Coupled biocatalysts applied to the synthesis of nucleosides

ROSARIO MEDICI; MARISA TAVERNA PORRO; ELIZABETH LEWKOWICZ; JAVIER MONTSERRAT; ADOLFO IRIBARREN

Hamburgo, Alemania

Congreso; International Congress on Biocatalysis; 2008

Hamburg University of Technology

In order to evaluate the production of natural and modified nucleosides by the use of the combined action of PPM and NPs, a general chemoenzymatic approach for the preparation of furanose 5-phosphates starting from ribose, arabinose and 2-deoxyribose was developed1. A recombinant PPM was obtained and to determine the better conditions for the catalyzed reaction, the dependence of enzyme activity and reaction yield on pH, temperature, glucose 1,6-diphosphate, mercaptoethanol and phosphate concentrations was studied. For this purpose, the conversion of ribose 5-phosphate to adenosine, catalyzed by PPM and commercial PNP, was selected as the model system. Over expressed PPM was further tested coupled to two different commercial NPs, PNP and TP, and a set of natural and modified nucleosides were obtained. An interesting example was the synthesis of Ribavirine (Virazole) from ribose 5-phosphate and 1,2,4-triazole-3-carboxamide. The reaction proceeded quantitatively and with high activity in 1.5 h. As mentioned above, the poor solubility of guanine is a limitation to carry out microbial transglycosylation2. To solve this, guanosine was used as the base source to obtain 2’-deoxyguanosine (dG), since it has higher solubility than guanine. In spite of the increase of dG productivity, the purification was difficult due to the presence of uridine, obtained as by-product. Moreover, non-natural guanosine analogues like arabinoguanosine (AraG), cannot be prepared by this methodology because long reaction times were required and the half-life time of guanosine in the media is very short. Then, microbial transglycosylation was coupled with ADA in order to obtain this kind of compounds. The last reaction was carried out by whole cells of Arthrobacter oxydans instead of isolated ADA from mammalian sources as usually. This bacteria, selected from our cell collection, showed high ADA activity and was explored as biocatalyst for the preparation of some inosine and guanosine derivatives from the corresponding adenine and 2-amino-6-substituted purine analogues. The reaction times must be rigorously controlled since the products are probably substrates of their endogenous PNP. In particular, purine arabinosides as well as purine dideoxynucleosides are poor substrates of the Arthrobacter oxydans PNP and consequently, using this tool, 9-b-D-arabinofuranosyl guanine (AraG) was obtained in high yield.