Method for synthesizing (1S, 5R) -dicyclic lactone by enzyme catalysis
阅读说明：本技术 一种酶催化的（1s,5r）-双环内酯的合成方法 (Method for synthesizing (1S, 5R) -dicyclic lactone by enzyme catalysis ) 是由 陈芬儿 竺科杰 黄则度 程荡 王佳琦 陶媛 于 2020-05-14 设计创作，主要内容包括：本发明属于生物制药技术领域,具体为酶催化的(1<I>S</I>,5<I>R</I>)-双环内酯的合成方法。本方法包括：制备含拜尔-维立格单氧化酶的工程菌；制备工程菌的静息细胞悬浊液,并经过超声破碎或者加压破碎后得含拜尔-维立格单氧化酶细胞上清液,再与底物消旋取代双环[3.2.0]-庚-2-烯-6-酮、氢供体、辅因子混合,进行不对称拜尔-维立格氧化,制得(1<I>S</I>,5<I>R</I>)-双环内酯；拜尔-维立格单氧化酶的氨基酸序列如SEQ IDNO.1所示；该方法可催化外消旋的取代双环[3.2.0]-庚-2-烯-6-酮(II)生成手性的(1<I>S</I>,5<I>R</I>)-双环内酯,所用工程菌方便易得,产物便于分离且对映选择性高。(The invention belongs to the technical field of biological pharmacy, in particular to enzyme catalytic (1) S ，5 R ) -synthesis of bicyclic lactone. The method comprises the following steps: preparing engineering bacteria containing Bayer-Virgo monooxygenase; preparing the suspension of the engineering bacteria, ultrasonic crushing or pressure crushing to obtain the cell supernatant containing Bayer-Virgo monooxygenase, and racemizing with substrate to substitute bicyclo [3.2.0 ]]Mixing the (E) -hept-2-en-6-one, hydrogen donor and cofactor, and performing asymmetric Bayer-Virgiger oxidation to obtain (1) S ，5 R ) -a bicyclic lactone; Bayer-VirgoThe amino acid sequence of the oxidase is shown as SEQ ID NO. 1; the method can catalyze racemic substituted bicyclo [ 3.2.0%]Production of chiral (1) from (II) -hept-2-en-6-one S ，5 R ) The bicyclic lactone has easy-to-obtain engineering bacteria, easy-to-separate product and high enantioselectivity.)
1. An enzyme-catalyzed (1)S，5R) The method for synthesizing the bicyclic lactone is characterized by comprising the following specific steps of:
(1) preparing engineering bacteria containing Bayer-Virgiger monooxygenase genes and engineering bacteria containing glucose dehydrogenase genes;
(2) respectively preparing resting cell suspension of two engineering bacteria;
(3) separately preparing cell supernatant containing Bayer-Virgo monooxygenase and cell supernatant containing glucose dehydrogenase gene
(4) Mixing the two cell supernatants, and mixing with racemic substituted bicyclo [ 3.2.0%]Mixing the (E) -hept-2-en-6-one, solvent, hydrogen donor and cofactor, and carrying out asymmetric Bayer-Virgiger oxidation reaction to obtain (1)S，5R) -a bicyclic lactone;
wherein the amino acid sequence of the Bayer-Virgiger monooxygenase gene is shown as SEQ ID NO.1, or the amino acid sequence with homology of more than 80 percent with the amino acid sequence shown as SEQ ID NO. 1.
2. The synthetic method of claim 1 wherein said engineered bacterium comprises the bayer-verriger monooxygenase gene of expression vector CHMO-Rhodo1 and the host cell is e.coli BL21(DE3).
3. The synthetic method of claim 1 wherein the cell supernatant is prepared by a method comprising: inoculating the engineering bacteria to a culture medium containing kanamycin, activating a shaking table, and expanding to OD600When the value reaches 0.8-1.2, adding an inducer, continuing culturing, centrifugally collecting cells, resuspending with a buffer solution, ultrasonically crushing, and centrifuging to obtain the cell supernatant.
