CN108220358A - The preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines - Google Patents
The preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines Download PDFInfo
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Abstract
The present invention discloses one kind（S）The preparation method of 1 tertbutyloxycarbonyl, 3 hydroxy piperidine, i.e., using Pichia yeastPichia Sp. SIT2014 is catalyzed 1 tertbutyloxycarbonyl, 3 piperidones as biocatalyst and carries out asymmetric reduction reaction generation（S）1 tertbutyloxycarbonyl, 3 hydroxy piperidine.The preparation method has the advantages of high conversion rate, optical purity are high, and total recovery is high, final gained（S）The yield of 1 tertbutyloxycarbonyl, 3 hydroxy piperidine reaches as high as 99.9%, and for optical purity more than 99%, total recovery is up to 95.1%.And the preparation process reaction time is short, and mild condition need not add a large amount of organic solvents, and production cost is low, is conducive to further industrialized production.
Description
Technical field
The present invention relates to a kind of (S) -1- is prepared by the use of Pichia yeast Pichia sp.SIT2014 as biocatalyst
The method of tertbutyloxycarbonyl -3- hydroxy piperidines.
Background technology
Chipal compounds have important role in human lives, since two kinds of enantiomers of same compound are in medicine
All there are huge difference in reason, toxicity and function, therefore optically pure chiral building block compound is prepared in medicine, agriculture
The fields such as medicine, environmental protection, material all have great importance.
(S) -1- tertbutyloxycarbonyls -3- hydroxy piperidines, structural formula are as follows：
It is that Rayleigh, BTK inhibitor do not replace woods, natural goods to synthesis non-natural drug congestion heart failure medicine card according to Shandong
The different white thorn quinoline leaching prodrugs such as amine and nitraria schoberi part of matter, therefore the compound is with a wide range of applications.
The synthesis of (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines can be divided into two kinds at present.
One kind is chemical resolution method.Chemical method is to obtain racemization 3- through 5% rhodium charcoal catalytic hydrogenating reduction by 3- pyridones
Hydroxy piperidine, then split, then three through D- tartaric acid derivatives (2S, 3S)-N- (4- chlorphenyls) -2,3- dihydroxy succinamic acid
(S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines [Shen Weiyi is made in lower reacted with di-tert-butyl dicarbonate " one kettle way " of ethamine effect
Deng, Chinese Journal of Pharmaceuticals, 2013,44 (5), 436-438], this method yield only up to 40%.There are yields for chemical resolution method
Low, the shortcomings of unit operation is more, of high cost.
Another kind is also to be obtained the tertiary fourths of optically pure 1- originally to 1- tertbutyloxycarbonyl -3- piperidones by reductase
Oxygen carbonyl -3- hydroxy piperidines.Zhu Wei etc. first passes through chemical method synthesis 1- tertbutyloxycarbonyl -3- piperidones, then by commercialized
Ketoreductase it is expeditiously realized asymmetric reduction [Zhu Wei, Wang Bo, Wu Hui, Li Binghao, Chinese Journal of Pharmaceuticals,
2015,46 (4), 349-350].Product (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines optical purity is more than 99%, yield
71%.But the preparation process needs to add additional glucose dehydrogenase enzyme powder glucose to be utilized to carry out coenzyme NADP 11 regeneration,
And commercialized glucose dehydrogenase enzyme preparation is expensive, limits the practicability of the catalytic process to a certain extent.
In addition to this, there is document report using 1- tertbutyloxycarbonyl -3- piperidones in the Carrot Roots mashed
(Daucuscarota) (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, yield 73%, ee values 95% can be converted under being catalyzed
[RomainLacheretz, Domingo Gomez Pardo, Janine Cossy, ORGANIC LETTERS, 2009,6 (11),
1245-1248], but since wild Daucuscarota is not easy largely to obtain, and product chiral purity is relatively low, this law does not have
Actual application value.
Bringing up within 2013 prosperous grade, [Ju Xin, Guo Hao, Li Bin, Tang are round, a kind of biological preparation side of chirality N-protected piperidine alcohols
Method, Chinese patent, 104059952 A of publication number CN] using reductase enzyme powder is recombinated, it can be with high yield, highly selective catalysis
1- tertbutyloxycarbonyl -3- piperidones is reduced to (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines.But genetic engineering bacterium was passing on
There are plasmid loss in journey to cause the poor risk of its mitotic stability, needs the derivant for adding in costliness different in incubation
Propyl-β-D- thiogalactosides, the addition of antibiotic cause the environmental improvement cost of fermented waste fluid, and the preparation of enzyme powder
Process is numerous and diverse.
Invention content
Present invention aims in order to solve to deposit in the preparation process of above-mentioned (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines
Catalyst it is at high price, catalytic activity is low, needs to add expensive co-factor or structure regenerating coenzyme system to co-factor
It carries out the technical problems such as in-situ regeneration and the preparation methods of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines is provided, the preparation
The conversion ratio of the final 1- tertbutyloxycarbonyls -3- piperidones of method is up to more than 99%, and optical purity is up to more than 99%, the reaction time
Short, reaction condition is mild, and overall yield of reaction does not need to the expensive co-factor of addition up to 95.1%.
