The scheme of enzymatic preparation of fludarabine-5′-monophosphate from 2-fluoroadenine and cyclocytidine as a donor of arabinose fragment was developed and experimentally proved. Acetylphosphate was used as a donor of phosphate group. Cytidine deaminase, uridine phosphorylase, purine nucleoside phosphorylase and deoxynucleoside kinase recovered from earlier designed strains of Escherichia coli were used as biocatalysts. The synthesized fludarabine-5′-monophosphate is planned for using as a pro-drug in enzymatic pro-medicinal approach to therapy of cancer.
Herein, the environmentally friendly synthesis of the earlier unknown phospholipid analog of kinetin riboside was carried out using phospholipase D of Streptomyces netropsis . Kinetin riboside and soybean lecithin served as substrates. A maximum degree of nucleoside conversion to 5′-phosphatidyl derivative of kinetin riboside at 37°С to 6 h exceeds 95%. The structure of target product was confirmed by UV and 1 Н NMR spectroscopy.
Recombinant full-length diguanylate cyclases (DGCs) of Thermotoga maritima with native and mutant allosteric sites were overexpressed in Escherichia coli cells and characterized. It has been shown that target enzymes are produced substantially in the form of active inclusion bodies. Introduction of the mutation in allosteric site resulted in 7-fold increase of the T. maritima DGC activity. Possibility of applying full-length DGC of T. maritima in the form of inclusion bodies for synthesis of c-di-GMP was originally demonstrated.
