Oxidation of non-phenolic substrates with the laccase/N-hydroxyacetanilide system: Structure of the key intermediate from the mediator and mechanistic insight

Article abstract of DOI:10.1039/b311907f

We have investigated the reactivity and mechanistic features in the oxidation of non-phenolic substrates by the enzyme laccase under mediation by N-hydroxyacetanilide, NHA. A radical route of hydrogen-abstraction by the 2 bonds on the righthand side signN

Full text of DOI:10.1039/b311907f

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P A P E R
Oxidation of non-phenolic substrates with the
laccase/N-hydroxyacetanilide system: Structure of
the key intermediate from the mediator and mechanistic insight
Gaetano Cantarella, Carlo Galli* and Patrizia Gentili*
Dipartimento di Chimica and IMC-CNR Sezione Meccanismi di Reazione,
`
Universita ‘La Sapienza’, P.le A. Moro 5, 00185, Roma, ITALY.
E-mail: carlo.galli@uniroma1.it; Fax: þ39 06 490421
Received (in Durham, UK) 26th September 2003, Accepted 13th January 2004
First published as an Advance Article on the web 9th February 2004
We have investigated the reactivity and mechanistic features in the oxidation of non-phenolic substrates by the
enzyme laccase under mediation by N-hydroxyacetanilide, NHA. A radical route of hydrogen-abstraction
by the QN–Oꢀ reactive intermediate of the mediator, formed in a preliminary oxidative interaction with the
enzyme, seems the more viable oxidation mechanism, in keeping with analogous conclusions reached for other
laccase/N–OH-type mediators. The evaluated value of the energy of the O–H bond of NHA corroborates the
occurrence of hydrogen-abstraction from the benzylic substrates by the QN–Oꢀ iþntermediate of NHA. The
=
occurrence of an alternative ionic route through the oxoammonium ion (QN O ) of mediator NHA is ruled
out by experimental evidence acquired through an Hammett structure/reactivity correlationþin the oxidation of
=
substituted benzyl alcohols, as well as by kinetic isotope effect determinations. The QN O species of NHA,
being a one-electron oxidant of moderate strength, could in principle even be responsible for an alternative
electron-transfer route of oxidation of the substrates. This hypothesis could also be dismissed through the use
of probe substrates and intermolecular selectivity determinations.
in bioremediation of soils and water,5c,11 or for an
environmental-benign bleaching process of kraft pulps for
paper making,7c,7d,12 which hopefully will enable to circumvent
the use of polluting chlorine-based treatments.
Introduction
Laccase, a family of ‘blue-copper’ oxidases containing four
copper ions, is excreted by white-rot fungi under ligninolytic
conditions.1,2 It cooperates with other enzymes, in particular
with lignin peroxidase (LiP)3 and manganese peroxidase
(MnP),4 in the degradation of the biopolymer lignin in woody
tissues. With respect to LiP and MnP, that are more powerful
oxidants,1 laccase has a lower redox potential (0.7–0.8 V/
NHE)1,5 and therefore is able to catalyse single-electron oxida-
tion steps only with the easy-to-oxidise phenolic constituents
of lignin, with the concomitant reduction of O2 to water.
Advantages of laccase are that it is more readily available
and easier to manipulate than LiP and MnP, and that its sub-
strate specificity is low, as long as a good match of oxidation
potentials is provided.1,2,5a,6 Additionally, the use of appropri-
ate mediators makes non-phenolic compounds, that are more
difficult to oxidise, to become substrates of laccase.5d,7 The role
of mediators in laccase oxidations is outlined in Scheme 1.
