CN113667702A - Biological synthesis method of miconazole chiral intermediate - Google Patents

Biological synthesis method of miconazole chiral intermediate Download PDF

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CN113667702A
CN113667702A CN202110777213.5A CN202110777213A CN113667702A CN 113667702 A CN113667702 A CN 113667702A CN 202110777213 A CN202110777213 A CN 202110777213A CN 113667702 A CN113667702 A CN 113667702A
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王普
张莹
桂铅
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Zhejiang University of Technology ZJUT
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Abstract

The invention provides a biological synthesis method of a miconazole chiral intermediate, which comprises the following steps: taking wet thalli obtained by fermenting and culturing verticillium terrestris (Cyberlindera saturnus) ZJPH1807 as an enzyme source, taking 2-chloro-1- (2, 4-dichlorophenyl) ethanone as a substrate, forming a conversion system in PBS (phosphate buffer solution) with the pH of 6.0-8.0, converting for 2-24 h at the temperature of 20-45 ℃ and the speed of 200rpm, extracting a conversion reaction liquid by using ethyl acetate after the reaction is finished, and taking an organic phase to obtain an ethyl acetate solution containing the miconazole chiral intermediate. The optical purity of the product prepared by the strain is high, and the ee value is more than 99.9%.

Description

Biological synthesis method of miconazole chiral intermediate
(I) technical field
The invention relates to a biological synthesis method of a miconazole chiral intermediate, belonging to the technical field of biological catalysis.
(II) background of the invention
(R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol (molecular weight: 225.5, CAS number: 114446-57-0) is a key chiral intermediate for the preparation of miconazole and has the following structural formula:
Figure BDA0003156038210000011
miconazole is a safe, highly effective, broad-spectrum antifungal agent, and has almost all effects on pathogenic fungi. The mechanism is to inhibit sterol synthesis of fungal cell membrane, influence cell membrane permeability, inhibit fungal growth and cause death. Most clinically isolated fungi were inhibited at concentrations below 4. mu.g/mL, with Cryptococcus neoformans, Candida and Coccidioides all being sensitive to them, Blastomyces dermatitidis and Histoplasma being highly sensitive to the drug, but Aspergillus is less preferred. In addition, miconazole also has antibacterial effect against Staphylococcus aureus, Streptococcus, gram-positive coccus, anthrax, etc.
At present, the preparation of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by catalytic asymmetric hydrogenation by using different catalysts including chiral oxazoloalkane catalyst, chiral metal catalyst and biocatalyst has been reported. The chiral oxazoleborane catalyst has a wide substrate spectrum, and a catalyst containing transition metals such as rhodium, ruthenium, iridium and the like is the most commonly used catalyst in the hydrogenation reaction of ketone substrates. However, the transition metal requires an additional chelating functional group to improve the enantioselectivity, the operation is difficult to control, and in addition, the high cost of the transition metal also limits its industrial application. Compared with chemical catalysts, biocatalysts have the advantage of high reaction selectivity and are environmentally friendly. With the increase of the market of novel antifungal drugs, the demand of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is rapidly increased, so that an excellent biocatalyst is developed, and a new process for preparing (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by biocatalysis has good application prospects.
Tangcloud et al (CN 108396040A) selects carbonyl reductase from Candida macedoniensis AKU4588, the concentration of a substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone is 1-5 g/L, the addition amount of the carbonyl reductase is 20-100U/L, and the 2-chloro-1- (2, 4-dichlorophenyl) ethanone is catalyzed in a buffer solution system to be reduced and synthesized into (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, and the ee value is more than 99%. Mangas et al (The Journal of Organic Chemistry,2011,76(7):2115-2122.) catalytically synthesized (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol using alcohol dehydrogenase under very mild reaction conditions, with a yield and ee value of greater than 99% at a substrate concentration of 23.86 mmol/L. Finally, chemical modification is carried out so as to comprehensively synthesize the required R enantiomer medicine in a single form. This novel chemoenzymatic route offers significant advantages without the need to use harmful or toxic catalysts to introduce chirality. Thiago et al (org.chem.,2018, (18): 2110-2116) catalyzed the biological reduction of 2,2',4' -trichloroacetophenone to (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol using the lipase Novozym 435, yield 46.5%, ee > 99%. Yuhui bud et al (CN 106701698A) disclose that carbonyl reductase SsCR expressed by xylose fermentation yeast CBS6054 and a mutant of the carbonyl reductase SsCR catalyze asymmetric reduction of a latent chiral carbonyl compound to prepare (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, the conversion rate is more than 99%, and the optical purity of a product is 99.9%.
