CN101864370B - Yeast strain converting quinuclidone into R-3-quinuclidinol and conversion method thereof - Google Patents

Abstract

The invention relates to a yeast strain for producing R-3-quinuclidinol by using quinuclidone and a transformation method thereof. The related yeast strain is rhodotorula rubra Rhodotorula rubra X15 which is preserved in China General Microbial Culture Preservation Management Center at present, and the preserving number of the yeast strain is CGMCC No: 3664. The transformation method adopts the following steps of: 1, activating and culturing seeds; 2, performing starter propagation; and 3, taking fermented culture solution, performing centrifugal collection and washing bacteria, adding a substrate of quinuclidone hydrochloride, adding glucose, reacting for 72 to 84 hours on a table, evaporating the solution to dryness, dissolving and stirring by using dichloromethane, filtering undissolved substances, performing distillation on the solvent under reduced pressure, and obtaining a white solid of R-3-quinuclidinol. Due to the adoption of the method, the high-yield and high-purity R-3-quinuclidinol can be obtained; and the transformation method has the advantages of no environmental disruption and low energy consumption.

Description

Transforming quininone is the yeast strain and the method for transformation thereof of R-3-quinuclidinol

Technical field

The present invention relates to a kind of quininone that ferments and produce the yeast strain and the method for transformation thereof of R-3-quinuclidinol.

Background technology

At present, most drug is main with the racemic modification medicine, and is the development trend of most chiral drugs as medicine with optically pure enantiomorph.

3-quininone hydrochlorate (C ₇H ₁₁NOHCl, CAS No:1193-65-3), be a kind of a kind of midbody that is used for synthetic azasetron, Palonosetron, contain the prochiral ketone base in the structure.

R-3-quinuclidinol, chemical name: R-(-)-1-chlorine dicyclo [2.2.2] suffering-3-alcohol of mixing, molecular formula is C ₇H ₁₃NO, molecular weight are 127.18, CAS accession number: 25333-42-0, and the pure article of R-3-quinuclidinol are white crystal, and its fusing point is 217～224 ℃, and boiling point is 120 ℃, and specific rotatory power is-44.5 °, the solubleness in water is 100g/100mL; The R-3-quinuclidinol is the important intermediate of a lot of anticholinergic agents, and for example Suo Linaxin, Revatropate etc. all are the up-to-date anticholinergic agents that contains R-3-quinuclidinol structure, and it has better curative effect for the treatment urinary incontinence and chronic obstructive pulmonary disease.R-3-quinuclidinol hydrochloride (C ₇H ₁₃NOHCl CAS No:42437-96-7); It is the chiral material that the reduction of 3-quininone obtains single opticity; Be a kind of important chiral alcohol that is used for synthetic various kinds of drug, as his sand that is used to treat senile dementia is halted (Talsaclidine) and is used to treat the Revatropate (Revatropate) etc. of chronic pulmonary embolism.

The method of existing synthetic R-3-quinuclidinol mainly contains chemosynthesis and the synthetic two kinds of methods of mikrobe.

Chemical process is synthetic:

1, with the 4-vinylpridine is the synthetic 3-quinuclidinol of raw material;

4-vinylpridine is oxidized to (4-pyridine)-1 in cold potassium permanganate salt brine solution; 2-terepthaloyl moietie; (4-pyridine)-1; 2-terepthaloyl moietie is hydrogenated in sour environment and is corresponding piperidines terepthaloyl moietie hydrochloride, and piperidines terepthaloyl moietie hydrochloride generates the 3-quinuclidinol under the catalysis at activated alumina under 300 ℃.

2, be the synthetic 3-quinuclidinol of raw material with the 3-quininone

The 3-quininone obtains the 3-quinuclidinol later on through sodium borohydride reduction in ethanol, this is reflected at and carries out very fastly in the ethanol, is to prepare the practical method of 3-quinuclidinol.

The chemosynthesis fado is reacting precursor with the quininone, and transition element is a chiral catalyst, obtains the R-3-quinuclidinol through polystep reaction, and reactions step is many, and side reaction is many, and theoretical yield has only 50%, and the optical activity of quinuclidinol is not high, and environmental pollution is serious.

Enzyme or mikrobe preparation method:

3, M.Ikunaka in 2003 has reported that a kind of proteolytic enzyme with honey aspergillus (Aspergillus melleus) splits the method for 3-quinuclidinol verivate, and transformation efficiency has reached 42%, and the ee value reaches 96%.But the theoretical maximum conversion rate that this racemize material enzyme kinetics splits is 50%, and derivatization reaction is arranged.

Summary of the invention

The purpose of this invention is to provide a kind of yeast strain and the method for transformation thereof that can produce the R3-quinuclidinol efficiently with quininone.