The mediator needs to be easily oxidised by laccase to the
Medox status: the structure of the latter is then crucial for
the mechanism of the ensuing non-enzymatic oxidation of
the substrate. In fact the Medox species may rely on a mechan-
ism of oxidation, radical for example, which differs from that
of the direct laccase oxidation.8 In this way the mediators
turn laccase into a much more versatile enzyme, as already
exemplified by various biotechnological applications, e.g. in
the textile dye bleaching,9 in selective organic transformations,10
We have undertaken a systematic mechanistic investigation
on the mediation phenomenon with laccase,8 and have already
reported on a comparative evaluation of a number of media-
tors in the oxidation of benzylic alcohols, taken as non-pheno-
lic lignin models.7e The mediators HBT, HPI, VLA (structures
and shortened names in Fig. 1), all endowed with the QN–OH
functionality, or TEMPO (2,20,6,60-tetramethylpiperidine-N-
oxyl), a stable QN–Oꢀ radical, presented the more promising
features as catalysts in this oxidation.8,13 For example, a sim-
ple and synthetically-attractive oxidation of alcohols to carbo-
nyl compounds by oxygen, induced by the laccase/TEMPO
system, has been developed.10f
In the present paper we investigate on N-hydroxyacetanilide
(NHA), another N–OH-type mediator of laccase. At variance
with the other mediators, NHA is not commercially available
Scheme 1 Catalytic cycle of a laccase/mediator oxidation system.
Fig. 1 Structures and abbreviated names of the mediators.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
366
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 6 6 – 3 7 2

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was rather slow (20 mV sꢁ1),15 we wanted to repeat the
and this has limited so far studies aimed at investigating its
efficiency and mechanism of oxidation.5d Previous work had
given firm indication that a radical route is followed by media-
tors HBT, HPI and VLA with laccase,5d,7b,7e,8,14 in their oxida-
tion of benzyl alcohols (and ethers) to carbonyl products. We
wished to confirm this behaviour with NHA, in view of the
common N–OH functionality. To this aim, the following
experimental evidence has been acquired in the oxidations with
the laccase/NHA system: (i) reactivity trends with significant
substrates; (ii) product patterns with suitable ‘scout’ precur-
sors; (iii) effect of substituents in the oxidation of benzyl alco-
hols (Hammett correlation); (iv) determination of the kinetic
isotope effect. An electrochemical investigation on the redox
features of NHA has been preliminarily carried out, and also
the energy of the O–H bond of its N–OH functionality eva-
luated, in order to acquire additional clues in support of the
various possible oxidation mechanisms. Purified laccase from
Trametes villosa (viz. Poliporus pinsitus) has been employed,
and mediator NHA synthesized according to the literature.
measurement at a faster scan rate (up to 5 V sꢁ1), in order
to verify how this could affect the irreversibility/reversibility
of the second electrochemical stage. Unfortunately, serious
adsorption problems of NHA on the surface of the glassy car-
bon electrode prevented us from any reliable determination.
We could detect only the presence of an irreversible anodic
peak at about 1.1 V/NHE, at 5 mV sꢁ1
.
This electrochemical discussion has general relevance with
respect to the mediation phenomenon by N–OH compounds
with laccase. In fact, mediators HBT, HPI and VLA are all
oxidised in CV experiments according to the first step of
Scheme 2,7e,18 with E1 values of 1.09, 1.08, and 0.92 V/
ꢂ
NHE, respectively, that are reversible at sweep scan faster than
1 V sꢁ1 7e Further oxidation of the corresponding QN–Oꢀ spe-
.
cies to the oxoammonium ion (second stage in Scheme 2)
ought to occur (if any) at E2 values more positive than 1.3
V, that cannot be experimentally attained due to the discharge
of the electrolytes in solution. Clearly, these higher redox
potentials are not accessible either to the oxidation capacity
of laccase (0.7–0.8 V),1,5 and therefore the oxidation mechan-
ism followed by these laccase/ QN–OH systems with non-
phenolic substrates (e.g., benzyl alcohols) is associated with
the unique formation of the QN–Oꢀ species, and proceeds
through the radical (HAT; Scheme 3) route extensively
documented.5d,7b,7e,8,14
Results and discussion
Electrochemical study
The electrochemical oxidation of NHA had been previously
investigated by cyclic voltammetry (CV), and two oxidation
steps detected.15 The first step was attributed to a quasi-rever-
sible one-electron transfer, occurring at 0.62 V, followed by a
second irreversible electron transfer at 0.97 V, both values
