Enzymatic Synthesis Of Glycosides And Their Ester Dernvatives

With the rapid development of glycobiology over the last decade, the diversity and complexity of glycoconjugate compounds have been well elucidated, and their biological functions have been gradually understood. So these compounds are attracting increasing attention in medicinal chemistry. Glycosides and their esters are an important group of candidates of glycoconjugate drugs. The conventional chemical approaches are characterized by low selectivity, the requirement of protection/deprotection, and being environmentally unfriendly. Doubtlessly, green enzymatic routes with mild reaction conditions, high efficiency and high selectivity are a promising alternative. Hence, in the dissertation, a variety of glycosidases of plant origin were screened to catalyze the synthesis of alkyl and aryl glycosides. Besides, the catalytic performance of glycosidase in ionic liquids-containing systems was examined. Then, with the acylation of arbutin as a model reaction, the catalytic performance of immobilized Penicillium expansum lipase (PEL) in various reaction media was examined. At last, the acyl donor recognition of immobilized PEL in the acylation of arbutin was explored.It was found that peach seed meal displayed the highest hydrolytic activity (75.2 U/g) among the seeds tested. The glycosidases from the seeds of black plum, prune, apple and almond exhibited moderate hydrolytic activities (20.3-34.7 U/g), while the activity of glycosidases from the seeds of watermelon, bunge cherry and pomelo was low (7.1-12.4 U/g). To the best of our knowledge, the seeds from black plum, prune, bunge cherry and pomelo as the sources ofβ-glucosidase have not been reported. In solvent-free systems, these glycosidases could catalyze the synthesis of alkyl glycosides via reverse hydrolysis with 14-78% yields. With increasing chain length of alcohols, the enzymatic glucosylation became unfavorable possibly due to the steric hindrance. The products were exclusivelyβ-glycosides as characterized by 13C NMR and 1H NMR.In ionic liquid (IL)-containing media, the effects of the nature and content of ILs on the catalytic performance of glycosidases were remarkable. It was revealed that the effects of ILs on the enzymactic reaction did not follow the Hofmeister series, and it could not be ascribed simply to the ion effect. With different aryl alcohols as glycosyl acceptors, the effects of various ILs on the enzymatic reaction were similar, but they were different with each other in the degree. Among ILs tested, the yields of aryl glucosides were efficiently improved by adding these ILs of moderate amount. In the BMIm·I (10%, v/v)-containing mixtures, prune seed could efficiently catalyze the synthesis of alyl glycosides via reverse hydrolysis with 15-28% yields (except for arbutin with a yield of 6%). It was worth noting that the yield of p-nitrobenzylβ-D-glucopyranoside in this medium was 15%, which is 1.56 times that in IL-free systems. The investigation of enzyme substrate recognition revealed that the structure of glycosyl acceptors exerted significant effects on the enzymatic reaction. It might be attributed to the structure, size and shape of the enzyme active center as well as steric hindrance of the substrates or nucleophilicity of.hydroxyl groups.In organic solvents, the characteristics of the regioselective butanoylation of arbutin catalyzed by inexpensive immobilized PEL were elucidated. The optimum reaction medium, enzyme dosage, molar ratio of vinyl butyrate to arbutin and reaction temperature were anhydrous THF,50 U/mL,7.5 and 35℃, respectively. Under the optimal conditions, the initial rate, the maximum substrate conversion and the 6'-regioselectivity were 75.1 mM/h, >99% and>99%, respectively. The structures of acyl donors showed a great influence on the reaction rate, while little effect on the regioselectivity was observered. The acylation reaction occurred exclusively on 6'-hydroxyl of arbutin (regioselectivity>99%) as characterized by 13C NMR and 1H NMR. Among the acyl donors tested [10 straight-chain fatty acid vinyl esters (C2-C18) and 6 other carboxylic acid vinyl esters bearing different substituents or functional groups], immobilized PEL showed the highest catalytic activity toward the regioselective acylation of arbutin with vinyl 10-undecenoate. Drastic decrease of the initial rate was observed in the acylation of arbutin with acyl donors bearing a substitute (i.e. methyl or ethyl) at the a-position or an unsaturated bond (i.e. C-C double bond) conjugated with the carbonyl group, due to steric hindrance or resonance effect. Among the synthesis of various aromatic acid esters of arbutin, the lipase was the most specific towards vinyl 3-phenylpropionate, with which a substrate conversion of 99% was recorded in 4 h. The reaction rate decreased significantly when the acyl donors had an unsaturated bond (i.e. C-C double bond) conjugated with the carbonyl group. Moreover, the substituents present in the phenyl moiety of the acyl donor exerted a negative impact on the reaction which could be attributed to the unfavorable resonance effect or steric hindrance. The enzymatic acylation route with immobilized PEL proved to be quite applicable and was successfully used for the synthesis of 26 novel ester derivatives of arbutin. It was noteworthy that the isolated yield of arbutin cinnamate was 88%, which is much higher than that (28%) reported previously. In addition, the lipase retained the residual activity of 63% when reused for 11 runs.In co-solvent mixtures, the acylation of arbutin with vanillic acid vinyl ester catalyzed by immobilized PEL was examined. In anhydrous THF-diisopropyl ether (20%, v/v), the lipase showed a high activitiy and an excellent regioselectivity (>99%). The effects of several key variables on the reaction were investigated in detail by response surface methodology of five-level-three-factors. The optimal enzyme dosage, the molar ratio of vinyl vanillic acid to arbutin and the reaction temperature were 93 U/mL,11 and 50℃, respectively, under which the initial reaction rate, the maximum arbutin conversion and the 6'-regioselectivity were 8.2 mM/h,93% and>99%, respectively. The maximum arbutin conversion of 93% was much higher in co-solvent system than that (30%) in THF. And the conversion in the p-hydroxylcinnamoylation,p-methoxylcinnamoylation and 3,4-dimethoxycinnamoylation were 97%,99% and 99%, respectively, which were much higher than those (36%,80% and 70%, respectively) in THF. Besides, the operational stability of the immobilized PEL in co-solvent mixtures was improved, as compared to that in THF. The lipase remained the residual activity of 71% when reused for 11 batches. It was revealed that using co-solvent system not only enhanced enzymatic acylation of arbutin, but also improved the operational stability of the enzyme.This study not only enriches the knowledge of fundamental enzymology, but also provides a novel, selective and efficient route to glucosides and their ester derivatives. Meanwhile, a group of novel glucosides and their ester derivatives have been successfully synthesized, which can be used for the investigation of structure-function of the glycoconjugate compounds and the screening of new glycoconjugate drugs.