4. The synthetic method of claim 3 wherein the inducer is IPTG and the concentration of the inducer is 0.05mM to 0.8 mM; the culture conditions after adding the inducer are as follows: the culture temperature is as follows: the culture time is 8-24 h at 15-25 ℃.
5. The method of claim 1, wherein the volume ratio of the cell supernatant containing bayer-virgine monooxygenase to the cell supernatant containing glucose dehydrogenase gene in the mixture of the two cell supernatants is 100:1 to 1: 2.
6. the method of synthesis according to claim 1, wherein the asymmetric oxidation reaction has the following reaction formula:
in the reaction process, on one hand, Bayer-Virgo monooxygenase CHMO catalyzes racemic substitution bicycle [3.2.0]Regioselective asymmetric oxidation of (II) -hept-2-en-6-one to give stereoisomerically pure (1)S，5R) Bicyclo lactone (I) accompanied by reduced cofactor FADH2Conversion to oxidized cofactor FADA process; on the other hand, glucose dehydrogenase oxidizes glucose into gluconolactone while consuming the oxidized coenzyme NADP+The reduced coenzyme factor NADPH is regenerated, and the reduced coenzyme factor NADPH reduces the oxidized coenzyme factor FAD to the reduced coenzyme factor FADH2Which is oxidized to oxidized coenzyme NADP+And a closed loop for coenzyme factor consumption and regeneration is formed to promote the main reaction.
7. The synthetic method of claim 6 wherein the hydrogen donor is glucose and the cofactor is NADP+/NADPH，FADH2/FAD。
8. The method of synthesis according to claim 6, wherein the asymmetric oxidation reaction is carried out at a substrate concentration of 1.0 to 100g/L and the cell supernatant containing Bayer-Virgo monooxygenase is prepared at a cell concentration of 0.1gDry weight of/L ~ 25gDry weight ofL, preparation of cell supernatant containing glucose dehydrogenase Gene at a concentration of 0.1gDry weight of/L ~ 25gDry weight ofThe concentration of the hydrogen donor is 5-750g/L, and the concentration of the cofactor is 0-0.5 mM.
9. The synthesis method according to claim 6, wherein the temperature of the asymmetric oxidation reaction is 20 to 40 ℃ and the pH of the reaction buffer is 6.0 to 8.0.
10. The synthesis method according to claim 1, wherein in the asymmetric oxidation reaction, the solvent used is a high dielectric constant solvent: dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide; aromatic solvent: benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene; non-polar solvent: n-hexane, cyclohexane; polar solvent: acetonitrile, ethyl acetate, dichloromethane, 1, 2-dichloroethane, methanol, ethanol, isopropanol, or a combination thereof.
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to enzyme catalysis (1)S，5R) -a process for the synthesis of bicyclic lactones (I).
Background
Known is a compound represented by the formula (1)S，5R) Bicyclic lactones are key intermediates for the synthesis of prostaglandins:
wherein R is hydrogen, halogen such as fluorine, chlorine, bromine, iodine, etc., C1-C8Alkyl or cycloalkyl, phenyl, mono-or poly-substituted aryl or aralkyl, thienyl, furyl, naphthyl, pyridyl, and the like. n is 1 to 10.
Its synthesis was first described by Tolstikov, G.A. (et al)Zhurnal Organicheskoi Khimii, 1989,25208) reports that the desired (1) is obtained by three-step synthesis of racemic substrate from cyclopentadiene, diastereomer crystallization resolution with (R) - (+) -methylbenzylamine and lactonizationS，5R) -a bicyclic lactone (I). The method has the common defects of low single resolution rate, complex operation, high cost and the like.
Veronique et al (Tetrahedron Lett., 1989,303663) from bicyclo [ 3.2.0) using microbially promoted enantioselective Baeyer-Villiger oxidation]One-step construction of (1) starting from (E) -hept-2-en-6-oneS，5R) -a bicyclic lactone (I). Furstoss et al (J. Org. Chem., 1992,571306) promoting stereoselectivity B with microorganismsaeyer-Villiger oxidation with high enantioselectivity (>95% ee) gave the lactone, but the unusual lactone products were also available with high ee values and were difficult to separate with similar polarities.