being obtained at a 20 mV sꢁ1 scan rate vs. NHE, in buffered
(pH 7.4) water/methanol (9/1 v/v) solution. This evidence was
attributed to the following electrochemical/chemical events
(Scheme 2).15
On the other extreme, TEMPO is a stable QN–Oꢀ species,19
þ
=
whose oxidation to a QN O ion is easier and occurs at
0.7 V.13,20 Such oxidation is possible for laccase, in view of
its redox potential,1,5 and consistently the oxidation of
benzyl alcohols with the laccase/TEMPO system can
take place through the oxoammonium ion, and it is ionic
(Scheme 4).7e,8,13
ꢀ
þ
The case of NHA could be somewhat intermediate. Its E1
value (0.7–0.8 V) is the lowest among the QN–OH mediators
and certainly at easy reach of laccase, but even the second oxi-
In the first electrochemical step the NHA species is gene-
rated, but its deprotonation to the amino-oxyl radical competes
with back-electron transfer, thereby making the E1 value lar-
gely irreversible. We have repeated this CV experiment and
scanned the potential up to sweep rates as fast as 5 V sꢁ1, cer-
tainly faster than the 20 mV sꢁ1 rate reported in the litera-
ture,15 without obtaining a well-structured reverse CV wave,
and therefore no full reversibility. We tentatively provide a
0.8 V vs. NHE value for the quasi Eꢂ1 of NHA in soꢀdium ace-
þ
=
dation step to the QN O ion (E2 of ca. 1 V) could be acces-
sible to the enzyme. Even though the oxoammonium ion of
NHA is short-lived (as suggested by the second anodic peak,
which is irreversible at 5 mV sꢁ1), the possibility could exist
that in a laccase/NHA oxidation of a non-phenolic substrate
þ
=
the transient QN O intermediate is partially stabilised by
proximity with the negatively charged enzymatic pocket.21
This could extend its life-time and make it responsible for
either the occurrence of an ionic oxidation route (according
to Scheme 4), or even an electron transfer (ET) route (cf.
Scheme 5) with suitably oxidisable non-phenolic substrates.
This could occur in combination/superposition with the radical
HAT route (Scheme 3) via the QN–Oꢀ species.
þ
tate buffer (pH 4.7), and point out that the NHA species
seems more short-lived than reported.16 In another electro-
chemical investigation on NHA and derivatives, an E1 value of
0.72 V/NHE (pH 6, phosphate buffer) was in fact given, at
scanning rates of about 20–500 mV sꢁ1, but described to be
reversible.16 A similar E1 value of 0.75 V/NHE at pH 4.5
was obtained in a recent CV determination, at a slower scan
rate (100 mV sꢁ1).17
We planned to investigate these possible mechanistic
alternatives, which depend on different reactive intermediates
originating from the NHA mediator.
A second oxidation wave is appreciable in the CV scan of
NHA, and it has been attributed to the oxidation of the QN–
Oꢀ species formed in the first electrochemical/chemical
stage (Scheme 2).15–17 The transient formation of an oxoam-
monium ion has been postulated, which easily loses acetylium
ion thereby explaining the irreversibility of the E2 value.
Once again, because the sweep scan employed in the study
Thermochemical considerations
Thermochemical data concerning the enthalpy of the O–H
bond (BDEOH) of N–OH mediators are accessible for a few
QN–OH compounds structurally similar to NHA (Fig. 2).22
Scheme 2 Electrochemical/chemical steps in the oxidation of NHA.
Scheme 3 The radical HAT route.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 6 6 – 3 7 2
367

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Fig. 2 BDEOH data, in kcal molꢁ1
.
However, and at variance with the results obtained with other
QN–OH mediators,8,25 the extent of oxidation by laccase/
NHA in this 4% MeCN mixed solvent resulted very meagre.
A 1:1 buffered-water:dioxane mixed solvent (i.e., 50% dioxane)
was then adopted,26 where the extent of oxidation became
higher and comparable with that of the other laccase/N–
OH-type systems towards the same substrates. Therefore, a
specific solubility problem of mediator NHA does emerge here.
Apart from the marked difference in conversion between the
two mixed solvents, the 50% dioxane solvent clearly shows that
the laccase/NHA system is able to oxidise even the unsubsti-
tuted precursor 1 (to 2) to a significant extent (32%), the con-
version slightly increasing with the electron-donor substituted
substrates 3 and 5, to the corresponding aldehydes 4 and 6.
Even the oxidation of an electron-poorer substrate, such as
4-chlorobenzyl alcohol (7), occurs appreciably under these
conditions (30%). Comparison between the yield of product
and the amount of substrate recovered (bracketed values, in
Table 1) ensures that the mass balance is good in all cases,
and that no other products are formed besides those indicated.
