2（It is set to 45 DEG C）, use
Conduit 4 is sequentially connected the import of reaction vessel 1, surge flask 6, peristaltic pump 5, the outlet of reaction vessel 1, composition closed circulation system
System, keeps constant temperature and opens peristaltic pump 5 and react.
Xi Gelieting is extracted：Reaction takes sample TLC detection (methyl alcohol in surge flask 6 after about 48 hours：Ethyl acetate：Just oneself
Alkane=1：3：3).Reaction is finished, and solution in surge flask 6 is poured out, then to 200mlDMSO is added in surge flask 6, opens peristaltic pump
5, circulate one hour, the entrance of surge flask 6 is disconnected, the liquid that will be collected into merges with solution before, adds 200ml water, adjusts molten
To 2,200 × 2 ethyl acetate extraction, water mutually adjusts pH value to 11, the extraction of 200 × 2 ethyl acetate, 200ml washings one to liquid pH value
Secondary, semi-saturation sodium chloride is washed once, 5 hours of anhydrous sodium sulfate drying, is concentrated to give white solid (Xi Gelieting) 83.5g,
Liquid phase detection purity is 91.3%, and conversion ratio is about 72%
Liquid phase color detection method is as shown in Figure 3 with spectrogram：
High performance liquid chromatograph：Shimadzu AAT-20 types, column temperature：30℃；
Detector：Ultraviolet detection/wavelength 267nm, flow velocity：1.0mL/min；
Chromatographic column：SB-C18 4.6*150mm；5um；
Dilution：Acetonitrile (HPLC)；
Standard items：Precursor ketone, X5, ethyl acetate, DMSO etc.；10mg/mL；
Mobile phase：A：Water containing 0.1% trifluoroacetic acid (TFA)；B：Acetonitrile containing 0.1%TFA；
The design of binary concentration gradient is shown in Table 1.
The HPLC binary concentration gradients of table 1
Claims (10)
1. a kind of method of Enzyme catalyzed synthesis Xi Gelieting, it is characterised in that comprise the following steps：There is catalysis to live for S1, cultivation
Transaminase somatic cells are crushed, aqueous suspension, centrifugation obtain crude enzyme liquid by the transaminase somatic cells of property；S2, using infiltration
Pressure stabilizer and sodium chloride solution are configured to the buffer solvent containing homeo-osmosis agent, to adding sodium alginate in buffer solvent
Sodium alginate soln is obtained, the crude enzyme liquid isometric with sodium alginate soln is taken, the crude enzyme liquid and sodium alginate soln that will be taken
It is well mixed to obtain mixed liquor, mixed liquor is added dropwise in calcium chloride solution, while also to adding phosphoric acid in calcium chloride solution
Pyridoxal obtains blend solution, and blended under agitation solution 3-48h obtains the calcium chloride solution containing spheroidizing immobilized enzyme；S3, Xiang Hanqiu
Diatomite is added in the calcium chloride solution of shape immobilized enzyme, the PH of control system is 7.5-9.0, obtains mixed system, extracted mixed
Moisture in zoarium system, the moisture in control mixed system is not more than 40%；S4, the water that substrate is dissolved in dimethyl sulfoxide (DMSO)
In solution, and isopropylamine hydrochloride is added to obtain reaction cushioning liquid；S5, mixed system are placed in isothermal reaction container, will
Reaction cushioning liquid is filled with isothermal reaction container in the way of circulating and is reacted with mixed system, obtains Xi Gelie
Spit of fland；Wherein, the structural formula of substrate is：。
2. a kind of method of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that Enzyme catalyzed synthesis west
Ge Lieting is carried out with device using Enzyme catalyzed synthesis Xi Gelieting, and the Enzyme catalyzed synthesis Xi Gelieting includes constant temperature with device
Heater, reaction vessel, peristaltic pump, surge flask and magnetic stirring apparatus；Mixed system is equipped with reaction vessel, reaction vessel
One end is provided with import, and the other end is provided with outlet；On magnetic stirring apparatus, surge flask is buffered the surge flask built with reaction
Solution；The import of reaction vessel is communicated in surge flask built with buffer portion using peristaltic pump, and the outlet of reaction vessel is passed through
Cross the air part that valve is communicated in cushioning liquid upper end in surge flask；Reaction vessel, peristaltic pump and surge flask form closing
The circulatory system；The reaction vessel is built in constent temperature heater；Opening peristaltic pump realizes reaction cushioning liquid in circulation
Circulated in system.