Technical scheme of the present invention
The preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, i.e., using Pichia yeast Pichia
For sp.SIT2014 as biocatalyst, it is not right to be carried out controlled at 20-50 DEG C of catalysis 1- tertbutyloxycarbonyl -3- piperidones
Claim reduction reaction 1-36h, obtained reaction solution is centrifuged, extracts successively, anhydrous sodium sulfate is dry and revolving processing to get
(S) -1- tertbutyloxycarbonyls -3- hydroxy piperidine crude products, the equation of reaction process are as follows：
The Pichia pastoris Pichia sp.SIT2014, are a kind of bacterial strains for belonging to fungi Parasaccharomyces, the bacterial strain
It is isolated from Chinese Soils of Shanghai Region, entrusted on 06 16th, 2014 in Chinese microorganism strain preservation management
Member can common micro-organisms center preservation, deposit number be CGMCC NO.9300, preservation organization address：The Chaoyang District, Beijing City North Star
The Institute of Microorganism, Academia Sinica of institute of West Road 1, postcode：100101.
The preparation method of above-mentioned one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specifically comprises the following steps：
(1), the preparation of Pichia yeast Pichia sp.SIT2014 cells
The Pichia pastoris Pichia sp.SIT2014 slant strains that 4 DEG C of refrigerators is taken to preserve, one ring strain of picking are linked into dress
Have in the 100mL shaking flasks of 15mL seed culture mediums, seed culture is carried out for 180rpm controlled at 30 DEG C, rotating speed on shaking table
13-15 h, obtain seed liquor；
Then seed liquor is inoculated into fermentation medium, the amount of seed liquor used is inoculated with, by zymotic fluid after inoculation
OD600=0.1 ratio calculates；
After inoculation, fermented and cultured is carried out for 180rpm for 24 hours controlled at 30 DEG C, rotating speed on shaking table, obtain zymotic fluid；
Above-described seed culture medium, fermentation medium are identical, by every liter of calculating, 15g containing glucose, and tryptone
5g, dusty yeast 5g, K2HPO40.5g, KH2PO40.5g, NaCl 1g, MgSO4·7H2O 0.5g, surplus are water；
The zymotic fluid of gained is centrifuged, the precipitation of gained is Pichia yeast Pichia sp.SIT2014 thin
Born of the same parents；
(2), the Pichia yeast Pichia sp.SIT2014 cells obtained by step (1) are placed in and mixing in solution are resuspended,
Obtain Pichia yeast Pichiasp.SIT2014 suspensions；
The resuspension solution be by buffer solution, methanol and glucose group into mixed liquor, be resuspended solution in methanol use
Amount, calculates, buffer solution by volume：Methanol is 95：5；The dosage of glucose, calculates in mass ratio, glucose：(buffer solution and
Methanol) it is 5：100；
The buffer solution is pH 5.0~9.0, preferably pH 6.0~8.0, and the sodium phosphate of more preferably pH 6.5 is water-soluble
Liquid；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichiasp.SIT2014
Cell and the amount that solution is resuspended, by Pichia yeast Pichiasp.SIT2014 dry cell weights：Resuspension solution is 1-20g：1L,
Preferably 10 g：The ratio of 1L calculates；
(3), 1- tertiary butyloxycarbonyls are added in into the Pichia yeast Pichia sp.SIT2014 suspensions obtained by step (2)
Base -3- piperidones, after mixing controlled at 20-50 DEG C, rotating speed carries out asymmetric reduction reaction 1- for 180r/min
36h obtains reaction solution；
The addition of above-mentioned 1- tertbutyloxycarbonyls -3- piperidones, by Pichia yeast Pichia sp.SIT2014 suspensions
Middle Pichia yeast Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 1-20g：50mmol, it is excellent
It is selected as 10g：The ratio of 50mmol calculates；
(4), the reaction solution of step (3) is centrifuged, obtained supernatant is extracted with isometric ethyl acetate
Twice, merge the extract liquor of extraction gained twice, then dried with anhydrous sodium sulfate, be then evaporated again with Rotary Evaporators, i.e.,
Obtain (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidine crude products；
The centrifugation preferably controls rotating speed to carry out 10 min of centrifugation for 10000r/min.
The yield of (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines of above-mentioned final gained reaches as high as 99.9%, optical voidness
Degree is more than 99%, and total recovery is up to 95.1%.
Beneficial effects of the present invention
The preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines of the present invention, due to the Pichia pastoris of selection
Bacterium Pichia sp.SIT2014, are calculated by stem cell, every gram of dry Pichia yeast Pichia sp.SIT2014 cells production carbonyl
Reductase vitality is about 22-25U, i.e., it has stronger reproducibility and selectivity to aryl hydroxy-ketone.Therefore it can be used for urging
Change 1- tertbutyloxycarbonyl -3- piperidones and carry out the anti-of asymmetric reduction reaction generation (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines
It should.
Further, the preparation method of one kind of the invention (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, due to preparation process
The Pichia yeast Pichia sp.SIT2014 cells of middle use have stronger reproducibility and selectivity to aryl hydroxy-ketone,
Therefore 1- tertbutyloxycarbonyl -3- piperidones is catalyzed using Pichia yeast Pichia sp. SIT2014 as biocatalyst to carry out
The reaction of asymmetric reduction reaction generation (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines has high conversion rate, optical purity height, always
The advantages of high income, up to 99%, optical purity exists the yield of (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines of final gained
More than 99%, total recovery is up to 95.1%.