It must be stressed that, in the absence of NHA, laccase does
not oxidise the non-phenolic substrates on its own, nor does
the mediator in the absence of the enzyme: it is therefore the
whole laccase/NHA system that is responsible for the oxida-
tion of the substrates, in consistency with the operation of
Scheme 1.7
The negligible relevance of substrate electron-richness upon
reactivity in these laccase/NHA oxidations is confirmed by a
competition experiment of 3 and 1, giving a k3/k1 relative rate
as small as 1.9. This complies with the operation of the radical
mechanism of oxidation (Scheme 3) common to the other N–
OH mediators.8 Further support to the operation of the HAT
route comes from use of another mechanistic test.27 Oxidation
of secondary benzyl alcohol 9 by laccase/NHA gives the
ketone 10 in good yield (67%), and 9 results easier to oxidise
than 3 in a competition experiment (k3/k9 ¼ 0.72). Abstrac-
tion of H-atom from secondary substrate 9 is indeed expected
to be easier than from primary 3 in a radical process, due to the
C–H bond energy value that is lower (85.4 kcal molꢁ1) for 9
than for 3 (88.0 kcal molꢁ1).8,27 Consistently, k3/k9 values
lower than 1 were obtained with all the other QN–OH media-
tors HBT (0.7), HPI (0.8) and VLA (0.4) in the same mixed
solvent, whereas the laccase/ABTS system, which follows an
ET route of oxidation, gave a k3/k9 value higher than 1 (i.e.,
1.5).8 However, because even the laccase/TEMPO system gave
a k3/k9 value lower than 1 (i.e., 0.78),13 the incursion of the
ionic route (Scheme 4) in competition with the HAT route
cannot be ruled out on this basis only.
Scheme 4 The ionic route.
Because the nitrogen atom of NHA is analogously bonded
to a single carbonyl group, and not to two carbonyl groups
that confer a BDEOH as high as 88.1 kcal molꢁ1 to HPI (cf.
Fig. 1),23 it is legitimate to infer a BDEOH value of roughly
79 kcal molꢁ1 for NHA as well. Accordingly, abstraction of
a benzylic H-atom from a benzyl alcohol (HAT route of
Scheme 3) by the amino-oxyl radical of NHA would be
approximately thermoneutral (Scheme 6), and possibly
proceed at an acceptable rate.
The corresponding HAT process is instead exothermic by ca.
6 kcal molꢁ1 for the QN–Oꢀ species deriving from HPI
(BDEOH of 88.1 kcal molꢁ1),23 and indeed it occurs easily,22
whereas it would be endothermic by ca. 12 kcal molꢁ1 with
TEMPO (BDEOH of 69.6 kcal molꢁ1),24 and therefore does
not occur and the ionic route (Scheme 4) takes over.13 Thermo-
chemistry (Scheme 6) confirms an intermediate position of
NHA among its congeners, and indicates the HAT route
(through QN–Oꢀ) as only a possible mechanistic alternative
þ
=
to the ET or ionic routes (through QN O ) for this mediator.
Reactivity data for laccase/NHA reactions were indeed
strongly needed in order to solve this mechanistic conundrum,
and be able to rule out the last two mechanistic hypotheses.
Reactivity of substrate
In keeping with our previous studies, it was deemed mechanis-
tically significant to investigate the efficiency of oxidation by
the laccase/NHA system with three specific benzyl alcohols,
i.e., PhCH2OH (1), p-anisyl alcohol (3) and veratryl alcohol
(5). Because these compounds widely differ in redox potential,
as reported in Table 1 (Eꢂ in V/NHE, in H2O),7e this should
cause a sizeable difference in reactivity among them, if the oxi-
dation of the substrate by the laccase/NHA system were to
proceed by the ET route (Scheme 5). In contrast, the redox fea-
tures of the substrate should have marginal importance on
reactivity both in the radical (Scheme 3) and ionic (Scheme
4) routes of oxidation.8,13,14c,14d
These mediated oxidations were performed at room tem-
perature in a stirred water solution buffered at pH 5, and
Oxidation of ‘scout’ substrates 11 and 13 with laccase/
NHA gives the corresponding carbonyl derivatives 12 and 14
purged with O2 prior to the addition of the reagents.7e,8
small amount of MeCN (i.e., 4% MeCN) was also added, in
order to ensure solubility of both substrate and mediator.
