Studies On The Stereoselective Hydrocyanation Catalyzed By Oxynitrilases To Synthesize The Pharmaceutical Intermediates

Research objectiveThe enantiopureα-cyanohydrins from aldehydes/ketones can be converted into a wide variety of products includingα-hydroxyketones,α-hydroxyesters,β-hydroxyamines,andα-aminonitriles etc..These compounds are versatile building blocks for the synthesis of fine chemicals,pharmaceuticals and agrochemicals.For more than three decades,the biotransformation has received great attention,high stereo-selectivity,no pollution and convenience being the main research objectives. Hydroxynitrile lyases(HNLs) are a family of versatile enzymes that catalyze the reversible cleavage ofα-hydroxynitriles and are utilized for the production of enantiopure cyanohydrins from aldehydes or ketones and HCN.This kind of biotransformation not only leads to the formation of new C-C bond but also brings the chiral centre into the product molecular.In this research,hydrocyanation of a series of carbonyl compounds catalyzed by oxynitrilases from seven kinds of plant sources are tested.These R-or S-α-cyanohydrins are all important pharmaceutical intermediates.Through the immobilization technology,the immobilized oxynitrilases are recyclable and more stable.It is the foundation of the oxynitrilases used for industrial applications.Explore new sources of oxynitrilases,especially screen the S-selective oxynitrilases whose contents in plants are relatively less compared with R-selective oxynitrilases from microorganisms,so as to expand the sources of S-selective oxynitrilases and expect S-oxynitrilases to be available which are cheap and easy to get in the synthesis of chiral pharmaceutical intermediates.Research Methods1.The substrates used in the research are as follows:phenylacetaldehyde, 1-benzothiophene-5-carbaldehyde,3,4-methylenedioxybenzaldehyde,3,4-methylenedioxyphenylacetaldehyde and 3,4-methylenedioxypropiophenone.Five kinds of racemic acetylatedα-cyanhydrins and a kind of racemic acetylatedβ-nitro alcohol from 3,4-methylenedioxybenzaldehyde and nitromethane were synthesized with high purity.Enantiomers of each racemic product mentioned above were separated well by selection of chiral column and optimization of HPLC conditions,which lay a foundation for the accurate analysis of ee value of biotransformation products.2.Taking acetone cyanohydrin as the source of HCN and using isopropyl ether containing citrate buffer as the solvent,the hydrocyanation of five substrates were catalyzed by various of oxynitrilases,including five R-selective oxynitrilases from bitter almond(PaHNL),apple,cherry,loquat and papaya(CsHNL) and two S-selective oxynitrilases from rubber(HbHNL) and cassava(MeHNL).The yield and ee value were selected as the index to identify the catalytic ability of different oxynitrilases with the same selectivity to substrates.3.Stereoselective addition of nitromethane to 3,4-methylenedioxybenzaldehyde in the presence of HbHNL was studied.Different reaction conditions including the organic solvent,buffer concentration,pH value,buffer content and nitromethane concentration were tested and the optimized conditions were obtained.4.Cross-linked and carrier-immobilized R-oxynitrilase from apple seed were obtained by using polyaldehyde dextran and Eupergit C as the cross-linker and immobilized agent respectively.Uniform experimental design of three factors and seven levels was adopted to research the influence of polyaldehyde dextran generated under different conditions on the activity of cross-linked enzyme.The influences of buffer pH,buffer concentration and the amount of Eupergit C on the activity of Eupergit C-immobilized enzyme were also investigated.Leakage of activity from the two kinds of immobilized enzymes was tested.We compared the catalytic results between the immobilized enzymes and free enzyme in biphasic and microaqueous systems using 1-benzothiophene-5-carbaldehyde as the substrate.Hydrocyanation of five substrates by recycling use of two kinds of immobilized enzymes were also attempted.5.HbHNL was immobilized with active magnetic particles.Leakage of activity from the immobilized enzyme and the storage stability were tested.We compared the catalytic results between immobilized enzyme