| Nucleoside analogs constitute a vital class of anticancer and antiviral agents. However, these nucleoside agents generally suffer from various severe side effects and poor bioavailability. It was reported that glycosylated derivatives of nucleoside drugs could mask their toxicity and/or improve their pharmacokinetic properties. Because the conventional chemical methods are characterized by low regioselectivity, the requirement of protection /deprotection, and being ernvironmentally unfriendly, an enzymatic approach with high efficiency and high selectivity was established in this dissertation. Firstly, a variety of glycosidases of different sources were screened. Then, with the glucosylation of 2′-deoxynucleosides mediated by the crudeβ-glycosidase extract of bovine liver as a model reaction, the effects of key parameters on the enzymatic reaction were examined. And enzyme substrate recognition in the glycosylation of nucleosides was discussed. At last, cross-linked enzyme aggregates (CLEAs) ofβ-glycosidase from bovine liver were prepared and characterized.The crudeβ-glycosidase extract of bovine liver is less expensive and can be simply prepared in a standard organic laboratory. In enzymatic regioselective glucosylation of 2'-deoxyuridine, the optimal glycosyl donor, buffer pH, temperature and enzyme dosage were p-nitrophenylβ-D-glucopyranoside (pNPGlc), 9.5, 42 oCand 0.05 U mL-1, respectively, under which, the initial reaction rate, the maximum yield and the 5′-regioselectivity were 1.5 mM h-1,67% and >99%, respectively. In addition, it was revealed that at least twoβ-glycosidases were presented in the crude extract, in which the major active fraction was an alkalineβ-glycosidase (pH 9.5). It suggested that the alkalineβ-glycosidase might be a novel one, which is quite different from the commercialβ-galactosidase from bovine liver (pH 6.5). Five novel glucosyl-containing disaccharide nucleosides (the glycosylated derivatives of 2′-deoxynucleoside, floxuridine,β-thymidine, 5-bromo-2′-deoxynucleoside, and idoxuridine) were successfully synthesized with the yields of 22-72% and exclusive 5'-regioselectivities (>99%). In addition, the crude extract of bovine liver was found to be a versatile biocatalyst for regioselective galactosylation of 2′-deoxynucleosides, which furnished the desired products with satisfactory yields (41-68%) and good to excellent 5'-regioselectivities (87->99%). Besides, the 5′-regioselectivity decreased with the increasing size of 5-group (H, F, CH3, Br and I), which might be attributed to unfavorable steric hindrance, thus destabilizing the conformation of 5′-glycosylation transition state.The effects of precipitating and cross-linking agents on the activity recovery of cross-linkedβ-glycosidase aggregates were examined, and the immobilizedβ-glycosidase (CLEAs) was characterized. The results revealed that the activity recovery of the aggregates was the highest via precipitation with 70% ammonium sulfate. The optimum cross-linking agent and its concentration, cross-linking time were dextran polyaldehyde, 50% (v/v) and 8 h, respectively. Under the optimum conditions, the activity recovery of CLEAs reached 58%. With pNPGlc as the substrate, the optimum pH, temperature, Km and Vmax were 8.5, 60 oC, 1.43 mM and 0.127μmol min-1 mg-1, respectively. The CLEAs had good operation stability and the aggregates clustered into less-defined structures by SEM.This study not only enriches the knowledge of fundamental enzymology, but also provides a novel and efficient route to regioselective glycosylation of nucleosides. In addition, several novel glycosylated derivatives of nucleosides have been successfully synthesized.
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