A
Scheme 5 The ET route.
Scheme 6 Hydrogen atom abstraction: thermochemical evaluation.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
368
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Table 1 Oxidation of selected substrates by the laccase/NHA system, at room temperature for 24 h
Product yield, in % vs. substrate
Substrate (Eꢂ in V/NHE, in H2O)a
Oxidation product
Solvent: 4% MeCN
Solvent: 50% dioxane
benzyl alcohol, 1 (2.4)
benzaldehyde, 2
3 (98)b
4 (97)b
16 (84)b
–
32 (67)b
54 (45)b
59 (42)b
30 (65)b
67 (31)b
4-MeO-benzyl alcohol, 3 (1.8)
3,4-dimethoxybenzyl alcohol, 5 (1.4)
4-Cl-benzyl alcohol, 7
4-MeO-benzaldehyde, 4
3,4-dimethoxy-benzaldehyde, 6
4-Cl-benzaldehyde, 8
1-(4-MeO-phenyl)-ethanol, 9
4-MeO-acetophenone, 10
–
11
12
4 (96)b
9 (91)b
13
14
–
25 (76)b
k3/k1 relative ratec
k3/k9 relative ratec
1.9
0.72
–
–
a
Conditions: [substrate] 20 mM, [mediator] 6 mM, with 15 units of laccase. The yield of oxidation was determined by GC or HPLC analyses.
c
Recovered substrate (%). In competition experiments.
b
as the unique products (Table 1). These two substrates are
Hammett correlation
viewed as mechanistic probes because they give different
products depending on the oxidation route,8,14c,28 and give
precisely the carbonyl products in a bona fide HAT process
(Scheme 7), but give (mostly) the Ca–Cb cleavage products in
a bona fide ET process. For example, with the laccase/ABTS
system, which follows the ET route,8 or with chemical mono-
electronic oxidants,28 or also under anodic oxidation,29 11
and 13 gave the cleavage products 4 and 6, respectively, which
are not observed with the laccase/NHA system nor with any
other laccase/N–OH-type mediator.8,14c Comparable mecha-
nistic dichotomies are known and have been reported with a
few other substrates.29,30
While this would seem to unambiguously indicate the occur-
rence of the HAT route with laccase/NHA, we must observe
that even the oxidation with laccase/TEMPO did give ketone
14 from precursor 13 as the unique product, albeit in low
yield.13 Because this took place through the ionic route
(Scheme 4), the mechanistic ambiguity between the latter and
the HAT route still persists!
In view of these subtleties, and for a more exhaustive charac-
terisation of the mechanism of oxidation with the laccase/
NHA system, a quantitative treatment of the effect of substitu-
ents upon reactivity has been performed through a Hammett-
type correlation. Four X-substituted benzyl alcohols, i.e.,
4-CF3-, 4-Cl- (7), 4-Me-, 4-MeO–C6H4CH2OH (3), have been
oxidised pair-wise in competition experiments vs. the unsubsti-
tuted precursor (1) as the relay compound,8 and the kX/kH
ratios reckoned by determining the relative amounts of the
corresponding aldehydes by GC analysis (Table 2). For a
closer comparison with the analogous studies performed with
the other QN–OH mediators, the 4% MeCN mixed solvent
has been employed.8 The consequent lower conversion (cf.
Table 1) is not a drawback in this contest, because a low extent
of oxidation is required for a kinetically significant application
of the competition treatment. The obtained log kX/kH ratios
give a better fit when plotted vs. the sþ substituent parameters
(Fig. 3) rather than vs. the s ones,31 and the resulting r corre-
lation parameter is given in Table 2. For the sake of compar-
ison, an analogous Hammett treatment has been conducted by
using the ET oxidant ABTS2þ (Eꢂ 1.1 V),7e independently
generated by a chemical oxidant (see Experimental) without
laccase,14c in view of the similarity of its redox potential with
the E2 value of NHA (1.0–1.1 V).