Further, the preparation method of one kind of the invention (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, due to entirely not right
Temperature into reduction reaction is 20-50 DEG C, and the reaction time only needs 1-36h, therefore the preparation method has the reaction time short, reacts
The characteristics of mild condition.
Further, the preparation method of one kind of the invention (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, due to life used
Object catalyst Pichia yeast Pichiasp.SIT2014, culture is convenient, and raw material is easy to get, and reaction process is without a large amount of organic molten
Agent so as to reduce production cost, is conducive to further industrialized production.
Description of the drawings
Fig. 1, differential responses temperature, differential responses time correspond to the conversion ratio feelings of substrate 1- tertbutyloxycarbonyl -3- piperidones
Condition curve.
Specific embodiment
The invention will now be described in further detail by way of examples and with reference to the accompanying drawings, but does not limit the present invention.
Reagent used in various embodiments of the present invention is all the analysis pure specifications of pure or HPLC.
Pichia yeast Pichiasp.SIT2014 dry cell weights are according to the OD of zymotic fluid in various embodiments of the present invention600
It calculates, according to experiment, measures the OD of zymotic fluid600With Pichia yeast Pichiasp.SIT2014 dry cell weights relational expression such as
Under：
Pichia yeast Pichiasp.SIT2014 dry cell weights=0.7660*OD600。
Gas is passed through to 1- tertbutyloxycarbonyl -3- piperidones asymmetric reduction reaction conversion ratios etc. in various embodiments of the present invention
Phase chromatography (GC2010, Shimadzu Corporation) is analyzed, and gas-chromatography pillar is 120 Capillary Column of Beta-DEX
(30m*0.25mm*0.25um, RS-Supelco company)
The conversion ratio of 1- tertbutyloxycarbonyl -3- piperidones calculates as follows:
Conversion ratio=(A0-A1)/A0* 100%
Wherein, A0For blank sample 1- tertbutyloxycarbonyl -3- piperidones peak areas, i.e., mixed in preparation process step (3)
1- tertbutyloxycarbonyl -3- piperidones is added in liquid but without the 1- tertbutyloxycarbonyl -3- piperidones peak areas before reaction；A1
Remaining 1- tertbutyloxycarbonyls -3- piperidones peak areas in the reaction solution of gained after being reacted for preparation process step (3).
In various embodiments of the present invention the yield of (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines according to actually generate product (S) -
The quality of 1- tertbutyloxycarbonyl -3- hydroxy piperidines divided by the quality of theoretical product (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines are come
It calculates；
The definition of signified cellular carbonyl reductase vitality unit is in the embodiment of the present invention：At 30 DEG C, the item of pH 6.5
Under part, (S) -1- tertbutyloxycarbonyls -3- of 1.0 μm of ol of catalysis substrate 1- tertbutyloxycarbonyl -3- piperidones reduction generation per minute
The enzyme amount of cellular carbonyl reductase needed for hydroxy piperidine is 1 unit of activity, i.e. 1U.
The detection of the optical voidness angle value of product (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines uses Shimadzu high performance liquid chromatography
Instrument LC-20A, from Shimadzu instrument company；Condition is as follows used by chromatography：
Mobile phase is n-hexane：Isopropanol=97:3, flow velocity 0.8mL/min, Detection wavelength be 210 nm, column temperature 27
DEG C, using chiral column, (4.6mm × 250mm, CHIRALCEL OD-H are purchased from Daicel medicine company chiral technology (Shanghai) limited public affairs
Department).
The optical purity ee value calculating methods of product (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines are as follows:
Ee (%)=[S (configuration)-R (configuration)]/[S (configuration)+R (configuration)] × 100%
S (configuration)：Represent the concentration of S (configuration) product enantiomer；
R (configuration)：Represent the concentration of R (configuration) product enantiomer.
Embodiment 1
The culture of Pichia yeast Pichia sp.SIT2014 cells：
Slant medium used calculates, 15.0g containing glucose, tryptone 5.0 g, dusty yeast 5.0g by every liter,
KH2PO40.5g,K2HPO40.5g, MgSO4·7H2O 0.5g, agar 20.0g, surplus are water；
Seed culture medium used calculates, 15.0g containing glucose, tryptone 5.0 g, dusty yeast 5.0g by every liter,
KH2PO40.5g,K2HPO40.5g, MgSO4·7H2O 0.5g, surplus are water；
Fermentation medium used calculates, 15.0g containing glucose, tryptone 5.0g, dusty yeast 5.0g by every liter,
KH2PO40.5g,K2HPO40.5g, MgSO4·7H2O 0.5g, surplus are water.
The Pichia pastoris Pichia sp.SIT2014 slant strains that 4 DEG C of refrigerators is taken to preserve, one ring strain of picking are linked into dress
Have in the 100mL shaking flasks of 15mL seed culture mediums, seed culture is carried out for 180rpm controlled at 30 DEG C, rotating speed on shaking table
15h obtains seed liquor；
Then seed liquor is inoculated into fermentation medium, the amount of seed liquor used is inoculated with, by zymotic fluid after inoculation
OD600=0.1 ratio calculates；
After inoculation, fermented and cultured is carried out for 180rpm for 24 hours controlled at 30 DEG C, rotating speed on shaking table, obtain zymotic fluid；
The zymotic fluid control rotating speed of gained is subjected to centrifugation 8min for 10000r/min, the precipitation of gained is Pichia pastoris
Bacterium Pichia sp.SIT2014 cells.