The (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol is prepared by biological reduction by using the whole microbial cells as a catalyst, and microbial enzyme source cells can be prepared by fermentation, so that the quality is stable, and the cost is low. Furthermore, in situ regeneration of the coenzyme during bioreduction can be achieved by adding inexpensive co-substrates.
Disclosure of the invention
The invention aims to provide a method for preparing (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by using Verticillium terrestris ZJPH1807 resting cells as a catalyst, and the method is used for preparing (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol by conversion, so that the influence of culture medium components on a biological conversion process can be eliminated, the composition of a conversion solution is effectively controlled, the conversion yield is improved, the subsequent separation and extraction of a conversion product are facilitated, strains are easy to culture, the preparation cost of cells containing enzyme sources is low, and compared with the traditional chemical reduction route, the method has the advantages of high stereoselectivity, low catalyst preparation cost, mild reaction conditions and environment-friendly conversion process.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a biological synthesis method of a miconazole chiral intermediate, which comprises the following steps: taking wet thalli obtained by fermenting and culturing verticillium terrestris (Cyberlindera saturnus) ZJPH1807 as an enzyme source cell, taking 2-chloro-1- (2, 4-dichlorophenyl) ethanone as a substrate, forming a conversion system in PBS (phosphate buffer solution) with the pH of 6.0-8.0 (preferably pH 7.2), converting for 2-24 h (preferably 5-24 h, more preferably 12h) at the conditions of 20-45 ℃ (preferably 30 ℃) and 200rpm, and obtaining a conversion solution containing a miconazole chiral intermediate after the reaction is finished, wherein the miconazole chiral intermediate is (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol; the dosage of the wet bacteria is 50-200 g/L (preferably 80g/L) based on the volume of the PBS buffer solution, and the dosage of the substrate is 5-10 g/L (preferably 5g/L) based on the volume of the PBS buffer solution;
the soil star crop yeast ZJPH1807 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019215, date of deposit 2019, 3 month 29, address of deposit: china, wuhan university, zip code 430072.
Note that: the conversion can occur under both aerated and unaerated conditions, but yields are higher under aerated conditions.
Further, the conversion solution containing the miconazole chiral intermediate is purified to obtain a target product according to the following method: centrifuging the transformation solution containing the miconazole chiral intermediate (centrifuging at 9000rpm for 10min for solid-liquid separation), taking supernatant, adding NaCl to saturation, extracting with ethyl acetate equal to the volume of the transformation solution containing the miconazole chiral intermediate for 3 times, combining organic extraction phases, and adding anhydrous Na2SO4Drying and removing water, then loading the obtained organic phase on a silica gel chromatographic column, and then adding petroleum ether: eluting with an eluant of 9:1(v/v), filtering the eluent with the target product, and concentrating under reduced pressure to obtain a yellow oily liquid, namely the (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol product.
Preferably, the concentration of the PBS buffer is 0.1M.
Furthermore, in order to promote coenzyme regeneration in the reduction process and improve the reaction yield, an auxiliary substrate is added into the conversion system, and the auxiliary substrate is one of the following substances: glucose, sucrose, maltose, lactose, trehalose, ethanol, glycerol, isopropanol, 1, 2-propanediol (preferably glucose, trehalose or maltose); the addition amount of the auxiliary substrate is 50-200 g/L (preferably 150g/L maltose) based on the volume of the PBS buffer solution.
