Biocatalyzed hydrolysis and transesterification of phosphotriesters. Synthesis of pronucleotides and identification of new phosphotriesterase activities

LUCAS DETTORRE; JULIA SANTILLAN; JULIETA BERTANA LAHOURCADE; ELIZABETH LEWKOWICZ; ADOLFO IRIBARREN

Manchester

Simposio; XI Simposio Internacional de Biocatalisis y Biotransformaciones; 2013

Universidad de Manchester

Phosphotriesterases (PTEs; E.C. 3.1.8.1) catalize the stereoselective hydrolysis of a large number of phosphotriesters. These enzymes have been found along the evolution scale with exception of insects. being PTEs from Brevundimonas diminuta and Flavobacterium sp., and human and rabbit serum paraoxonases the best characterized ones. Since phosphotriesters are extensively used as pesticides and are also the basis of some chemical weapon, PTEs are potential detoxifying agents . With the aim of identifying new sources of bacterial PTEs, we screened our cell collection using a fluorogenic substrate, and performed further studies concerning the performance of the selected hydrolytic activities using different substrates, temperatures and pHs. In this way, we have identified new PTE activities belonging to strains of the genera Arthrobacter, Nocardia and Streptomyces . Although PTE hydrolytic activity is well stablished there is only one previous report regarding transesterification reactions. Therefore, we evaluated the phosphotriesterase catalyzed transesterification reaction in anhydrous solvent to obtain nucleoside phosphotriesters. Nucleoside analogues have been used as antiviral and anticancer drugs, however, these compounds present desadvantages, since they are rapidly catabolised to inactive derivatives, have poor bioavailability and in some cases the first enzymatic phosphorylation necessary to produce the active drug (nucleoside 5?-triphosphate) has been shown to be the rate limiting step. Thus, nucleoside 5?-phosphotriesters provide masked phosphates with enhanced lipophilicity and also make possible the intracellular release of the corresponding 5?-nucleoside phosphate thus skipping the first phosphorylation step. This strategy, called ?kinase bypass?, is essential in those cases where the nucleoside does not exert the expected biological property while its triphosphate derivative proved to be active. Different nucleoside 5?-dimethylphosphate phosphotriesters were obtained by the PTE catalyzed transesterification reaction in anhydrous solvent. The results showed that PTE was capable of recognizing pyrimidine and purine nucleosides as substrates, even when sugar moieties are modified, and that the transesterification occurs regioselectively through the 5? position