The present invention adopts following technical scheme to realize: it is the yeast strain of R-3-quinuclidinol that a strain transforms quininone; Called after rhodothece rubra Rhodotorula rubra X15; In the preservation of specified depositary institution of State Intellectual Property Office; Depositary institution's name is called Chinese common micro-organisms culture presevation administrative center, and deposit number is CGMCC No:3664.

A kind of method of utilizing yeast strain conversion quininone for the R-3-quinuclidinol, it adopts following steps:

(1), seed activation is cultivated;

(2), fermentation enlarged culturing;

(3), get the rhodothece rubra fermented liquid of fermentation enlarged culturing gained, centrifugal collection thalline, wet thallus, in Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution, be suspended in again in the same damping fluid after the washing; Add the substrate quininone hydrochlorate again, add glucose, on 30 ℃, shaking table, react; React after 72～84 hours, evaporate to dryness solution stirs with the methylene dichloride dissolving; The filtering insolubles removes solvent under reduced pressure, obtains white solid R-3-quinuclidinol.

Rhodothece rubra bacterium colony characteristic: bacterium colony is a scarlet, circle, and protuberance, opaque, smooth surface, moistening, neat in edge; The rhodothece rubra cell is circular or oval, through the positive bacterium of gramstaining, single-ended gemmation; No pseudohypha generates; Identify through Physiology and biochemistry and 18S rRNA, be accredited as rhodothece rubra Rhodotorula rubra, called after Rhodotorula rubra X15.

The invention has the beneficial effects as follows that the present invention utilizes keto reductase or has the mikrobe resting cell catalytic substrate quininone of reductase activity, substrate need not derivatization treatment; Only need can obtain reactant once the step reduction reaction, theoretical yield 100% is more because the high specific of enzyme; The optical purity of R-3-quinuclidinol is at 97%e.e.; Adopt immobilization rhodothece rubra whole-cell catalytic reduction quininone hydrochlorate, reaction conversion ratio reaches 86%, and the optical purity of R-3-quinuclidinol reaches 99.3%; This method reaction conditions is gentle, reactions step is few, consuming little energy, productive rate is high, product purity is high, belongs to environmental friendliness, the sustainable type mode of production.

Embodiment

Embodiment 1

It is the yeast strain of R-3-quinuclidinol that one strain transforms quininone; Called after rhodothece rubra Rhodotorula rubra X15; In the preservation of specified depositary institution of State Intellectual Property Office, depositary institution's name is called Chinese common micro-organisms culture presevation administrative center, and deposit number is CGMCC No:3664.

A kind of method of utilizing described yeast strain conversion quininone for the R-3-quinuclidinol, adopt following steps:

(1) seed activation: rhodothece rubra Rhodotorula rubra X15 bacterial strain is transferred in the seed liquid nutrient medium from the glycerol stock of preserving, cultivate 24h in 30 ℃, 150～250rpm.Get cultured bacteria suspension dilution; Evenly be applied in the seed solid plate substratum; After 25 ℃ of incubators are cultivated 48h, choose single bacterium colony with toothpick and be inoculated in the seed liquid nutrient medium test tube 30 ℃, 150～250rpm overnight cultures once more; Get the cultured bacteria suspension of 5mL and be transferred to 30 ℃ of shaking culture of the mid-shaking table of the 500mL triangular flask that 150mL seed liquid nutrient medium is housed, rotating speed is 150～250rpm.

Said seed solid medium: peptone 5g/L, yeast extract paste 15g/L, sodium-chlor 4g/L, glucose 10g/L, agar 20g/L, pH are 5.0, the seed liquid nutrient medium is the seed solid medium that lacks agar.

(2) fermentation culture, culture medium prescription: glucose 12g/L, peptone 0.5g/L, yeast extract paste 0.5g/L; Sal epsom 0.1g/L, potassium primary phosphate 0.1g/L, ammonium sulfate 0.15g/L, it is 5.0 that 2mol/L hydrochloric acid is transferred pH; In inoculum size is 6%, and rotating speed 450r/min, air flow are under the condition of 6L/min; Through 30 ℃, 3 days fermentation culture gets the rhodothece rubra fermented liquid;

(3) get 6L rhodothece rubra fermented liquid, centrifugal collection thalline obtains the 10g wet thallus; After using 20mmol/L, pH to be 7.0 Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution washing 2 times, be suspended in again in the same damping fluid of 100ml, add 10g/L, pH again and be the substrate quininone hydrochlorate of 7.0 100ml, add glucose 0.2g; At 30 ℃, react on the shaking table of 160r/min, react after 72 hours; Evaporate to dryness solution under 100 ℃ of conditions stirred the filtering insolubles with purity 1 hour greater than 99.0% methylene dichloride dissolving;-0.85Mpa, remove solvent under reduced pressure under 35～40 ℃ of conditions, obtain white solid R-3-quinuclidinol 0.866g; Utilize HPLC to detect solid purity, quinuclidinol purity is 99.0%, and quininone and other impurity are 1%.

