INVESTIGADORES
IRIBARREN Adolfo Marcelo
congresos y reuniones científicas
Título:
Chemical and enzymatic synthesis of modified nucleosides. Applications in functional oligonucleotides
Autor/es:
ADOLFO M. IRIBARREN
Reunión:
Conferencia; ESF-COST High-Level Research Conference NATURAL PRODUCTS CHEMISTRY, BIOLOGY AND MEDICINE II; 2009
Resumen:
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.