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J. A. Trelles et al. / Tetrahedron Letters 44 (2003) 2605–2609
2. Experimental
3.4. Analytical methods
The synthesis of nucleosides was qualitatively followed
by TLC analysis. Silica gel plates were used with
Cl3CH/methanol 80:20 (v/v) as development solvent.17
2.1. Materials
Agar and agarose from Glacillaria were a generous gift
from HISPANAGAR S.A. (Spain). E. gergoviae CECT
875 was kindly supplied by the ‘Coleccio´n Espan˜ola de
Cultivos Tipo (CECT)’, Universidad de Valencia
(Spain). All chemicals were analytical or HPLC grade
quality and were obtained from commercial sources.
The production of purine nucleosides was quantita-
tively measured by HPLC from LDC Analytical, model
CM 4000. Samples from the supernatants were diluted
10 times with 30 mM phosphate buffer and analysed
with a C-18 column (250×4 mm) at a flow rate of 0.9 ml
min−1. The mobile phase was water/methanol 85/15
(v/v). The UV detector was set at 254 nm and the
column was operated at room temperature.
3. Growth conditions
E. gergoviae CECT 875 was cultured in 250 ml Erlen-
meyer flasks containing 50 ml culture medium: 1%
(w/v) meat extract, 0.5% (w/v) yeast extract and 0.5%
(w/v) NaCl in deionised water adjusted to pH 7 with
KOH. The cells were shacked at 37°C for 16 h and then
harvested by centrifugation for 10 min at 12 000 g.
The H NMR spectra of isolated compounds were com-
pared to those of commercial nucleosides and of the
ribo and 2%-doxyribo adenosines obtained in the labora-
tory, in order to confirm the b-configuration previously
described for nucleosides obtained with whole cells.5,7
3.1. Immobilisation of cells
Acknowledgements
3.1.1. Entrapment in agar or agarose. The whole cell
pellet was mixed with 10 ml of previously sterilised 2%
(w/v) agar or agarose. The homogeneous mixture was
then added dropwise to stirred sunflower oil (20 ml) at
25°C for 5 min. The resulting gel beads (mean diameter:
4 mm, load) were cooled, filtered, washed with hexane
and then with physiological solution to obtain free
solvent beads.
This work was supported by Project BQU 2001-1301
from Ministerio de Educacio´n y Ciencia of Spain and
by the Agencia Espan˜ola de Cooperacio´n Internacional
(Project PR158/01-100 65) and by Universidad
Nacional de Quilmes, Secretar´ıa de Ciencia y Te´cnica
de la Nacio´n, Argentina. AMI is research member of
CONICET, Argentina.
3.2. Biotransformation conditions
References
3.2.1. Free cells. The standard reaction mixture com-
prising: wet cell paste, 0.15 mmol of uridine, 0.05 mmol
of adenine and 30 mM pH 7 potassium phosphate
buffer (5 ml), was stirred at 200 rpm in a orbital shaker
and 60°C for 1 h. Samples were centrifuged at 10 000 g
for 30 s and the supernatants were analysed by both
TLC and HPLC.
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versidad de Valencia, Spain.