【作者】 方方；

【导师】 吉爱国； 孟昭力；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2008， 博士

【摘要】 研究目的:手性α-氰醇作为手性源可以衍生得到手性α-羟基酮、α-羟基酸酯、β-羟基胺和α-氨基腈等诸多重要的药用、农用和精细化工产品的手性中间体。以氧腈酶为生物催化剂立体选择性的催化合成手性α-氰醇,在形成新的C-C键的同时,产物的分子结构中也被引入了手性。本论文以多种来源的氧腈酶为生物催化剂,催化生成在药物合成中具有重要应用价值的手性α-氰醇的药用中间体;通过固定化技术对不同选择性和不同结构的氧腈酶进行固定后使用,为氧腈酶的工业化应用打下基础;探索新的氧腈酶源,尤其是从微生物中筛选在植物中含量相对较少的S-选择性的氧腈酶,扩大S-型氧腈酶的来源,以期在合成手性药物中间体时,具有廉价易得的S-型氧腈酶可利用。研究方法:1.本研究选取的底物分别为:苯乙醛、5-苯并噻吩甲醛、3,4-亚甲二氧基苯甲醛、3,4-亚甲二氧基苯乙醛和3,4-亚甲二氧基苯丙酮。通过化学法合成出相应的五种外消旋乙酰化α-氰醇和一种由3,4-亚甲二氧基苯甲醛与硝基甲烷反应生成的相应外消旋乙酰化β-硝基醇。以上述产物为标准品探索合适的色谱条件,为准确分析生物转化产物的ee值奠定基础。2.在含有柠檬酸缓冲液的IPE反应体系中,以丙酮氰醇为HCN源,使用苦杏仁(PaHNL)、苹果、樱桃、枇杷和木瓜(CsHNL)五种R-选择性的氧腈酶和橡胶(HbHNL)、木薯(MeHNL)两种S-选择性的氧腈酶为生物催化剂,分别催化五种底物的手性羟腈化反应。以收率和ee值的高低为指标,比较具有相同选择性的氧腈酶转化以上底物的能力。3.以HbHNL为催化剂,催化3,4-亚甲二氧基苯甲醛与硝基甲烷的缩合反应,分别考察有机溶剂的种类、缓冲溶液的浓度、pH值、缓冲溶液的百分含量和硝基甲烷的浓度对生物转化的影响,确定该类生物转化反应的最佳反应条件。4.分别使用多醛基葡聚糖和Eupergit C为固定化载体,与苹果氧腈酶共价结合后,得到相应的固定化酶。进行三因素七水平的均匀设计实验,研究不同条件下生成的多醛基葡聚糖对固定化酶活性的影响。探讨Eupergit C与苹果氧腈酶结合时,缓冲溶液的pH值、浓度和Eupergit C用量对固定化酶活性的影响。分别检测两种方法对苹果氧腈酶固定化的牢固程度。使用5-苯并噻吩甲醛为底物,以收率和ee值的大小为指标,对比固定化酶与游离酶在双相和微水相反应体系中的催化结果。探讨底物分别被固定化苹果氧腈酶循环催化的情况。5.使用磁性微球对HbHNL进行固定化,检测HbHNL被固定化的牢固程度及放置时间对固定化酶活性的影响,探讨固定化酶被循环使用的次数,以3,4-亚甲二氧基苯甲醛为底物,以收率和ee值的大小为指标,对比固定化酶与游离酶在双相和微水相反应体系中的催化结果,探讨底物分别被磁性微球固定化HbHNL循环催化的情况。6.以外消旋2-(苯并噻吩-5-基)-2-羟基乙腈为HCN的供体,以3,4-亚甲二氧基苯甲醛为底物,在磁性微球固定化HbHNL催化下,尝试一次生物转化得到两种具有重要用途的手性α-氰醇。7.从反应体系pH值、酶制剂的种类和用量、丙酮氰醇的用量等方面探讨3,4-亚甲二氧基苯丙酮被手性羟腈化时ee值低下的原因。8.以苯乙酮为底物,优化来源于木瓜氧腈酶(以前未见报道)的催化反应条件,探讨其催化一系列甲基酮底物手性羟腈化的能力。尝试使用多种植物组织粗粉为催化剂,以苯甲醛为转化底物,以期寻找到新的植物来源的氧腈酶。以多份土壤为待筛样品,以扁桃腈为唯一碳源,从中筛选仅产S-型氧腈酶的微生物。结果:1.以Chiralcel OB-H(4.6×250 mm,5μm)为手性固定相,以不同体积百分比的正己烷和异丙醇溶液为流动相,对五种外消旋乙酰化α-氰醇和一种乙酰化β-硝基醇进行了较好的分离。2.苹果氧腈酶催化五种底物生物转化的收率介于24-91%之间,催化生成R-型产物的ee值介于41-97%之间。HbHNL催化五种底物生物转化的收率介于35-85%之间,催化生成S-型产物的ee值介于29-98%之间。3.在含柠檬酸缓冲液(40%v/v,0.2 M,pH 7.5)的MTBE溶液(10 mL)中,当加入的HbHNL量为200μL,硝基甲烷与3,4-亚甲二氧基苯甲醛初始浓度分别为2.4 M和0.3 M时,20-25℃反应24 h后生物转化的收率为71%,ee值为83%。4.