Further, the enzyme source cell of the present invention is prepared as follows: (1) slant culture: inoculating Verticillium terrestris (Cyberlindera saturnus) ZJPH1807 to slant culture medium, and culturing at 30 deg.C for 24 hr to obtain slant strain; the final concentration composition of each component in the slant culture medium is as follows: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO4 1.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 15-20 g/L (preferably 20g/L) of agar powder, water as a solvent and pH of 6.5;
(2) seed culture: selecting a ring of thalli from the slant strains in the step (1) and inoculating the selected thalli into a seed culture medium, culturing at 25-30 ℃ and 150-250 rpm for 10-24 h (preferably culturing at 30 ℃ and 200rpm for 12h) to obtain a seed solution; the final concentration composition of each component in the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCl l g/L，MgSO4·7H2O is 0.5g/L, the solvent is water, and the pH value is 6.5;
(3) fermentation culture: inoculating the seed solution in the step (2) into a fermentation culture medium by an inoculation amount with the volume concentration of 8%, fermenting and culturing for 32h at 30 ℃ and 200rpm, centrifuging the obtained fermentation liquor after the fermentation is finished, washing the obtained precipitate by using 0.1M phosphate buffer solution with the pH value of 7.5, and centrifuging to collect wet bacteria, namely the enzyme-containing source cells; the final concentration composition of each component in the fermentation medium is as follows: 34.36g/L glucose, 14.89g/L yeast extract, NH4Cl 30.34g/L，KH2PO4 1.01g/L，CaCl20.11g/L, solvent is water, pH 7.5.
The soil star crop yeast (Cyberlindera saturnus) ZJPH1807 is disclosed in the prior patent application (publication No. CN 110283733A, published as 2019, 9 and 27) of the applicant, is preserved in China center for type culture Collection, and has the preservation number of CCTCC NO: m2019215, date of deposit 2019, 3 month 29, address of deposit: china, wuhan university, zip code 430072.
Compared with the prior art, the invention has the following beneficial effects: the invention uses the resting cells of the prorocentrum terrestris ZJPH1807 strain as a catalyst to biologically catalyze the asymmetric reduction of 2-chloro-1- (2, 4-dichlorophenyl) ethanone to obtain the corresponding (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol. The optical purity of the product prepared by the strain is high, and the ee value is more than 99.9%. In a phosphate buffer system with pH 7.2, the added amount of the bacterial cells is 80g/L, 5g/L (22.37 mM) of a substrate is added, the conversion is carried out for 12h, and the yield of the product is 79.48%.
(IV) description of the drawings
FIG. 1 is a gas chromatogram of standard n-tetradecane (A), 2-chloro-1- (2, 4-dichlorophenyl) ethanone (B), product (S) -2-chloro-1- (2, 4-dichlorophenyl) ethanol (C) and (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol (D) in example 2.
FIG. 2 is a gas chromatogram of an extract from the bioreduction reaction of Verticillium terrestris ZJPH1807 according to example 3; a: n-tetradecane; b: 2-chloro-1- (2, 4-dichlorophenyl) ethanone; c: (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: obtaining of Wet cells
The slant culture medium comprises: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO43g/L，KH2PO41.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 20g/L of agar powder, water as a solvent and pH of 6.5;
the seed culture medium comprises the following components: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO42g/L，KH2PO42g/L，NaCl l g/L，MgSO4·7H2O is 0.5g/L, the solvent is water, and the pH value is 6.5;
the fermentation medium comprises the following components: 34.36g/L glucose, 14.89g/L yeast extract, NH4Cl 30.34g/L，KH2PO41.01g/L，CaCl20.11g/L, solvent is water, pH 7.5.
