Nucleosides represent one class of fundamental building blocks of life systems. Their analogues are extensively used as therapeutic agents for cancer and viral diseases; as precursors of oligonucleotides for therapeutic or diagnostic use; and as molecular tools in research. However, many biologically active nucleosides are difficult to synthesize chemically, which hinders biological trials and studies as well as the application of these compounds. The chemoenzymatic synthesis in comparison to the pure chemical synthesis offers great benefits as a simple, efficient and “green” technology in view of simplicity, costs and yield. The focus of this study was on the application of novel nucleoside phosphorylases (NPs) for the biocatalytic synthesis of nucleoside analogues. The applied enzymes originate from thermophilic microorganisms (Deinococcus geothermalis, Geobacillus thermoglucosidasius, Thermus thermphilus, and Aeropyrum pernix). Five thermostable NPs (TtPyNP, GtPyNP, DgPNP, GtPNP and ApMTAP) were successfully recombinantly expressed in soluble and active form in the Gram-negative bacterium Escherichia coli, and were characterised with respect to temperature optimum, stability, substrate specificity, kinetic behaviour, pH optimum and in view of their similarity by sequence alignments. The results demonstrate their promising biocatalytic properties, especially for TtPyNP and GtPNP, which displayed particularly high catalytic activity towards natural substrates (3-6 times higher than the commonly used enzymes from E. coli) under the experimental conditions. Furthermore, they showed high promiscuity, i.e. they were able to catalyse a broad range of modified substrates. Both enzymes accept nucleosides modified in the sugar moiety, e.g. 2’- or 3’-NH2, 2’-F (ribo- or arabino-) and 2’-OH (arabino); GtPNP recognizes 2,6-Cl or F substituted purine. In coupled reactions with TtPyNP and GtPNP as catalysts various modified purine nucleosides were successfully synthesized. For the synthesis of purine nucleoside analogues, TtPyNP and GtPNP were successfully immobilized on magnetic microsphere beads with high residual enzyme activity, high enzyme loading, and further enhanced enzyme stability. The application of the immobilized enzymes in the synthesis of 2,6-dichloropurine riboside and 6-chloro-2-fluoropurine riboside (6C2FP-R) resulted in product yields of 78.5 % and 85.5 % (HPLC), the latter molecule was isolated and purified ( >98 %) by silica chromatography (normal-phase column) and the compound’s structure was confirmed. These results reveal the great practical potential of the studied biocatalysts. Hence, it is conceivable to produce a number of nucleoside analogues by the described method, which appears more efficient than other synthetic routes described in literature so far.
|Advisor:||Neubauer, PeterMikhailopulo, Igor A|
|School:||Technische Universitaet Berlin (Germany)|
|Source:||DAI-C 81/1(E), Dissertation Abstracts International|
|Subjects:||Bioengineering, Chemical engineering|
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