使用100 kDa的葡聚糖、葡聚糖和高碘酸钠的用量分别为1.2 g和1.8g、反应130 min时制得的多醛基葡聚糖与1 mL苹果氧腈酶粗提液反应,交联后的固定化酶与游离酶相比保留了53%的酶活。循环催化苯乙醛羟腈化反应10次也未见明显的酶活丧失。循环催化5-苯并噻吩甲醛、3,4-亚甲二氧基苯甲醛和3,4-亚甲二氧基苯乙醛的手性羟腈化反应8次时,分别从不同的使用轮次开始多醛基葡聚糖固定化酶的催化能力开始有了显著降低。在1.2 M,pH 5.0磷酸二氢钠缓冲溶液中,以350 mg的Eupergit C与200μL苹果氧腈酶粗提液反应,制得的Eupergit C固定化酶与游离酶相比保留了69%的酶活。循环催化3,4-亚甲二氧基苯乙醛的手性羟腈化反应8次以上时,产物的ee值下降明显。循环催化苯乙醛、5-苯并噻吩甲醛和3,4-亚甲二氧基苯甲醛手性羟腈化反应10次以上未见明显的酶活降低。以上两种固定化苹果氧腈酶在0℃微水相体系中,表现出了比游离酶更高的转化率和立体选择性。5.磁性微球固定化HbHNL,与游离酶相比酶活保留了44%。放置时间为60天后,固定化酶的活性降低至原游离酶活力的38%,相同条件下的游离酶在放置60天后,酶活降低至原来的17%。固定化酶与游离酶相比,在微水相和双相反应体系中反应的收率无明显变化,但立体选择性则有不同程度提高。在循环催化苯乙醛、3,4-亚甲二氧基苯甲醛和3,4-亚甲二氧基苯乙醛手性羟腈化反应8次的过程中,均未见明显的酶活丧失。循环催化5-苯并噻吩甲醛手性羟腈化反应第6次时,ee值开始下降显著。6.以磁性微球固定化HbHNL为催化剂,外消旋2-(苯并噻吩-5-基)-2-羟基乙腈为HCN供体,催化3,4-亚甲二氧基苯甲醛的手性羟腈化,反应最终的体系中含有7%的(S)-2-(3,4-亚甲二氧基苯基)-2-乙酰氧基乙腈,ee值为97%,体系中63%的2-(苯并噻吩-5-基)-2-羟基乙腈,ee值为27%。7.以等物质的量的丙酮氰醇为HCN供体、加入10 g/L的磁性微球固定化HbHNL,在0℃微水相体系中,催化3,4-亚甲二氧基苯丙酮的羟腈化反应,反应80 min时ee值达到83%。8.新来源的木瓜氧腈酶在含18%的柠檬酸缓冲液(0.1 M、pH 4.0)的MTBE溶液中,具有最佳催化性能。对于苯乙酮和碳链少于7个碳原子的脂肪族甲基酮都具有良好的催化结果,但对于体积较大的芳酮和碳链多于7个碳原子的脂肪族甲基酮催化效果较差甚至不能转化。从土壤中筛选出一株仅产S-选择性氧腈酶的菌株,菌株的归属待定。结论及意义:1.以合成手性药物中间体—手性α-氰醇为目的,以多种来源、不同立体选择性的氧腈酶为生物催化剂,对苯乙醛、5-苯并噻吩甲醛、3,4-亚甲二氧基苯甲醛、3,4-亚甲二氧基苯乙醛和3,4-亚甲二氧基苯丙酮五种底物的手性羟腈化反应进行了系统研究,总体上以苹果氧腈酶和HbHNL催化分别得到R-和S-型手性α-氰醇的立体选择性最高。2.首次对HbHNL催化3,4-亚甲二氧基苯甲醛与硝基甲烷的反应进行了研究,探索了最适反应条件。3.分别以多醛基葡聚糖和Eupergit C为交联剂和固定化载体,首次对苹果氧腈酶粗蛋白进行了较好的固定。不仅酶活保留较好,而且与游离酶相比固定化酶催化底物手性羟腈化的收率和ee值均有不同程度的提高。在循环使用的次数上,以Eupergit C为载体的固定化酶要优于以多醛基葡聚糖为交联剂的固定化酶。为苹果氧腈酶的批量催化及工业化应用奠定了基础。4.首次以磁性微球固定化法对HbHNL进行了固定化研究,固定化的HbHNL仍然保留了较高的催化活性,循环使用性能良好。与游离酶相比固定化HbHNL催化底物的立体选择性更高。为HbHNL的应用提供了新的方便、价廉的固定化方法。5.以外消旋2-(苯并噻吩-5-基)-2-羟基乙腈为HCN供体,以3,4-亚甲二氧基苯甲醛为底物,以磁性微球固定HbHNL为生物催化剂。通过改变氢氰酸的供体,对一次生物转化得到两种手性分子的可行性做了有益的尝试。6.首次探讨了通过抑制手性甲基酮氰醇的分解而提高手性氰醇ee值的方法,对改善甲基酮底物被转化时普遍偏低的ee值具有重要意义。7.首次发现了新来源的CsHNL,对CsHNL催化甲基酮的底物谱做了探讨。筛选到了一株仅产S-型氧腈酶的菌株,对于扩展目前报道较少S-型氧腈酶的来源具有重要意义。更多还原

【Abstract】 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.更多还原