After testing, Pichia yeast Pichia sp.SIT2014 cells produce carbonyl reduction enzyme activity to the zymotic fluid of above-mentioned gained
Power is about 220~230U/L, and Pichia yeast Pichia sp. SIT2014 cell concentrations press Pichia yeast in zymotic fluid
Pichia sp.SIT2014 dry cell weights calculate about 8~9g/L, i.e. unit Pichia yeast Pichia sp.SIT2014 cells
It is about 22-25U/g stem cells to produce carbonyl reduction enzyme activity.
The above results show that Pichia yeast Pichia sp.SIT2014 cells have aryl hydroxy-ketone stronger go back
Originality and selectivity.
Application Example 1
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), the Pichia yeast Pichia sp.SIT2014 cells of the gained of embodiment 1 are respectively placed in be resuspended solution 1,
Solution 2 is resuspended, is resuspended in solution 3, respectively obtains corresponding resuspension solution 1, solution 2 is resuspended and the Pichia yeast of solution 3 is resuspended
Pichiasp.SIT2014 suspensions；
The resuspension solution 1 is buffer solution, and the buffer solution is the sodium phosphate aqueous solution of pH 7.0；
The resuspension solution 2 is the mixed liquor being made of buffer solution and methanol, and the buffer solution is the phosphorus of pH 7.0
Acid sodium aqueous solution；The dosage of methanol in solution 2 is resuspended, calculates by volume, buffer solution：Methanol is 95：5；
It is described be resuspended solution 3 be by buffer solution, methanol and glucose group into mixed liquor, the buffer solution is pH
7.0 sodium phosphate aqueous solution；The dosage of methanol in solution 3 is resuspended, calculates by volume, buffer solution：Methanol is 95：5；It is resuspended molten
The dosage of glucose, calculates in mass ratio in liquid 3, glucose：(buffer solution and methanol) is 5：100；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned corresponding resuspension solution 1, Pichia yeast Pichia
Sp.SIT2014 cells and the dosage that solution 1 is resuspended, by Pichia yeast Pichia sp.SIT2014 dry cell weights：It is resuspended molten
Liquid 1 is 10g：The ratio of 1L calculates；
In the Pichia yeast Pichia sp.SIT2014 suspensions of above-mentioned corresponding resuspension solution 2, Pichia yeast Pichia
Sp.SIT2014 cells and the dosage that solution 2 is resuspended, by Pichia yeast Pichia sp.SIT2014 dry cell weights：It is resuspended molten
Liquid 2 is 10g：The ratio of 1L calculates；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned corresponding resuspension solution 3, Pichia yeast Pichia
Sp.SIT2014 cells and the dosage that solution 3 is resuspended, by Pichia yeast Pichia sp.SIT2014 dry cell weights：It is resuspended molten
Liquid 3 is 10g：The ratio of 1L calculates；
(2), to the corresponding Pichia yeast that solution 1, resuspension solution 2 and resuspension solution 3 is resuspended obtained by step (1)
1- tertbutyloxycarbonyl -3- piperidones is separately added into Pichia sp.SIT2014 suspensions, after mixing controlled at 30
DEG C, rotating speed carries out asymmetric reduction reaction 20h for 180r/min, respectively obtains corresponding resuspension solution 1, solution 2 is resuspended and is resuspended
The reaction solution of solution 3；
The above-mentioned corresponding Pichia yeast Pichiasp.SIT2014 that solution 1 is resuspended, solution 2 is resuspended and solution 3 is resuspended hangs
The addition of 1- tertbutyloxycarbonyl -3- piperidones in liquid, it is red by finishing in Pichia yeast Pichia sp.SIT2014 suspensions
Saccharomycete Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 10g：The ratio meter of 50mmol
It calculates；
(3), by the corresponding reaction solution that solution 1 is resuspended, solution 2 is resuspended and solution 3 is resuspended obtained by step (2) respectively into
Row centrifuges, and corresponding to for obtaining is resuspended solution 1, solution 2 is resuspended and the supernatant of solution 3 is resuspended respectively with isometric acetic acid
Ethyl ester is extracted twice, and is merged the extract liquor of extraction gained twice, is then dried respectively with anhydrous sodium sulfate, gas-chromatography
(GC2010, Shimadzu Corporation) carries out the conversion ratio of 1- tertbutyloxycarbonyls -3- piperidones in analysis different solutions.
Corresponding above-mentioned resuspension solution 1 is resuspended solution 2 and solution 3 is resuspended, and utilizes the Pichia yeast of 1 gained of embodiment
Pichia sp.SIT2014 carry out asymmetric reduction reaction system as biocatalyst catalysis 1- tertbutyloxycarbonyl -3- piperidones
Standby (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, the conversion ratio of final substrate 1- tertbutyloxycarbonyls -3- piperidones see the table below：
As can be seen from the above table, when being resuspended in solution containing cosolvent first alcohol and glucose, it is right to significantly improve
Into the conversion ratio of reduction reaction substrate 1- tertbutyloxycarbonyl -3- piperidones, this, which is primarily due to methanol, can promote substrate uncle 1-
The dissolving of butoxy carbonyl -3- piperidones, and on the one hand glucose has intracellular enzyme certain protective effect, meanwhile, for
Cell interior cofactor regeneration process has certain facilitation.