The prorocentrum terrestris (Cyberlindera saturnus) ZJPH1807 is inoculated to a slant culture medium and cultured for 24h at 30 ℃ to obtain slant thalli. A ring of thalli is picked from a mature culture inclined plane and inoculated into a 250mL shake flask filled with 100mL of seed culture medium, the culture is carried out for 12h at 30 ℃ and 200rpm to obtain seed liquid, then the seed liquid is transferred into the 250mL shake flask filled with 90mL of fermentation culture medium by the inoculation amount with the volume concentration of 8 percent, and the culture is carried out for 32h at 30 ℃ and 200 rpm. And after the culture is finished, centrifugally separating the fermentation liquor, washing the obtained precipitate by using a phosphate buffer solution with the pH of 7.0, and centrifugally collecting the precipitate to obtain wet bacterial cells for later use.
Example 2: gas phase detection method of product
After the reaction, the conversion solution was extracted by adding ethyl acetate of equal volume, and the product and unreacted substrate concentrations in the extract were analyzed by gas chromatography and quantified by internal standard method. The gas chromatography detection method comprises the following steps: the gas chromatograph is Agilent 7820A, and the chromatographic column is Varian CP-Chirasil-Dex chiral capillary gas chromatographic column (25m × 0.25mm × 0.25 μm, d)f0.25). Quantitative analysis of the substrate and product was performed using tetradecane as an internal standard, nitrogen as the carrier gas, and a hydrogen Flame Ionization Detector (FID) was used.
Gas phase (GC) detection conditions: the carrier gas flow is 2mL/min, the sample feeding amount is 1 mul, the split ratio is 15:1, the sample feeding port temperature is 250 ℃, the detector temperature is 250 ℃, the chromatographic column temperature is 160 ℃, the temperature is kept for 1min, and then the temperature is increased to 180 ℃ at the speed of 2 ℃/min. The retention time of each substance is respectively as follows: tetradecane for 2.4min, 2-chloro-1- (2, 4-dichlorophenyl) ethanone for 5.3min, (S) -2-chloro-1- (2, 4-dichlorophenyl) ethanol for 9.1min, and (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol for 9.7min, and the gas phase detection chromatogram is shown in FIG. 1.
The calculated yield is:
Y(yield)＝CProduct of/CSubstrate×100％
In the formula, CProduct ofTo convert the mass concentration of the resulting product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, CSubstrateThe mass concentration of the substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone at the beginning of the reaction.
The optical purity of the product is represented by enantiomeric excess (ee):
ee＝(CR-CS)/(CR+CS)×100％
in the formula, CRIs the concentration of (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol, CSThe concentration of (S) -2-chloro-1- (2, 4-dichlorophenyl) ethanol.
Example 3: effect of conversion reaction temperature on catalytic results
1.0g of wet cells obtained according to the method of example 1 was resuspended in 10mL of PBS buffer (0.1M, pH 7.0), 50mg of substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 1.0g of co-substrate glucose were added, and the mixture was transformed at 200rpm for 24 hours at different temperatures (20-45 ℃), analyzed and detected by the method of example 2 after the reaction was completed, and the yield and ee value of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were calculated, and the results are shown in Table 1.
TABLE 1 Effect of conversion reaction temperature on catalytic results
Figure BDA0003156038210000061
The preferred conversion temperature is 30 ℃ under which conditions the yield is 60.78% and the ee is > 99.9%.
Example 4: effect of aeration conditions on catalytic results
1.0g of wet cells obtained according to the method of example 1 was resuspended in 10mL of PBS buffer (0.1M, pH 7.0), 50-100 mg of substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 1.0g of co-substrate glucose were added, the mixture was transformed under aeration conditions and non-aeration conditions with a stoppered vial, the mixture was transformed at 30 ℃ and 200rpm for 24 hours, and the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was analyzed and detected by the method of example 2 after the reaction was completed, and the yield and ee value were calculated, and the results are shown in Table 2.
TABLE 2 Effect of different aeration conditions on the catalytic results
Figure BDA0003156038210000062
Figure BDA0003156038210000071
Preference is given to vented conversion, under which conditions the yield is 60.78% and the ee is > 99.9%.
Example 5: effect of cell addition on catalytic results
0.5 to 2.0g of wet cells obtained according to the method of example 1 were resuspended in 10mL of PBS buffer (0.1M, pH 7.0), 50mg of substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 1.0g of co-substrate glucose were added, the mixture was transformed at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the yield and ee value of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were determined by the method of example 2 and calculated, and the results are shown in Table 3.