R-3-quinuclidinol ee value adopts the 3-quinuclidinol to be derivatized to phenylformic acid 3-quinine ester, detects with HPLC then.At first the Benzoyl chloride 99min. with 0.123mL splashes in the methylene dichloride that contains a small amount of triethylamine; 0.5 add the R-3-quinuclidinol after this reaction of 0.1g is accomplished after hour; Stirring at normal temperature 4～6 hours; Solvent is removed under reduced pressure, directly be dissolved in purity, and measure the ee value with HPLC greater than 99.0% ETHYLE ACETATE.Chromatographic condition is following, column type: Chiralpak AD-H, column temperature: 25 ℃, moving phase speed: 1mL/min detects wavelength: 254nm, moving phase ratio: 97% normal hexane, 3% Virahol.Measure through HPLC, the ee value of the R-3-quinuclidinol after this reaction is accomplished is 99.3%.

Embodiment 2

A kind of method of utilizing described yeast strain conversion quininone for the R-3-quinuclidinol, adopt following steps:

(1) seed activation: rhodothece rubra Rhodotorula rubra X15 bacterial strain is transferred in the seed liquid nutrient medium from the glycerol stock of preserving, cultivate 24h in 30 ℃, 130～270rpm.Get cultured bacteria suspension dilution; Evenly be applied in the seed solid plate substratum; After 30 ℃ of incubators are cultivated 48h, choose single bacterium colony with toothpick and be inoculated in the seed liquid nutrient medium test tube 30 ℃, 150～250rpm overnight cultures once more; Get the cultured bacteria suspension of 5mL and be transferred to 30 ℃ of shaking culture of the mid-shaking table of the 500mL triangular flask that 150mL seed liquid nutrient medium is housed, rotating speed is 150～250rpm.

Said seed solid medium: peptone 5g/L, yeast extract paste 15g/L, sodium-chlor 4g/L, glucose 10g/L, agar 20g/L, pH are 5.0, the seed liquid nutrient medium is the seed solid medium that lacks agar.

(2) fermentation culture, culture medium prescription: glucose 15g/L, peptone 0.5g/L, yeast extract paste 0.5g/L; Sal epsom 0.1g/L, potassium primary phosphate 0.1g/L, ammonium sulfate 0.15g/L, transferring pH is 5.0; In inoculum size is 6%, and rotating speed 450r/min, air flow are under the condition of 6L/min; Through 30 ℃, 3 days fermentation culture gets the rhodothece rubra fermented liquid;

(3) get 6L rhodothece rubra fermented liquid, centrifugal collection thalline obtains the 10g wet thallus; After using 20mmol/L, pH to be 7.0 Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution washing 2 times, be suspended in again in the same damping fluid of 100ml, add 10g/L, pH again and be the substrate quininone hydrochlorate of 7.0 100ml, add glucose 0.2g; At 30 ℃, react on the shaking table of 160r/min, react after 84 hours; Evaporate to dryness solution under 100 ℃ of conditions; Stirred 1.5 hours greater than 99.0% methylene dichloride dissolving with purity, the filtering insolubles is at 67Kgf/cm ², remove solvent under reduced pressure under 35～40 ℃ of conditions, obtain white solid R-3-quinuclidinol 0.867g, utilize HPLC to detect solid purity, quinuclidinol purity is 99.1%, quininone and other impurity are 1%.

The detection method of R-3-quinuclidinol ee value is with embodiment 1, and recording R-3-quinuclidinol ee value is 99.4%.

Claims (2)

1. strain conversion quininone is the yeast strain of R-3-quinuclidinol; It is characterized in that its called after rhodothece rubra Rhodotorula rubra X15; In the preservation of specified depositary institution of State Intellectual Property Office; Depositary institution's name is called Chinese common micro-organisms culture presevation administrative center, and deposit number is CGMCC No:3664.

2. one kind is utilized the described yeast strain of claim 1 to transform the method for quininone for the R-3-quinuclidinol, it is characterized in that it adopts following steps:

(1), seed activation is cultivated;

(2), fermentation enlarged culturing;

(3), get the rhodothece rubra fermented liquid of fermentation enlarged culturing gained, centrifugal collection thalline, wet thallus, in Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution, be suspended in again in the same damping fluid after the washing; Add the substrate quininone hydrochlorate again, add glucose, on 30 ℃, shaking table, react; React after 72～84 hours, evaporate to dryness solution stirs with the methylene dichloride dissolving; The filtering insolubles removes solvent under reduced pressure, obtains white solid R-3-quinuclidinol.