Application Example 2
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), by the Pichia yeast Pichia sp.SIT2014 cells of the gained of embodiment 1 be respectively placed in 9 parts it is different
It is resuspended in solution, respectively obtains the Pichia yeast Pichia sp.SIT2014 suspensions of corresponding different resuspension solution；
Described 9 parts of resuspensions solution, be by buffer solution, methanol and glucose group into mixed liquor；
Above-mentioned every part is resuspended in solution, and the dosage of methanol calculates, buffer solution by volume：Methanol is 95：5；Grape
The dosage of sugar, calculates, glucose in mass ratio：(buffer solution and methanol) is 5：100；
The buffer solutions of 9 parts of resuspension solution of above-mentioned correspondence be respectively pH5.0,5.5,6.0 sodium citrate aqueous solution,
Sodium phosphate aqueous solution, pH8.0, the 9.0 Tris-HCl solution of pH6.0,6.5,7.0,8.0；
The correspondence of above-mentioned gained is different to be resuspended in the Pichia yeast Pichia sp. SIT2014 suspensions of solution, Bi Chi
Saccharomycete Pichia sp.SIT2014 cells and the amount that solution is resuspended, are done by Pichia yeast Pichiasp.SIT2014 cells
Weight：Solution is 10g：The ratio of 1L calculates；
(2), the different Pichia yeast that solution is resuspended of 9 parts of correspondences of gained into step (1)
1- tertbutyloxycarbonyl -3- piperidones is separately added into Pichiasp.SIT2014 suspensions, controls temperature respectively again after mixing
It it is 30 DEG C, rotating speed carries out asymmetric reduction reaction 20h for 180r/min, obtains 9 parts of reaction solutions；
The tertiary fourths of 1- in the Pichia yeast Pichia sp. SIT2014 suspensions of the different resuspension solution of above-mentioned 9 parts of correspondences
The addition of oxygen carbonyl -3- piperidones, by Pichia yeast Pichia in Pichia yeast Pichia sp.SIT2014 suspensions
Sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 10g：The ratio of 50mmol calculates；
(3), 9 parts of reaction solutions of step (2) are centrifuged respectively, obtained supernatant is respectively with isometric second
Acetoacetic ester is extracted twice, and is merged the extract liquor of extraction gained twice, is then dried respectively with anhydrous sodium sulfate, respectively gas-chromatography
(GC2010, Shimadzu Corporation) is analyzed in the asymmetric reduction reaction, and the buffer solution of difference pH in solution is resuspended to the tertiary fourths of substrate 1-
The influence of the conversion ratio of oxygen carbonyl -3- piperidones, as a result see the table below：
As can be seen from the above table, the substrate 1- tertbutyloxycarbonyls -3- piperidones in the range of the pH of buffer described in upper table
Different degrees of asymmetric reduction reaction can occur, preferable reaction effect is in pH 6.0-8.0, and best is pH 6.5
Sodium phosphate aqueous solution.
Application Example 3
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), the Pichia yeast Pichia sp.SIT2014 cells of 1 gained of embodiment are placed in and be resuspended in solution, obtained
Pichia yeast Pichia sp.SIT2014 suspensions；
It is described be resuspended solution for buffer solution, methanol and glucose group into mixed liquor, the dosage of methanol in solution is resuspended,
It calculates by volume, buffer solution：Methanol is 95：5；The dosage of glucose in solutions is resuspended, calculates in mass ratio, glucose：It is (slow
Fliud flushing and methanol) it is 5：100；The buffer solution is the sodium phosphate aqueous solution of pH 6.5；
In the Pichia yeast Pichia sp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichia
The amount of sp.SIT2014 cells and buffer solution, by Pichia yeast Pichia sp.SIT2014 dry cell weights：Buffer solution is
10g：The ratio of 1L calculates；
(2), the Pichia yeast Pichia sp.SIT2014 suspensions obtained by step (1) are divided into quarter；
1- tertbutyloxycarbonyl -3- piperidones, mixing are added in into every part of Pichia yeast Pichia sp.SIT2014 suspension
Again respectively controlled at 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C after uniformly, rotating speed carries out asymmetric reduction reaction 1- for 180r/min
36h obtains reaction solution；
The addition of 1- tertbutyloxycarbonyl -3- piperidones in above-mentioned every part of Pichia yeast Pichiasp.SIT2014 suspensions
Amount, by Pichia yeast Pichia sp.SIT2014 dry cell weights in Pichia yeast Pichia sp.SIT2014 suspensions：
1- tertbutyloxycarbonyl -3- piperidones is 10g：The ratio of 50mmol calculates；
(3), during step (2) asymmetric reduction reaction, each reaction system reaction 1h, 4h, 8h, 12h, 16h,
20h, 25h, 30h, 36h extract reaction solution sample respectively, and after being extracted twice respectively with isometric ethyl acetate, merging extracts institute twice
Extract liquor, then dried with anhydrous sodium sulfate, be then evaporated again to get corresponding differential responses temperature with Rotary Evaporators and
(S) -1- tertbutyloxycarbonyl -3- hydroxy piperidine crude products of differential responses time；GC analyzes substrate 1- tertbutyloxycarbonyl -3- piperidines
The conversion ratio of ketone；
Above-mentioned differential responses temperature, differential responses time correspond to the conversion ratio feelings of substrate 1- tertbutyloxycarbonyl -3- piperidones
Condition curve graph is as shown in Figure 1, as can be seen from Figure 1 slower in 20 DEG C of reaction rates, during end reaction 36h, the tertiary fourths of substrate 1-
The conversion ratio of oxygen carbonyl -3- piperidones is significantly lower than the conversion ratio that reaction temperature is 30 DEG C；In 40 DEG C and 50 DEG C of initial reaction stage bottoms
The conversion ratio of object 1- tertbutyloxycarbonyl -3- piperidones is very fast, but later transformation rate significantly slows down, it may be possible to because of higher temperature
Lower enzyme inactivation causes.
