β-Galactosidase-catalyzed Regioselective Galactosylation Of Floxuridine And Its Analogs

Floxuridine (FUdR) is a fluorinated deoxynucleoside analog, which has been used extensively in the treatment of a variety of tumors. Even though, it suffers from poor oral bioavailability and various side effects in the clinical application. It has been proven that glycosylated derivatives of nucleoside drugs show higher antitumor activity than the parent compounds. In addition, disaccharide nucleosides are an important group of natural antibiotics. The conventional chemical approach is characterized by low regioselectivity, the requirement of protection/deprotection, and being environmentally unfriendly. Doubtlessly, green enzymatic approach with mild reaction conditions, high efficiency and high selectivity is a promising alternative. Hence, in the dissertation, with the galactosylation of FUdR mediated byβ-galacosidase from bovine liver as a model reaction, the effects of key parameters on the enzymatic reaction were examined. Then, we focused on enzyme substrate recognition in the galactosylation of nucleosides. At last, the effects of organic solvents, ionic liquids and deep eutectic solvents (DES) on the enzymatic galactosylation of FUdR were investigated in the co-solvents mixture.It was revealed thatβ-galacosidase from bovine liver was an excellent catalyst for the glycosylation of FUdR with excellent 5′-regioselectivity and high activity in phosphate buffer. The optimal glycosyl donor, buffer pH, temperature, molar ratio of FUdR to o-nitrophenyl-β-D-galactoside (oNPG) and enzyme dosage were oNPG, 6.5, 45℃, 2 and 0.15 U mL-1, respectively, under which, the initial reaction rate, the maximum yield and the 5′-regioselectivity were 0.28 mM h-1, 75 % and >99%, respectively.Five novel disaccharide nucleosides (the glycosylated derivatives of 2′-deoxynucleoside, FUdR,β-thymidine, 5-bromo-2′-deoxynucleoside, and idoxuridine) were successfully synthesized with the yields of 45-85% and 5′-regioselectivities of 92->99%. An examination into enzyme-substrate recognition showed that the yield of the enzymatic glycosylation of nucleoside decreased with the enlargement of 5-group (H, F, CH3 and Br) of the nucleoside with the exception of idoxuridine. Also, the 5′-regioselectivity decreased with the increasing size of 5-group. It might be attributed to unfavorable steric hindrance, which destabilizes the conformation of 5′-glycosylation transition state.In the co-solvent mixtures, it was shown that the addition of nonaqueouse solvents such as organic solvents, ionic liquids and DES would result in the inactivation ofβ-galacosidase from bovine liver. Among 7 organic solvent-containing systems, the enzyme displayed the highest catalytic activity in DMSO-containing systems. The yields of enzymatic glycosylation reached more than 66% when the content of DMSO was less than 10% (v/v) in the medium. And the yield was 75% in buffer-[MMIm]MeSO4 (1%, v/v) system, which is comparable to that in buffer. Among 9 DES (25%, v/v)-containing systems, the enzyme showed the highest activity in the choline chloride/glycerol-containing system, in which the initial reaction rate and the yield were 0.15 mM h-1 and 22%, respectively. With the decrease of the nonaqueous solvent content, its detrimental effect on the enzyme was reliefed significantly, while.the chemical selectivity of enzymatic galactosylation of FUdR was enhanced.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.