and free enzyme in biphasic and microaqueous system using 3,4-methylenedioxybenzaldehyde as the substrate. Hydrocyanation of five substrates by recycling use of magnetic particles-immobilized HbHNL were attempted.6.Using racemic 2-(benzothiophene-5-yl)-2-hydroxyacetonitrile as the donor of HCN and 3,4-methylenedioxybenzaldehyde as the receptor,we try to obtain two kinds of chiralα-cyanohydrins catalyzed by magnetic particles-immobilized HbHNL at one time.7.The reason for the low ee of chiral hydrocyanation of 3,4-methylenedioxy propiophenone from different aspects such as pH value,types of enzyme preparations, concentration of acetone cyanohydrin and amount of enzyme preparation were investigated.8.Establish the optimum reaction conditions for the CsHNL(never be reported before),using acetophenone as the substrate and a serious of methyl ketones were biotransformed in the optimized reaction conditions.We tried to find new source oxynitrilase from plants by taking many kinds of plant tissue powders as the catalyst and using benzaldehyde as the substrate.Screening of microorganisms that can produce S-oxynitrilase were conducted using soil as samples and taking mandelonitrile as the sole carbon source.Results1.Five kinds of racemic acetylatedα-cyanhydrins and one kind of acetylatedβ-nitro alcohol were well separated with chiral OB-H(4.6×250mm,5μm) as the chiral solid phase,and hexane and isopropanol solution in different ratio as the mobile phase.2.The yields of five kinds of products catalyzed by apple oxynitrilase were between 24%and 91%,and the ee values of R-products were between 41%and 97%. The yields of five kinds of substrates catalyzed by HbHNL were between 35%and 85%and the ee values of S-products were between 29%and 98%. 3.We determined that HbHNL displays its best biocatalytic activity in the addition of CH₃NO₂ to 3,4-methylenedioxybenzaldehyde in the following solvent system, MTBE as the solvent,which contained citric acid buffer(40%v/v,0.2 M,pH 7.5). When the initial concentrations of nitromethane and 3,4-methylenedioxybenzaldehyde were 2.4 M and 0.3 M,the stereoselective reaction proceeded smoothly in room temperature by employing 200μL HbHNL as the biocatalyst.The yield and the product ee were 71%and 83%respectively after 24 h.4.Cross-linking process was carried out by reacting polyaldehyde dextran with 1 mL extraction of apple seed meal.The polyaldehyde dextran with best cross-linking performance was prepared from dextran(1.2 g,100 kDa) and sodium periodate(1.8 g) in 150 min at room temperature.Compared with the free enzyme,the cross-linked enzyme kept 53%activities.Even after reusing the cross-linked catalysts 10 times,no decrease of enantioselectivity was observed when phenylacetaldehyde was used as substrate.In the same conditions,recycling use of the cross-linked enzyme to catalyze the hydrocyanations of 1-benzothiophene-5-carbaldehyde,3,4-methylenedioxybenzaldehyde and 3,4-methylenedioxyphenyl-acetaldehyde,catalytic activities of the enzyme became lost at different recycle times during 8 consecutive batch reactions.In sodium dihydrogen phosphate buffer solution(1.2 M,pH 5.0),the apple seed oxynitrilase(in 200μL solution) reacted with Eupergit C(350 mg) and the immobilization resulted in 69%relative activity.The catalytic ability of the immobilized enzyme began to decrease after more than 8 consecutive batch reactions, when 3,4-methylenedioxyphenylacetaldehyde was used as substrate.Even after reusing Eupergit C-immobilized enzyme over 10 times,no decrease of enantioselectivity was observed in the process of hydrocyanation of phenylacetaldehyde, 1-benzothiophene-5-carbaldehyde,3,4-methylenedioxybenzaldehyde. Compared with free enzyme,the two kinds of immobilized preparations afforded higher conversions and ee values during the hydrocyanation of substrates in microaqueous system at 0℃. 