TABLE 3 Effect of cell addition on catalytic results
Figure BDA0003156038210000072
Preferably, the cell loading is 0.8g, under which conditions the yield is 63.54%, ee > 99.9%
Example 6: effect of initial pH of buffer on catalytic results
0.8g of wet cells obtained according to the method of example 1 was resuspended in 10mL of PBS buffer (0.1M, pH 6.0-8.0), 50mg of substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 1.0g of co-substrate glucose were added, the mixture was transformed at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the yield and ee value of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were determined by the method of example 2, and the results are shown in Table 4.
TABLE 4 influence of initial pH of buffer on the catalysis results
Figure BDA0003156038210000073
Figure BDA0003156038210000081
Preferably, the buffer has an initial pH of 7.2, under which conditions the yield is 65.79% and the ee is > 99.9%
Example 7: effect of co-substrate species on catalytic results
0.8g of wet cells obtained according to the method of example 1 was resuspended in 10mL of PBS buffer (0.1M, pH 7.2), 50mg of the substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 1.0g of different cosubstrates were added, the mixture was transformed at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the yield and ee value of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were determined by the method of example 2 and calculated, and the results are shown in Table 5.
TABLE 5 Effect of co-substrate species on catalytic results
Figure BDA0003156038210000082
Glucose, trehalose, maltose are preferred as cosubstrates, and the corresponding addition amounts are continuously examined.
Example 8: effect of additional substrate addition on catalytic results
0.8g of wet cells obtained according to the method of example 1 was resuspended in 10mL of PBS buffer (0.1M, pH 7.2), 50mg of substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 0.5-2.0 g of different cosubstrates were added, and the mixture was transformed at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was analyzed and detected by the method of example 2, and the yield and ee value were calculated, and the results are shown in Table 6.
TABLE 6 Effect of co-substrate addition on catalytic results
Figure BDA0003156038210000091
Preference is given to a maltose addition of 1.5g, under which conditions the yield is 77.17% and the ee value > 99.9%
Example 9: effect of conversion time on catalytic results
0.8g of wet cells obtained according to the method of example 1 was resuspended in 10mL of PBS buffer (0.1M, pH 7.2), 50mg of substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone and 1.5g of co-substrate maltose were added, the mixture was transformed at 30 ℃ and 200rpm for 2-24 hours, and after the reaction was completed, the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was analyzed and detected by the method of example 2, and the yield and ee value were calculated, and the results are shown in Table 7.
TABLE 7 Effect of conversion time on catalytic results
Figure BDA0003156038210000092
The preferred conversion time is 12h, under which conditions the yield is 79.48%, ee > 99.9%
Comparative example 1: examination of the ability of Candida parapsilosis ZJPH1305 to biocatalytically produce (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol
(1) Candida parapsilosis (Candida palrapsilosis) ZJPH1305, deposited in the chinese type culture collection with a date of 11/8 in 2013, address: china, wuhan university, accession number: CCTCC NO: m2013559. This strain has been disclosed in a prior patent application (publication No. CN103849574A, published: 6/11/2014). The culture method of the bacterial strain and the preparation process of the enzyme-derived cells were carried out according to the prior patent application (publication No. CN103849574A, published: 6/11/2014).
(2) Biocatalytic preparation of miconazole chiral intermediate
1.0g of wet cells of Candida parapsilosis ZJPH1305, 11.3mg of the substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone, 1.0g of the co-substrate glucose were added to 10mL of PBS buffer (0.1M, pH 7.0), and the mixture was transformed at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was analyzed and detected by the method of example 2, and the yield and ee value of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were calculated.
And (4) conclusion: candida parapsilosis ZJPH1305 catalyzed (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was obtained in 15.56% yield and 45.61% ee.