3. the method for a kind of Enzyme catalyzed synthesis Xi Gelieting according to claim 2, it is characterised in that also filtered including core
Net, the position that the core filter screen is close to the exit in reaction vessel.
4. the method for a kind of Enzyme catalyzed synthesis Xi Gelieting according to claim 2, it is characterised in that reaction vessel is post
Shape structure, the blade diameter length ratio of the reaction vessel of column structure is 1：(3.5-5.0).
5. the method for a kind of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that in the crude enzyme liquid
The concentration of enzyme is 13.5-18g/L, and the mass concentration of calcium chloride solution is 1%-15%.
6. a kind of method of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that spheroidizing immobilized enzyme
A diameter of 0.1-1.0mm.
7. a kind of method of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that homeo-osmosis agent
It is inose alcohol.
8. the method for a kind of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that to solid containing spheroidizing
When determining to add diatomite in the calcium chloride solution of enzyme, diatomite is 1 with the mass ratio of spheroidizing immobilized enzyme：（0.15-1.8）.
9. the method for a kind of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that dimethyl sulfoxide (DMSO)
The mass content of dimethyl sulfoxide (DMSO) is 10%-70% in the aqueous solution.
10. a kind of method of Enzyme catalyzed synthesis Xi Gelieting according to claim 1, it is characterised in that isopropylamine hydrochloric acid
Salt is diisopropylamine hydrochloride, isopropanolamine hydrochloride, triethylamine hydrochloride or triethanolamine hydrochloride.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109082450A (en) * | 2018-08-07 | 2018-12-25 | 江苏恒盛药业有限公司 | A method of applying continuous Flow Technique production Xi Gelieting free alkali |
| CN110791538A (en) * | 2019-11-14 | 2020-02-14 | 湖北省宏源药业科技股份有限公司 | Production method suitable for synthesizing sitagliptin phosphate by enzyme method |
| CN112852620A (en) * | 2020-12-30 | 2021-05-28 | 苏州汇通色谱分离纯化有限公司 | Universal quasi-immobilized enzyme reactor |
| CN113943292A (en) * | 2020-07-15 | 2022-01-18 | 浙江医药股份有限公司新昌制药厂 | Recovery method of sitagliptin phosphate key intermediate |
| CN117467733A (en) * | 2023-12-27 | 2024-01-30 | 北京元延医药科技股份有限公司 | High chiral purity sitagliptin and method for preparing same by using immobilized transaminase |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109082450A (en) * | 2018-08-07 | 2018-12-25 | 江苏恒盛药业有限公司 | A method of applying continuous Flow Technique production Xi Gelieting free alkali |
| CN109082450B (en) * | 2018-08-07 | 2021-10-26 | 江苏恒盛药业有限公司 | Method for producing sitagliptin free base by using continuous flow technology |
| CN110791538A (en) * | 2019-11-14 | 2020-02-14 | 湖北省宏源药业科技股份有限公司 | Production method suitable for synthesizing sitagliptin phosphate by enzyme method |
| CN113943292A (en) * | 2020-07-15 | 2022-01-18 | 浙江医药股份有限公司新昌制药厂 | Recovery method of sitagliptin phosphate key intermediate |
| CN113943292B (en) * | 2020-07-15 | 2023-04-14 | 浙江医药股份有限公司新昌制药厂 | Recovery method of sitagliptin phosphate key intermediate |
| CN112852620A (en) * | 2020-12-30 | 2021-05-28 | 苏州汇通色谱分离纯化有限公司 | Universal quasi-immobilized enzyme reactor |
| CN112852620B (en) * | 2020-12-30 | 2023-11-21 | 苏州汇通色谱分离纯化有限公司 | Universal quasi-immobilized enzyme reactor |
| CN117467733A (en) * | 2023-12-27 | 2024-01-30 | 北京元延医药科技股份有限公司 | High chiral purity sitagliptin and method for preparing same by using immobilized transaminase |
| CN117467733B (en) * | 2023-12-27 | 2024-03-12 | 北京元延医药科技股份有限公司 | High chiral purity sitagliptin and method for preparing same by using immobilized transaminase |
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