N-Arylhydroxamic Acids as Novel Oxidoreductase Substrates
FULL PAPER
Cs-1087, Cypress Systems, Inc., USA). As a reference, a saturated
calomel electrode (SCE, saturated with KCl, mod. K-401, Radio-
tions was set to 50. The maximum number of energy evaluations
that the genetic algorithm could make was 250 000. The maximum
meter, Denmark), and as auxiliary electrode a Pt wire (diameter number of generations was 27000. The number of the top indi-
0.2 mm, length 4 cm), mounted on the end of the reference elec-
trode, were used. The electrode potential was varied from 200 to
800 mV. The potential scan rate was 100 mV/s. The formal redox
viduals that are guaranteed to survive into the next generation was
1. Crossover rate and mutation rate was 0.02 and 0.80, respectively.
potential (E) was estimated by using the relationship E ϭ (Ep,a
ϩ
Ep,c)/2, where Ep,a and Ep,c are the anodic and the cathodic peak
potentials, respectively. The differential-pulse voltammetry (DPV)
was performed by using the same electroanalytical system and elec-
trodes. The formal redox potential was calculated as E ϭ (EpϩEh/
2), where Ep is a peak potential and Eh is a height of the pulse.
(10)
The Electron Spin Resonance (ESR) investigations of the radicals
were performed with a Bruker EMX 113 ESR spectrometer with a
ST4102 cavity. A borosilicate capillary tube was filled with 95 µL
of the reaction mixture and placed in the cavity. Care was taken
that the sample was placed at the same position each time. The
measurements were performed at 22.5 °C. The ESR spectrum of
the radical of 1a was recorded after 4.7 min incubation of a 0.1 m
solution of 1a, with 25 µ H2O2 and 16.7 n rCiP in 10 m phos-
phate buffer at pH ϭ 7.0. The parameters were: sweep time 20.9 s,
sweep width 41.720 G, center field 3373.66G, modulation ampli-
tude 0.1 G, modulation frequency 100 kHz, frequency
9.469545 GHz, and power 2.007 mW. Simulation of the ESR spec-
trum was performed using the WINEPR SimFonia program by
Bruker. The time profile of the radical was observed by monitoring
the amplitude of the ESR signal after preparing the reaction mix-
ture and introducing it into the instrument. The typical delay time
from mixing to recording was 30Ϫ40 s. Radical production and
decomposition was followed in a mixture containing 500 n rCiP,
0.5 m H2O2 and 0.2 m 1a in three buffer solutions: 50 m acet-
ate pH ϭ 5.5, 50 m phosphate pH ϭ 7.0, and 50 m borate pH ϭ
8.5. The signal amplitude was monitored in the static field of
3460.78 G and with the parameters: frequency 9.7459 GHz, modu-
lation frequency 100 kHz, modulation amplitude 2.5 G, power
0.6362 mW, receiver gain 1.12e5, time constant 1.31072.
Acknowledgments
The authors express their sincere thanks to MEMPHYS Ϫ Mem-
brane and Statistical Physics Group, Department of Chemistry, the
Technical University of Denmark, for kindly letting us use their
ESR instrument. We also wish to thank Rie Kristine Schjeltved
and Christine Ludvigsen for technical assistance and Arthur J. Ol-
son for supplying AutoDock 3.0 for the substrates docking calcula-
tions.
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3483