In conclusion the Pichia yeast Pichia sp.SIT2014 of the present invention are catalyzed the tertiary fourths of 1- as biocatalyst
Oxygen carbonyl -3- piperidones carries out the optimum response temperature that asymmetric reduction reaction prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines
Spend is 30 DEG C.
Application Example 4
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), the Pichia yeast Pichia sp.SIT2014 cells of 1 gained of embodiment are placed in and be resuspended in solution, obtained
Pichia yeast Pichiasp.SIT2014 suspensions；
It is described be resuspended solution for buffer solution, methanol and glucose group into mixed liquor；The dosage of methanol in solution is resuspended,
It calculates by volume, buffer solution：Methanol is 95：5；The dosage of glucose in solutions is resuspended, calculates in mass ratio, glucose：It is (slow
Fliud flushing and methanol) it is 5：100；The buffer solution is the sodium phosphate aqueous solution of pH 6.5；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichiasp.SIT2014
Cell and the amount that solution is resuspended, by Pichia yeast Pichiasp.SIT2014 dry cell weights：Resuspension solution is 10g：The ratio of 1L
Example calculates；
(2), added in into the Pichia yeast Pichiasp.SIT2014 suspensions obtained by step (1) 1- tertbutyloxycarbonyls-
3- piperidones, after mixing controlled at 30 DEG C, rotating speed carries out asymmetric reduction reaction 20h for 180r/min, obtains anti-
Answer liquid；
The addition of above-mentioned 1- tertbutyloxycarbonyls -3- piperidones, by Pichia yeast Pichia sp.SIT2014 suspensions
Middle Pichia yeast Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 10g：The ratio of 50mmol
It calculates；
(3), the reaction solution of step (2) is centrifuged, obtained supernatant is extracted with isometric ethyl acetate
Twice, merge the extract liquor of extraction gained twice, then dried with anhydrous sodium sulfate, be then evaporated again with Rotary Evaporators, i.e.,
Obtain (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidine crude products；
The centrifugation control rotating speed carries out centrifugation 30min for 10000r/min.
To (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines crude products of above-mentioned gained weigh promoting the circulation of qi phase, the liquid phase point of going forward side by side
Analysis, the yield of target product (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines are>99%, ee value is>99%, the total recovery of reaction
It is 95.1%.
Application Example 5
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), the Pichia yeast Pichia sp.SIT2014 cells of 1 gained of embodiment are placed in and be resuspended in solution, obtained
Pichia yeast Pichiasp.SIT2014 suspensions；
It is described be resuspended solution for buffer solution, methanol and glucose group into mixed liquor；The dosage of methanol in solution is resuspended,
It calculates by volume, buffer solution：Methanol is 95：5；The dosage of glucose in solutions is resuspended, calculates in mass ratio, glucose：It is (slow
Fliud flushing and methanol) it is 5：100；The buffer solution is the sodium phosphate aqueous solution of pH 6.5；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichiasp.SIT2014
Cell and the amount that solution is resuspended, by Pichia yeast Pichiasp.SIT2014 dry cell weights：Resuspension solution is 20g：The ratio of 1L
Example calculates；
(2), added in into the Pichia yeast Pichiasp.SIT2014 suspensions obtained by step (1) 1- tertbutyloxycarbonyls-
3- piperidones, after mixing controlled at 30 DEG C, rotating speed carries out asymmetric reduction reaction 12h for 180r/min, obtains anti-
Answer liquid；
The addition of above-mentioned 1- tertbutyloxycarbonyls -3- piperidones, by Pichia yeast Pichia sp.SIT2014 suspensions
Middle Pichia yeast Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 20g：The ratio of 50mmol
It calculates；
(3), the reaction solution of step (2) is centrifuged, obtained supernatant is extracted with isometric ethyl acetate
Twice, merge the extract liquor of extraction gained twice, then dried with anhydrous sodium sulfate, be then evaporated again with Rotary Evaporators, i.e.,
Obtain (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidine crude products；
The centrifugation control rotating speed carries out centrifugation 30min for 10000r/min.