5.HbHNL was immobilized covalently onto active magnetic particles and the immobilization resulted in 44%relative activity.When stored for 60 days,the relative activity of the immobilized enzyme decreased to 38%,whereas the free enzyme retained only 17%of its full activity at room temperature.The immobilized enzyme remained good activity during 8 times consecutive batch reactions when phenylacetaldehyde,3,4-methylenedioxybenzaldehyde,3,4-methylenedioxyphenylacetaldehyde were used as substrates.As to the substrate 1-benzothiophene-5-carbaldehyde,the ee value became decreased after over 6 times recycling.Yields of the biotransformation catalyzed by immobilized and free enzyme in the microaqueous and biphasic systems were almost the same but the enantioselectivity of immobilized enzyme increased slightly in microaqueous system.6.Taking magnetic particles-immobilized HbHNL as the biocatalyst and using racemic 2-(benzothiophene-5-yl)-2-hydroxyacetonitrile as the donor of HCN,the hydrocyanation of 3,4-methylenedioxybenzaldehyde proceeded slightly.We detected 0.1 g(S)-2-(3,4-methylenedioxyphenyl)-2-acetoxyacetonitrile and 1.2 g 2-(benzothiophene-5-yl)-2-acetoxyacetonitrile in the final mixture after acetylation, and the ee value were 97%and 27%respectively.7.Hydrocyanation of 3,4-methylenedioxypropiophenone using acetone cyanohydrin as the donor of HCN and 1:1 in molar ratio of acetone cyanohydrin to substrate,the maximum ee value was 83%after 80 min at 0℃in microaqueous system catalyzed by using magnetic particles-immobilized HbHNL as the biocatalyst.8.Oxynitrilase from Chaenomeles Speciosa seed meal had the optimum activity in the methyl tert-butyl ether solution(0.1 M,pH 4.0) containing 18%citric acid buffer solution.The catalytic results were well to acetophenone and aliphatic methyl ketone which contained less than seven carbon atoms.But the catalytic results were not so well and even could not biotransform aromatic ketone which had large size and aliphatic methyl ketone which contained more than seven carbon atoms.One S-selectivity oxynitrilase producing strain was separated from the soil,and its category was uncertain.Conclusion and significance1.Aiming at getting the chiral a-cyanohydrins used as pharmaceutical intermediates,we employed oxynitrilases from different plant sources as biocatalyst to study the chiral hydrocyanation of five kinds of substrates systematically (phenylacetaldehyde,5-thionaphthenaldehyde,3,4-methylenedioxybenzaldehyde, 3,4-methylenedioxyphenylacetaldehyde and 3,4-methylenedioxypropiophenone).We found that the stereoselectivity of R-and S-α-cyanohydrin catalyzed by apple oxynitrilase and HbHNL was the maximum respectively.2.We studied systematically the reaction of 3,4-methylenedioxy benzaldehyde with nitromethane catalyzed by HbHNL for the first time and optimized the reaction conditions.3.We immobilized the apple oxynitrilase well using the polyaldehyde dextran and Eupergit C as the cross-linker and the carrier respectively for the first time.In microaqueous system the yield and the ee value of hydrocyanation catalyzed by immobilized enzyme increased compared with free enzyme.The immobilized enzyme with Eupergit C as the carrier was superior to the immobilized enzyme with polyaldehyde dextran as the cross-linker for the times of recycling use.It laid a foundation for the batch catalysis and industrial application of apple oxynitrilase.4.We studied the immobilization of HbHNL using the active magnetic particles as the carrier for the first time.The immobilized HbHNL kept high catalytic activities, and the catalytic performance remained good when it was recycled,which supplied a convenient and cheap method for the industrial application of HbHNL.5.Small quantity of(S)-2-(3,4-methylenedioxyphenyl)-2-acetoxyacetonitrile was obtained with high ee value under the following conditions:using racemic 2-(benzothiophene-5-yl)-2-hydroxyacetonitrile as the donor of HCN,the 3,4-methylenedioxybenzaldehyde as the substrate,catalyzed by magnetic particlesimmobilized HbHNL.We made a beneficial attempt of getting two kinds of chiral moleculars with one biotransformation by changing the donor of HCN. 6.We studied the method for improving the product ee through inhibiting the decomposition of chiral methyl ketone cyanohydrin for the first time,making a beneficial exploration for the improvement of the low ee value when the substrate of methyl ketone was biotransformed.7.The spectrum of methyl ketones catalyzed by oxynitrilase in Chaenomeles Speciosa seed meal was discussed.We got a strain producing S-oxynitrilase,which had great significance to extend the source of S-oxynitrilase rarely reported at present.