Comparative example 2: examination of transformation Capacity for the biocatalytic preparation of Miconazole chiral intermediate by Candida tropicalis 104
(1) Candida tropicalis (Candida tropicalis)104, deposited in the chinese type culture collection with a date of 11/8/2013, address: china, Wuhan and Wuhan university, the preservation number is CCTCC No. M209034, and the preservation date is 2009, 2 months and 27 days. This strain has been disclosed in a prior patent application (publication No. CN 101519674a, published: 2009, 9/2). The culture method of the bacterial strain and the preparation process of the enzyme-derived cells were carried out according to the prior patent application (publication No. CN 101519674A, published: 2009, 9/2).
(2) Biocatalytic preparation of miconazole chiral intermediate
1.0g of wet Candida tropicalis (Candida tropicalis)104 cells, 11.3mg of the substrate 2-chloro-1- (2, 4-dichlorophenyl) ethanone, 1.0g of the co-substrate glucose were added to 10mL of PBS buffer (0.1M, pH 7.0), the mixture was converted at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol was analyzed and detected by the method of example 2, and the yield and ee value of the product (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol were calculated.
And (4) conclusion: candida tropicalis 104 catalyzed (R) -2-chloro-1- (2, 4-dichlorophenyl) ethanol in a yield of only 9.4% and an ee of 99%.
Comparative example 3: capacity examination of 2-chloro-1- (3, 4-difluorophenyl) ethanone by Turkish-head yeast (Cyberlindera saturnus) ZJPH1807 biocatalysis
(1) The structural formula of the 2-chloro-1- (3, 4-difluorophenyl) ethanone is as follows:
Figure BDA0003156038210000111
(2) detection method of 2-chloro-1- (3, 4-difluorophenyl) ethanone and corresponding alcohol thereof
The gas chromatograph is Agilent 7820A, and the chromatographic column is Varian CP-Chirasil-Dex chiral capillary gas chromatographic column (25m × 0.25mm × 0.25 μm, d)f0.25). Substrate and product using dodecane as internal standard substanceQuantitative analysis of the substances was carried out using a hydrogen Flame Ionization Detector (FID) with nitrogen as the carrier gas. Gas phase (GC) detection conditions: the carrier gas flow is 2mL/min, the sample injection amount is 1 mu L, the split ratio is 15:1, the sample injection port temperature is 260 ℃, the detector temperature is 280 ℃, and the chromatographic column temperature is 120-165 ℃; temperature rise rate: 5 ℃/min, and 165 ℃ for 1 min. The retention time of each substance is respectively as follows: substrate 5.1 min; r-configuration product for 8.4 min; s-configuration product 7.9 min; dodecane 2.7 min.
(2) Biotransformation of 2-chloro-1- (3, 4-difluorophenyl) ethanone
10mL of PBS buffer (0.1M, pH 7.0) was added with 1.0g of Verticillium alternatum (Cyberlindera saturnus) ZJPH1807 wet cells, 9.5mg of substrate 2-chloro-1- (3, 4-difluorophenyl) ethanone, 1.0g of glucose as an auxiliary substrate, and the mixture was converted at 30 ℃ and 200rpm for 24 hours, and after the reaction was completed, the yield and ee value of the product 2-chloro-1- (3, 4-difluorophenyl) ethanol were analyzed and detected by the method of example (2).
And (4) conclusion: the yield of (R) -2-chloro-1- (3, 4-difluorophenyl) ethanol prepared by biocatalyzing 2-chloro-1- (3, 4-difluorophenyl) ethanone with Verticillium terrestris (Cyberlindera saturnus) ZJPH1807 was 73.1%, and the ee value was 56.6%.

