C. D’Alfonso et al. / Tetrahedron 70 (2014) 3049e3055
3053
favored by electron withdrawing substituents that increase the
3. Conclusions
BDENOeH of 6-X-HBTs as shown by the results of the UVevis radical
equilibration experiments (Table 1). Accordingly, the rate constants
for the HAT process from several classes of organic compounds by
6-CF3-BTNO are significantly higher than those observed in the
reactions promoted by BTNO.24,30 This conclusion has been con-
firmed by the results of the kinetic studies of the hydrogen atom
abstraction from 4-methoxybenzyl alcohol (6) and 1-(3,4-
dimethoxyphenyl)-2-phenoxyethanol (8) to the 6-X-BTNOs in
MeCN (Scheme 7).
The introduction of substituents in the aromatic ring of 1-
hydroxybenzotriazole caused a significant variation of the media-
tion efficiency in the laccase promoted oxidation of benzylic sub-
strates. A bell shaped profile is observed for the yields of the
oxidation products on going from the strong electron withdrawing
CF3 substituent to the electron releasing CH3O group with a maxi-
mum value observed for 6-CH3-HBT, which is by far the most effi-
cient mediator, even more efficient than the unsubstituted HBT.
This result can be rationalized considering that, for most of the 6-X-
HBT investigated, the overall reaction rate is governed by the oxi-
dation of the ring substituted HBTs to the corresponding N-oxyl
radical (6-X-BTNOs) by laccase with the exception of 6-CH3O-HBT
for which the substrate oxidation step becomes rate determining.
The particularly high mediation efficiency found with 6-CH3-
HBT represents an indication of a possible application of this
compound in combination with laccase, either for synthetic pur-
poses or for the environmentally benign oxidative delignification of
wood pulps and degradation of organic pollutants.
Scheme 7.
The kinetic studies were carried out by UVevis spectropho-
tometry generating the 6-X-BTNOs by oxidation of 6-X-HBTs with
cerium(IV) ammonium nitrate as reported in the literature.24,30,31
Using an excess of 6 or 8 under pseudo first-order conditions, the
observed rate constants (kobs) were measured by following the
decay of the 6-X-BTNOs at their absorption maxima. Clean first
order decays were observed and excellent linear fits were obtained
by plotting kobs as a function of the concentration of the substrates.
From the slope of these plots, the second order rate constants for
the HAT processes (kH) were determined. The kH values are re-
ported in Table 4. A significant increase of reactivity of the 6-X-
BTNOs in the HAT process from both 6 and 8 is observed by in-
creasing the electron withdrawing power of the substituent
thereby confirming the previous statement.
4. Experimental section
4.1. Instrumentation
1H NMR spectra were recorded on a Bruker AC300P spectrom-
eter in CDCl3. GCeMS analyses were performed on a HP5890 GC
(OV 1 capillary column, 12 mꢃ0.2 mm) coupled with a HP5970
MSD. GC analyses were carried out on a Varian CP-3800 (OV 1701
capillary column, 30 mꢃ0.25 mm). UVevis measurements were
performed on a HP Vectra 8453 Diode Array spectrophotometer.
4.2. Starting materials
CH3CN (spectrophotometric grade) was used for all the spectro-
photometric studies. Aqueous solutions were prepared using
Table 4
18.2 M
U
cmꢁ1 Milli-Q waterat25ꢀC obtainedfrom a Milliporesystem
Second order rate constants (kH) for the hydrogen atom transfer reaction from 4-
methoxybenzyl alcohol (6) and 1-(3,4-dimethoxyphenyl)-2-phenoxyethanol (8) to
aryl substituted benzotriazolyl N-oxyl radicals (6-X-BTNOs) in MeCN
(0.22
mm filter). Commercial samples of 1-hydroxybenzotriazole
(HBT), 6-trifluoromethyl-1-hydroxybenzotriazole (6-CF3-HBT), cer-
ium(IV) ammonium nitrate, citric acid, 4-methoxybenzyl alcohol,
3,4-dimethoxybenzyl alcohol were used as received. 6-Methyl-1-
hydroxybenzotriazole (6-CH3-HBT), 6-chloro-1-hydroxy-benzo-
triazole (6-Cl-HBT), 6-methoxy-1-hydroxybenzotriazole (6-MeO-
HBT), and 1-(3,4-dimethoxyphenyl)-2-phenoxyethanol, 1-(3,4-
dimethoxy-phenyl)-2-phenoxyethanone were synthesized accord-
ing to the literature.26,28a Laccase from a strain of Trametes villosa (viz.
Poliporus pinsitus) (Novo Nordisk Biotech) was employed. It was pu-
rified by ion-exchange chromatography on Q-Sepharose by elution
with a mixture of 10 mM TriseHCl and 0.2 M NaCl (linear gradient
1e70 mM NaCl)32 and its activity (9000 U/ml) was determined
spectrophotometrically by the standard reaction with ABTS.33
kH (Mꢁ1 sꢁ1
)
6
8
X¼CF3
X¼Cl
73ꢂ3
44ꢂ2
38ꢂ2
25ꢂ1
X¼H
9.3ꢂ0.3
5.8ꢂ0.5
7.9ꢂ0.3
4.8ꢂ0.5
X¼CH3
The significant increase in the HBTs mediation efficiency ob-
served with the electron donor substituent would seem to indicate
that the effect of the HBTs aryl substituent on step a of Scheme 5 is
more important than that on step b. With 6-CH3O-HBT, the rela-
tively low BDENOeH value likely determines a significant contribu-
tion of the hydrogen atom transfer step from the substrate to 6-
CH3O-BTNO thus decreasing the product yields. It is interesting to
note that 4-CH3O-NHPI resulted instead the more efficient media-
tor in the oxidation of lignin model compounds promoted by the
laccase/4-X-NHPIs/O2 systems.14 This difference can be rationalized
by considering that HBTs are characterized by lower redox poten-
tials and BDENOeH values with respect to NHPIs,14,16 thus the con-
tribution of the HAT step to the overall reactivity is more significant
in the series of HBTs mediators where 6-CH3O-HBT displayed
a lower efficiency than 6-CH3-HBT whereas 4-CH3O-NHPI was the
best mediator among the aryl substituted NHPIs.
4.3. Spectrophotometric studies
4.3.1. Determination of BDENOeH. In a 3 mL quartz cuvette 150 mL of
a 2 mM solution of cerium(IV) ammonium nitrate (CAN) in CH3CN
was added to 2.85 mL of a solution of the N-hydroxylamine (4-
MeO-HPI, 6-CF3-HBT or HBT, final concentration 10 mM) in
CH3CN. Then a 10 mM solution of N-hydroxyderivative (6-CF3-HBT,
HBT, 6-Me-HBT, or 6-Cl-HBT) in MeCN was added (final concen-
trations: 1 mM, 2 mM, and 3.3 mM). After the addition the UVevis
spectra were immediately recorded.
4.3.2. Kinetic studies of the HAT reactions from 4-methoxybenzyl
alcohol and 1-(3,4-dimethoxyphenyl)-2-phenoxyethanol. In a 3 mL
quartz cuvette 150 mL of a 5 mM CAN solution in CH3CN was added