To (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines crude products of above-mentioned gained weigh promoting the circulation of qi phase, the liquid phase point of going forward side by side
Analysis, the yield of target product (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines are>99%, ee value is>99%, the total recovery of reaction
It is 90.5%.
Application Example 6
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), the Pichia yeast Pichia sp.SIT2014 cells of 1 gained of embodiment are placed in and be resuspended in solution, obtained
Pichia yeast Pichiasp.SIT2014 suspensions；
It is described be resuspended solution for buffer solution, methanol and glucose group into mixed liquor；The dosage of methanol in solution is resuspended,
It calculates by volume, buffer solution：Methanol is 95：5；The dosage of glucose in solutions is resuspended, calculates in mass ratio, glucose：It is (slow
Fliud flushing and methanol) it is 5：100；The buffer solution is the sodium phosphate aqueous solution of pH 6.5；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichiasp.SIT2014
Cell and the amount that solution is resuspended, by Pichia yeast Pichiasp.SIT2014 dry cell weights：Resuspension solution is 5g：The ratio of 1L
Example calculates；
(2), added in into the Pichia yeast Pichiasp.SIT2014 suspensions obtained by step (1) 1- tertbutyloxycarbonyls-
3- piperidones, after mixing controlled at 30 DEG C, rotating speed carries out asymmetric reduction reaction 20h for 180r/min, obtains anti-
Answer liquid；
The addition of above-mentioned 1- tertbutyloxycarbonyls -3- piperidones, by Pichia yeast Pichia sp.SIT2014 suspensions
Middle Pichia yeast Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 5g：The ratio of 50mmol
It calculates；
(3), the reaction solution of step (2) is centrifuged, obtained supernatant is extracted with isometric ethyl acetate
Twice, merge the extract liquor of extraction gained twice, then dried with anhydrous sodium sulfate.
The centrifugation control rotating speed carries out centrifugation 30min for 10000r/min.
To the extract liquor of above-mentioned gained, respectively with GC and HPLC analysis yield and ee values, the tertiary fourth oxygen of target product (S) -1-
The yield of carbonyl -3- hydroxy piperidines is that 47%, ee values are>99%.
Application Example 7
Pichia yeast Pichia sp.SIT2014 cells by the use of 1 gained of embodiment are catalyzed 1- as biocatalyst
Tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, specific to wrap
Include following steps：
(1), the Pichia yeast Pichia sp.SIT2014 cells of 1 gained of embodiment are placed in and be resuspended in solution, obtained
Pichia yeast Pichiasp.SIT2014 suspensions；
It is described be resuspended solution for buffer solution, methanol and glucose group into mixed liquor；The dosage of methanol in solution is resuspended,
It calculates by volume, buffer solution：Methanol is 95：5；The dosage of glucose in solutions is resuspended, calculates in mass ratio, glucose：It is (slow
Fliud flushing and methanol) it is 5：100；The buffer solution is the sodium phosphate aqueous solution of pH 6.5；
In the Pichia yeast Pichiasp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichiasp.SIT2014
Cell and the amount that solution is resuspended, by Pichia yeast Pichiasp.SIT2014 dry cell weights：Resuspension solution is 1g：The ratio of 1L
Example calculates；
(2), added in into the Pichia yeast Pichiasp.SIT2014 suspensions obtained by step (1) 1- tertbutyloxycarbonyls-
3- piperidones, after mixing controlled at 30 DEG C, rotating speed carries out asymmetric reduction reaction 20h for 180r/min, obtains anti-
Answer liquid；
The addition of above-mentioned 1- tertbutyloxycarbonyls -3- piperidones, by Pichia yeast Pichia sp.SIT2014 suspensions
Middle Pichia yeast Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 1g：The ratio of 50mmol
It calculates；
(3), the reaction solution of step (2) is centrifuged, obtained supernatant is extracted with isometric ethyl acetate
Twice, merge the extract liquor of extraction gained twice, then dried with anhydrous sodium sulfate.
The centrifugation control rotating speed carries out centrifugation 30min for 10000r/min.
To the extract liquor of above-mentioned gained, respectively with GC and HPLC analysis yield and ee values, the tertiary fourth oxygen of target product (S) -1-
The yield of carbonyl -3- hydroxy piperidines is that 13%, ee values are>99%.
It is in conclusion provided by the invention a kind of by the use of Pichia yeast Pichia sp.SIT2014 as living things catalysis
Agent catalysis 1- tertbutyloxycarbonyl -3- piperidones carries out asymmetric reduction reaction and prepares (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines
Method.The preparation method has many advantages, such as that high conversion rate, optical purity are high, and reaction condition is mild.(S) -1- of final gained
The yield of tertbutyloxycarbonyl -3- hydroxy piperidines is up to 99%, and more than 99%, separation yield reaches optical purity ee values
95.1%.
The above is only the citing of embodiments of the present invention, it is noted that for the ordinary skill of the art
For personnel, without departing from the technical principles of the invention, several improvement and modification can also be made, these improve and become
Type also should be regarded as protection scope of the present invention.