Masami et al describe a stereoselective hydrolysis preparation of an endo-racemic diester using pig liver esterase as a catalyst (1)S,5R) Process for the bicyclic lactones (I) ((I))Organic ReactionsNJ, United States, 37, No pp.given; 1989); ogasawara et al (Synlett1996,319) reports the construction of asymmetric desymmetrization reactions catalyzed by lipases (1)S，5R) -bicyclic lactone (I). However, these methods have disadvantages such as a small production scale and troublesome post-treatment.
Disclosure of Invention
The invention aims to overcome the defects of the existing microbial catalysis technology and provide (1) which is convenient to separate, high in purity and simple to operateS，5R) -bicyclic lactone (I) synthesis method.
The invention provides a method for synthesizing (1S, 5R) -bicyclic lactone catalyzed by enzyme, which comprises the following steps:
(1) preparing engineering bacteria containing Bayer-Virgiger monooxygenase genes and engineering bacteria containing glucose dehydrogenase genes;
(2) respectively preparing resting cell suspension of two engineering bacteria;
(3) separately preparing cell supernatant containing Bayer-Virgo monooxygenase and cell supernatant containing glucose dehydrogenase gene
(4) Mixing the two cell supernatants, and mixing with racemic substituted bicyclo [ 3.2.0%]Mixing the (E) -hept-2-ene-6-one (II), solvent, hydrogen donor and cofactor, and performing asymmetric Bayer-Virgiger oxidation reaction to obtain (1)S，5R) -a bicyclic lactone (I);
the amino acid sequence of the Bayer-Virgiger monooxygenase gene (CHMO gene for short) is shown in SEQ ID NO.1,
or an amino acid sequence with homology of more than 80 percent with the amino acid sequence shown in SEQ ID NO. 1.
Wherein the engineering bacteria contain an expression vector pET-28a, and the host cell is Escherichia coli BL21(DE3).
The Bayer-Virgiger monooxygenase gene is expressed in Escherichia coli BL21(DE3) to obtain Bayer-Virgiger monooxygenase and glucose dehydrogenase.
The preparation method of the cell supernatant comprises the following steps: inoculating the engineering bacteria to a culture medium containing kanamycin, activating a shaking table, and expanding to OD600And when the value reaches 0.8-1.2, adding an inducer, continuing to culture, centrifugally collecting cells, resuspending with a buffer solution, ultrasonically crushing, and centrifuging to obtain the cell supernatant.
Preferably, the inducer is IPTG, and the concentration of the inducer is 0.05mM-0.8 mM.
Preferably, the culture conditions after addition of the inducer are: the culture temperature is as follows: the culture time is 8-24 h at 15-25 ℃. The buffer solution is sodium dihydrogen phosphate-disodium hydrogen phosphate; the concentration of the buffer solution is 30-300 mM.
The reaction formula of the asymmetric oxidation reaction of the invention is as follows:
in the whole process of asymmetric oxidation reaction, on one hand, Bayer-Virgo monooxidase CHMO catalyzes racemic substituted bicyclo [3.2.0]Regioselective asymmetric oxidation of (II) -hept-2-en-6-one to give stereoisomerically pure (1)S，5R) Bicyclo lactone (I) accompanied by reduced cofactor FADH2The process of conversion to oxidized cofactor FAD; on the other hand, glucose dehydrogenase oxidizes glucose into gluconolactone while consuming the oxidized coenzyme NADP+The reduced coenzyme factor NADPH is regenerated, and the reduced coenzyme factor NADPH reduces the oxidized coenzyme factor FAD to the reduced coenzyme factor FADH2Which is oxidized to oxidized coenzyme NADP+And a closed loop of coenzyme factor consumption and regeneration is formed to promote the main reaction.
In the asymmetric oxidation reaction, glucose is used as a hydrogen donor, and the related cofactorIs NADP+/NADPH，FADH2/FAD。
The concentration of the bayer-verriger monooxygenase and glucose dehydrogenase during the reaction has an effect on the yield of the final product. Preferably, the volume ratio of the cell supernatant containing bayer-verge monooxygenase to the cell supernatant containing glucose dehydrogenase in the mixture of the two cell supernatants is 20:1 to 1:2, and more preferably, the volume ratio of the cell supernatant containing bayer-verge monooxygenase to the cell supernatant containing glucose dehydrogenase is 10:1 to 8: 1.