Claims (10)

1. A biological synthesis method of a miconazole chiral intermediate is characterized by comprising the following steps: taking wet thalli obtained by fermentation culture of the alternaria alternata ZJPH1807 as enzyme source cells, taking 2-chloro-1- (2, 4-dichlorophenyl) ethanone as a substrate, forming a conversion system in a PBS (phosphate buffer solution) with the pH of 6.0-8.0, converting for 2-24 hours at the temperature of 20-45 ℃ and at 200rpm, and obtaining a conversion solution containing the miconazole chiral intermediate after the reaction is finished; the dosage of the wet bacteria is 50-200 g/L calculated by the volume of PBS buffer solution, and the dosage of the substrate is 5-10 g/L calculated by the volume of PBS buffer solution;
the soil star crop yeast ZJPH1807 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019215, date of deposit 2019, 3 month 29, address of deposit: china, wuhan university, zip code 430072.
2. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 1, wherein: the pH of the PBS buffer was 7.2.
3. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 1, wherein: the conversion condition is 30 ℃ and 5-24 h.
4. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 1, wherein: the dosage of the wet thallus is 80g/L based on the volume of the PBS buffer solution.
5. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 1, wherein: the amount of the substrate used was 5g/L based on the volume of PBS buffer.
6. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 1, wherein: the concentration of the PBS buffer was 0.1M.
7. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 1, wherein: an auxiliary substrate is also added into the conversion system, and the auxiliary substrate is one of the following substances: glucose, sucrose, maltose, lactose, trehalose, ethanol, glycerol, isopropanol, 1, 2-propanediol; the adding amount of the auxiliary substrate is 50-200 g/L based on the volume of the PBS buffer solution.
8. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 7, wherein: the auxiliary substrate is glucose, trehalose or maltose.
9. The method for the biosynthesis of a chiral intermediate of miconazole as claimed in claim 8, wherein: the auxiliary substrate is maltose, and the addition amount of the auxiliary substrate is 150g/L based on the volume of the PBS buffer solution.
10. A process for the biosynthesis of a chiral intermediate of miconazole as defined in claim 1, wherein the enzyme-derived cell is prepared by: (1) slant culture: inoculating the soil star crop yeast ZJPH1807 to a slant culture medium, and culturing at 30 ℃ for 24h to obtain slant strains; the final concentration composition of each component in the slant culture medium is as follows: 15g/L glucose, 7.5g/L peptone, 6g/L yeast extract, (NH)4)2SO4 3g/L，KH2PO41.5g/L，NaCl 0.75g/L，MgSO4·7H20.75g/L of O, 15-20 g/L of agar powder, water as a solvent and pH of 6.5;
(2) seed culture: selecting a ring of thalli from the slant strains in the step (1) and inoculating the selected thalli into a seed culture medium, and culturing at 25-30 ℃ and 150-250 rpm for 10-24 hours to obtain a seed solution; the final concentration composition of each component in the seed culture medium is as follows: 15g/L glucose, 20g/L peptone, 10g/L yeast extract, (NH)4)2SO4 2g/L，KH2PO4 2g/L，NaCll g/L，MgSO4·7H2O is 0.5g/L, the solvent is water, and the pH value is 6.5;
(3) fermentation culture: inoculating the seed solution in the step (2) into a fermentation culture medium in an inoculation amount with the volume concentration of 8%, fermenting and culturing for 32 hours at 30 ℃ and 200rpm, centrifuging the obtained fermentation liquor after the fermentation is finished, washing the obtained precipitate with 0.1M phosphate buffer solution with the pH value of 7.5, and centrifuging to collect wet bacteria, namely enzyme source cells; the final concentration composition of each component in the fermentation medium is as follows: 34.36g/L glucose, 14.89g/L yeast extract, NH4Cl 30.34g/L，KH2PO4 1.01g/L，CaCl20.11g/L, solvent is water, pH 7.5.
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CN110982757A (en) * 2019-12-30 2020-04-10 浙江工业大学 Enterobacter cloacae ZJPH1903 and application
CN112048538A (en) * 2020-08-21 2020-12-08 浙江工业大学 Method for preparing (S) - [3, 5-bis (trifluoromethyl) phenyl ] ethanol by using Verticillium terrestris

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CN112048538A (en) * 2020-08-21 2020-12-08 浙江工业大学 Method for preparing (S) - [3, 5-bis (trifluoromethyl) phenyl ] ethanol by using Verticillium terrestris

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