Claims (8)
1. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines, it is characterised in that the preparation method is red using finishing
Saccharomycete Pichia sp.SIT2014 are as biocatalyst, controlled at 20-50 DEG C of catalysis 1- tertbutyloxycarbonyl -3- piperazine
Pyridine ketone carries out asymmetric reduction reaction 1-36h, obtained reaction solution is centrifuged, extracts successively, anhydrous sodium sulfate is dry and
Revolving processing is to get (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines；
The Pichia yeast Pichia sp.SIT2014, on 06 16th, 2014 in Chinese microorganism strain preservation pipe
The common micro-organisms center preservation of the reason committee, deposit number be CGMCC NO.9300, preservation organization address：Chaoyang District, Beijing City
The Institute of Microorganism, Academia Sinica of institute of North Star West Road 1, postcode：100101.
2. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as described in claim 1, it is characterised in that tool
Body includes the following steps：
(1), the preparation of Pichia yeast Pichia sp.SIT2014 cells
The Pichia pastoris Pichia sp.SIT2014 slant strains that 4 DEG C of refrigerators is taken to preserve, one ring strain of picking, which is linked into, to be equipped with
In the 100mL shaking flasks of 15mL seed culture mediums, seed culture is carried out for 180rpm on shaking table controlled at 30 DEG C, rotating speed
13-15h obtains seed liquor；
Then seed liquor is inoculated into fermentation medium, the amount of seed liquor used is inoculated with, by the OD of zymotic fluid after inoculation600
=0.1 ratio calculates；
After inoculation, fermented and cultured is carried out for 180rpm for 24 hours controlled at 30 DEG C, rotating speed on shaking table, obtain zymotic fluid；
Above-described seed culture medium, fermentation medium are identical, by every liter of calculating, 15g containing glucose, tryptone 5g, ferment
Female powder 5g, K2HPO40.5g, KH2PO40.5g, NaCl 1g, MgSO4·7H2O 0.5g, surplus are water；
The zymotic fluid of gained is centrifuged, the precipitation of gained is Pichia yeast Pichia sp.SIT2014 cells；
(2), the Pichia yeast Pichia sp.SIT2014 cells obtained by step (1) are placed in and mixing in solution is resuspended, obtained
Pichia yeast Pichia sp.SIT2014 suspensions；
The resuspension solution be by buffer solution, methanol and glucose group into mixed liquor, be resuspended solution in methanol dosage, press
Volume ratio calculates, buffer solution：Methanol is 95：5；The dosage of glucose in solutions is resuspended, calculates in mass ratio, glucose：(buffering
Liquid and methanol) it is 5：100；The buffer solution is the sodium phosphate aqueous solution of pH 5.0~9.0；
In the Pichia yeast Pichia sp.SIT2014 suspensions of above-mentioned gained, Pichia yeast Pichia sp.SIT2014 are thin
Born of the same parents and the amount that solution is resuspended, by Pichia yeast Pichia sp.SIT2014 dry cell weights：Resuspension solution is 1-20g：The ratio of 1L
Example calculates；
(3), 1- tertbutyloxycarbonyl -3- piperazines are added in into the Pichia yeast Pichia sp.SIT2014 suspensions obtained by step (2)
Pyridine ketone, after mixing controlled at 20-50 DEG C, rotating speed carries out asymmetric reduction reaction 1-36h for 180r/min, obtains anti-
Answer liquid；
The addition of above-mentioned 1- tertbutyloxycarbonyls -3- piperidones, it is red by finishing in Pichia yeast Pichia sp.SIT2014 suspensions
Saccharomycete Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 1-20g：The ratio meter of 50mmol
It calculates；
(4), the reaction solution of step (3) being centrifuged, obtained supernatant is extracted twice with isometric ethyl acetate,
Merge twice extraction gained extract liquor, then dried with anhydrous sodium sulfate, then again with Rotary Evaporators be evaporated to get (S)-
1- tertbutyloxycarbonyl -3- hydroxy piperidine crude products.
3. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as claimed in claim 2, it is characterised in that step
Suddenly the sodium phosphate aqueous solution that buffer solution is pH 6.0-8.0 in (2).
4. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as claimed in claim 3, it is characterised in that step
Suddenly the sodium phosphate aqueous solution that buffer solution is pH 6.5 in (2).
5. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as claimed in claim 4, it is characterised in that step
Suddenly in (2) in the Pichia yeast Pichia sp.SIT2014 suspensions of gained, Pichia yeast Pichia sp.SIT2014 are thin
Born of the same parents and the amount that solution is resuspended, by Pichia yeast Pichia sp.SIT2014 dry cell weights：Resuspension solution is 10g：The ratio of 1L
It calculates.
6. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as claimed in claim 5, it is characterised in that step
Suddenly in (3) 1- tertbutyloxycarbonyls -3- piperidones addition, by Pichia yeast Pichia sp.SIT2014 suspensions finish it is red
Saccharomycete Pichia sp.SIT2014 dry cell weights：1- tertbutyloxycarbonyl -3- piperidones is 10g：The ratio of 50mmol calculates.
7. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as claimed in claim 6, it is characterised in that step
Suddenly controlled at 30 DEG C in (3), rotating speed carries out asymmetric reduction reaction 20h for 180r/min.
8. the preparation method of one kind (S) -1- tertbutyloxycarbonyl -3- hydroxy piperidines as claimed in claim 7, it is characterised in that step
Suddenly the centrifugation control rotating speed described in (4) carries out centrifugation 10min for 10000r/min.
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