Specifically, in the asymmetric oxidation reaction, the concentration of the substrate is 1.0-100 g/L, and the cell concentration of the cell supernatant containing the Bayer-Virgiger monooxygenase is 0.1gDry weight of/L ~ 25gDry weight ofL, preparation of cell supernatant containing glucose dehydrogenase Gene at a concentration of 0.1gDry weight of/L ~ 25gDry weight ofThe concentration of the hydrogen donor is 5-750g/L, and the concentration of the cofactor is 0-0.5 mM.
Preferably, the temperature of the asymmetric oxidation reaction is 20-40 ℃, and the pH value of the reaction buffer solution is 6.0-8.0. More preferably, the reaction temperature is 20-25 ℃, the pH of the reaction buffer is 6.5-7.5, and the buffer concentration is 100-250 mM.
The asymmetric oxidation reaction is carried out by using solvents with high dielectric constants such as dimethyl sulfoxide, N-dimethylformamide, N-dimethylacetamide and the like as solvents; aromatic solvents such as benzene, toluene, ethylbenzene, chlorobenzene, bromobenzene, and the like; nonpolar solvents such as n-hexane and cyclohexane; one or more of polar solvents such as acetonitrile, ethyl acetate, dichloromethane, 1, 2-dichloroethane, methanol, ethanol, isopropanol and the like, and phosphoric acid buffer solution. The preferred solvent is a mixed solvent of toluene, 1, 2-dichloroethane, and phosphate buffer at a mixing ratio of 3:2: 95.
In the whole reaction process, after the reaction is carried out until a GC detection substrate is completely consumed, extracting for 3-4 times by using ethyl acetate with the same volume, combining organic phases, washing for 2 times by using saturated sodium bicarbonate, washing for 1 time by using water, washing for 1 time by using saturated salt, drying by using anhydrous sodium sulfate, and removing an organic solvent by reduced pressure distillation to obtain a target product.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention constructs engineering bacteria containing Bayer-Virgiger monooxygenase CHMO and glucose dehydrogenase GDH, and applies the engineering bacteria to the catalysis of racemic substitution bicyclo [3.2.0]-hept-2-en-6-one (II) is (1)S，5R) The production of the bicyclic lactone (I) provides a new biological production route;
(2) the invention adopts the combination of Bayer-Virgiger monooxygenase CHMO and glucose dehydrogenase GDH, so that the consumption and the regeneration activities in the reaction process are matched, and the self-isomerization effect of regioisomers in the system is utilized, so that the final product has great polarity difference, and the optimal preparation (1) is obtainedS，5R) -bicyclic lactone (I) process, improving the separation conditions of the product;
(3) the Bayer-Virgiger monooxygenase which is convenient and easy to obtain is adopted as the catalyst, so that the use of expensive chemical catalytic reagents in the catalytic process is avoided.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis of CHMO-Rhodo1 cell supernatant protein induced by genetically engineered bacteria. Wherein the content of the first and second substances,
m nucleic acid Marker, 1: CHMO-Rhodo1 cell supernatant expressed by genetic engineering bacteria.
FIG. 2 isRacemicHPLC analysis of (E) -7, 7-dichloro-bicyclolactone (I).
FIG. 3 is (1)S，5R) HPLC analysis of ee value for 7, 7-dichloro-bicyclolactone (I).
FIG. 4 is (1)S，5R) -7, 7-dichloro-bicyclolactone (I) XRD single crystal diffractogram (CCDC: 1978566).
FIG. 5 shows the reaction substrate 7, 7-dichlorobicyclo [3.2.0 ]]Process for producing (II) hept-2-en-6-one1HNMR spectrogram.
FIG. 6 shows the reaction product (1)S，5R) Process for preparing (I) -7, 7-dichloro-bicyclolactone1HNMR spectrogram.
FIG. 7 shows the reaction product (1)R，2S) Of cyclopentene (IV) o-dicarboxylates1H NMR spectrum.
Detailed Description