Study On Enzymatic Regioselective Acylation Of Gemcitabine

Gemcitabine is an unnatural nucleoside which possesses a high antitumor activity.However, it has some disadvantages in clinical use, such as poor liphilicity, shorthalf-life in human plasma. To solve these problems, its monoesters are prepared byacylation, which shows a higher antitumor activity than Gemcitabine itself.Nevertheless, most of the present strategies for the acylation of Gemcitabine wereconcentrated on chemistry methods, which have some difficult problems, such as thelow regioselectivity, the cumbersome reaction procedures and unfriendly toenvironment. Up to now, there has been only one report on the enzymatic acylation ofGemcitabine with Lipase CAL-B as a catalyst and divinyl dicarboxylates as acyldonor in acetone. In this report, however, the efficiency of the reaction wasdisappointing low. Thus, the possibility of the enzymatic regioselective acylation ofGemcitabine with carboxylic acid vinyl esters was explored for the first time in thisdissertation. At the same time, the effects of the various factors on the enzymaticreaction in different media were comparatively studied, and the catalyticperformances of the lipases were exhibited in various media. In addition, novelenzymatic reaction systems have been established which could be used for highlyefficient and regioselective acyltion of Gemcitabine.Lipozyme TL IM (lipase from Thermomyces lanuginosus) and Lipozyme RM IM(lipase from Rhizomucor miehei) could both catalyze the regioselective acylation ofGemcitabine at5’-OH with high efficiency. While Lipozyme TL IM with a lowerprice was a better catalyst for the preparation of Gemcitabine5’-esters with mediumchain (C12), Lipozyme RM IM displayed higher activity for the synthesis ofGemcitabine5’-esters with longer chain (C18).The appropriate solvent was30%(v/v) DMF-acetonitrile for the Lipozyme TLIM-mediated regioselective acylaton of Gemcitabine with respect to the initial rate,the maximum substrate conversion and the regioselectivity. And30%(v/v)pentanol-acetone was better as a medium for the Lipozyme RM IM-catalyedregioselective acylation of Gemcitabine.In30%(v/v) DMF-acetonitrile, both the initial rate and the maximum substrateconversion of Lipozyme TL IM-catalyzed regioselective acylation of Gemcitabinewere influenced by many factors, while the regioselectivity of the reaction remainedalmost unchanged and was constantly higher than99%within the scope examined. In the case of the enzymatic acylation of Gemcitabine with vinyl laurate, the optimalinitial water activity (αw), the Gemcitabine concentration, the molar ratio ofgemcitabine to vinyl laurate, reaction temperature and shaking rate were0.07,40mmol/L,1:9,45℃and200r/min in30%(v/v)DMF-acetonitrile, under which theinitial rate and the maximum substrate conversion were25.52mM/h and74.9%,respectively.Similar to the Lipozyme TL IM-catalyzed acylation of Gemcitabine in30%(v/v)pentanol-acetone, the initial rate and the maximum substrate conversion of LipozymeRM IM-mediated regioselective acylation of Gemcitabine with vinyl stearate wereaffected by many factors, while the regioselectivity of the reaction was also constantlyhigher than99%. The optimal initial water activity (αw), the molar ratio ofgemcitabine to vinyl stearate, reaction temperature and shaking rate were0.07,1:12,40℃and200r/min. Under the optimized conditions, the initial rate and the maximumsubstrate conversion were10.13mM/h and83.1%, respectively.The effect of the acyl donors’ structure on the enzymatic regioselective acylation ofGemcitabine varied from one enzyme to another. Generally, foe the Lipozyme TLIM-catalyzed acylation of Gemcitabine in the reaction medium, an increasing initialreaction rate or the maximum substrate conversion with the elongation of chain lengthof acyl donors from C2to C12was observed. With the chain of acyl donors beingfurther lengthened (from C12to C18), however, the initial reaction rate and themaximum substrate conversion declined. Investigation of Lipozyme RM IM-mediatedacylation of Gemcitabine showed that in the reaction medium, the initial reaction rateand the maximum substrate conversion increased with the elongation of chain lengthof the acyl donors from C2to C18. The5’-regioselectivity of the reaction wasconstantly above99%with all the acyl donors tested.This study not only enriches our knowledge of enzymatic acylation andfundamental enzymology, but also provides a novel and efficient route toregioselective acylation of Gemcitabine.