Lipase-catalyzed Regioselective Undecylenoylation Of Nucleosides

Nucleoside analogues constitute an important type of antitumor/antiviral drugs. However, this class of drugs suffers from poor lipophilicity, short half-lives in human plasma and various side effects, etc. It has been reported that their ester prodrugs via the regioselective acylation can effectively overcome the aforementioned problems. Furthermore, undecylenic acid has antifungal and antiviral activities. Therefore, undecylenic acid esters of nucleosides not only could overcome the inherent drawbacks of the individual compounds, but also might have therapeutic efficiency of twin drugs. And these nucleoside esters can be used as starting materials for the synthesis of functional polymeric prodrugs with controlled release because of the presence of an unsaturated bond. The conventional chemical approaches are characterized by low regioselectivity, multi reaction steps and being environmentally unfriendly, etc. Therefore, the enzymatic approaches with high efficiency, excellent selectivity and mild reaction conditions have attracted considerable interest. In the dissertation, various lipases were examined for the regioselective undecylenoylation of purine nucleosides, and the immobilized Candida antarctica lipase B (CAL-B, Novozyme435)-catalyzed regioselective undecylenoylation of purine nucleosides was explored. Then, the catalytic performance of the immobilized, home-made and cheap Penicillium expansum lipase (PEL) in undecylenoylation of purine and pyrimidine nucleosides in biomass-based solvent2-methyltetrahydrofuran (MeTHF) was studied for the first time. The reason for the higher activity of immobilized PEL in MeTHF was explained based on the kinetic studies. Through rational design of the structures of nucleoside molecules, the substrate recognition of PEL in regioselective acylation of nucleosides was elucidated for the first time.Of lipases tested, the immobilized Candida antarctica lipase B (CAL-B) exhibited the highest activity in regioselective undecylenoylation of adenosine. Among the six organic solvents tested, anhydrous THF was the most suitable for this reaction. The optimal molar ratio of vinyl ester to nucleoside, enzyme dosage, reaction temperature and molecular sieves amount were5,20U/mL,45℃and75mg/mL, respectively. Under the above-mentioned conditions, the initial reaction rate, yield and5’-regioselectivity were1.1mM/h,90%and >99%, respectively. The enzymatic acylation of various purine nucleosides by CAL-B furnished the desired5’-ester derivatives with the yields of60-96%and5’-regioselectivities of>99%. In addition, the immobilized CAL-B displayed excellent operational stability in THF; after reused for5batches, its relative activity of96%remained.Although the immobilized CAL-B has high activity and good operational stability in the acylation of nucleosides, this enzyme is very expensive. In addition, THF is a non-renewable organic solvent derived from fossil resources. Therefore, the possibility of using cheap PEL in renewable and biomass-based MeTHF with low toxicity was examined for undecylenoylation of purine nucleosides. Immobilized PEL displayed much higher activity and thermostability in MeTHF than in other reaction media. The kinetic studies revealed that the apparent activation energy of the enzymatic reaction in MeTHF was much lower than those in other organic solvents (24.5vs.43.3-57.1kJ/mol), and the catalytic efficiency of the enzyme was much higher in the former (Vmax/Km,5.8vs.1.1-2.9h-1), which could rationally account for the higher activity of PEL in MeTHF. The optimal molar ratio of vinyl ester to nucleoside, enzyme dosage, reaction temperature for immobilized PEL-mediate acylation of8-chloroadenosine (8-Cl-Ado) were7.5,35℃and25U/mL, respectively. Under the conditions described above, the desirable5’-O-undecylenoyl-8-Cl-Ado was furnished with a yield of95%and a regioselectivity of>99%in3h. After reused for6runs, the relative activity of this immobilized enzyme decreased to33%. So its operational stability needs to be improved. In addition, this lipase was capable of catalyzing regioselective undecylenoylation of various purine nucleosides, thus affording5’-esters with the yields of63-95%. The effects of the substituents in purine moiety and2’-substituents in sugar moiety on PEL-catalyzed regioselective acylation of purine nucleosides were studied. It was revealed that within the range of purine nucleosides tested, the substituents in purine moiety exerted no effect on5’-regioselectivities that kept>99%, but they played a major role in the solubility of nucleosides and initial reaction rates. The enzymatic reaction rates of purine2’-deoxynucleosides were much higher than those of the corresponding ribonucleosides, but the absence of2’-hydroxyl group resulted in slight reduction in the5’-regioselectivity.Then, the immobilized PEL-catalyzed regioselective acylation of pyrimidine nucleosides was investigated. The immobilized PEL-catalyzed undecylenoylation of5-iodouridine was optimized by response surface methodology. Under the optimal conditions (the optimal molar ratio of10.4, enzyme dosage of37.7U/mL and reaction temperature of41℃), the initial reaction rate and5’-regioselectivity were10.1mM/h and>99%, respectively. Maximum substrate conversion reached98%in6h. The effects of5-,2’-and3’-substituents in pyrimidine nucleosides on PEL-catalyzed acylation were studied. The enzymatic acylation of a group of pyrimidine ribonucleosides afforded the desirable5’-esters with excellent5’-regioselectivities (96->99%). The effect of5-substituents on5’-regioselectivities was slight, but the reaction rates showed a clear dependence on5-substituents (10.1-60.1mM/h). In the enzymatic acylation of pyrimidine2’-deoxynucleosides, however, the5’-regioselectivities of the enzyme decreased significantly with increasing bulk of5-substituents. Similarly, the enzymatic reaction rates of pyrimidine2’-deoxynucleosides were much higher than those of the corresponding ribonucleosides. Interestingly, the lack of the3’-hydroxyl group significantly improved the solubility of nucleosides in the organic medium, but had a negative effect on the enzymatic reaction rates. It is the first report on the effect of3’-groups in nucleosides on the enzymatic acylation of nucleosides.This study not only enriches the knowledge of fundamental enzymology, but also provides a novel, highly efficient and regioselective route for undecylenoylation of nucleosides. Meanwhile, many novel ester derivatives of nucleosides (38esters) have been successfully synthesized and a compound library has been established, from which nucleoside derivatives of various biological activities could be screened.