The r parameter for the laccase/NHA system (ꢁ0.42) is
small in value and very comparable with those of the other lac-
case/ Q N–OH-type systems (r values in the ꢁ0.4 to ꢁ0.9
range),8,14d as expected for a HAT route through the QN–Oꢀ
Table 2 Determination of the r value for the laccase/NHA system,
and with ‘pre-formed’ ABTS2þ, by oxidations of p-X-C6H4CH2OH
vs. PhCH2OH, in competition experiments at room temperaturea
Oxidant
kp-NO /kH kp-CF /kH kp-Cl/kH kMe/kH kMeO/kH qb
2 3
laccase/NHA
ABTSþþ
—
0.59
—
0.96
0.85
1.3
8.7
2.2
26
ꢁ0.42
ꢁ1.3
0.35
a
Determined by duplicated GC analyses; typical error ꢃ4%. See Experimental
for conditions. Obtained vs. the sþ parameters.
b
Scheme 7 Performance of the mechanistic probes.
T h i s j o u r n a l i s Q T h e R o y a l S o c i e t y o f C h e m i s t r y a n d t h e
C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 6 6 – 3 7 2
369

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Fig. 3 Hammett structure–reactivity correlation with the laccase/
NHA system: oxidation of 4-X-substituted-benzyl alcohols in competi-
tion experiments.
Scheme 8 Intramolecular (above) and intermolecular (below) kinetic
isotope effect studies.
intermediate (Schemes 3 and 6); in contrast, the slightly larger
r value of ABTS2þ is more in line with r values obtained for
bona-fide chemical ET oxidants.14d More importantly, how-
ever, the analogous Hammett treatment for the oxidation of
the same substrates by the laccase/TEMPO system gave a V-
shaped plot vs. the sI parameter, consistent with the operation
of the ionic oxidation route (Scheme 4).13 In this case the alco-
hol substrate behaves as a nucleophile in its rate-determining
addition to the oxammonium ion, as long as the substituent
is electron-withdrawing; moving to electron-donor substitu-
ents, the addition step becomes increasingly faster and the
rate-determining step shifts to the deprotonation of the
adduct, which is indeed retarded by electron-donor groups.13
This outcome makes a noteworthy difference with the present
linear plot of the NHA case (Fig. 3), and definitely rules out
the operation of the ionic route.
indeed due to the deprotonation of the adduct of the ionic
route (Scheme 4),19 formed by the addition of the alcohol to
the TEMPO-oxammonium ion, which is rate-determining (cf.
previous section) with electron-rich alcohols,13 such as 5.
Conclusions
The present work investigates reactivity and mechanistic
features of the laccase/NHA system in the oxidation of non-
phenolic substrates (i.e., benzyl alcohols) (cf. Scheme 1), and
attempts to discriminate between the involvement of either
one the oxidation forms (Medox) of mediator NHA, i.e., the
ꢀ
þ
=
QN–O or the QN O species (cf. Scheme 2). Experimental
evidence acquired through the Hammett correlation, and the
kinetic isotope effect (inter- and intra-molecular), contrasts
with the possible occurrence of an ionic route (Scheme 4) of
=
þ
Kinetic isotope effect determinations
oxidation through the QN O species of NHA. The reactive
behaviour of two mechanistic-probe substrates (11 and 13),
as well as the value of the k3/k9 intermolecular selectivity, con-
trast with the occurrence of an ET route of oxidation (Scheme
5). The radical HAT route (Scheme 3) through the QN–Oꢀ
species is therefore the more likely possibility for the laccase/
NHA system, in keeping with the analogous conclusion
reached in the oxidations with other laccase/N–OH-type med-
iators.8 The evaluated enthalpy of the O–H bond of NHA is
consistent with the occurrence of an almost thermoneutral
radical H-abstraction route of oxidation via the QN–Oꢀ inter-
mediate. It can be finally observed that laccase oxidations
mediated by N–OH-type compounds, which proceed through
the QN–Oꢀ species, appear promising for a selective delignifica-
tion of wood pulp for paper manufacture, because they are
respectful of the integrity of cellulose.33 In contrast, the
In the HAT mechanism of Scheme 3, cleavage of a Ca–H bond
is the rate-determining step of the oxidation process, whereas
in the concurrent ET route Ca–H deprotonation of the radical
cation of the substrate comes after the rate-determining mono-
electronic oxidation of the substrate (cf. Scheme 5).14c,27,32
This difference in rate-determining steps had already enabled
us to corroborate the HAT route of oxidation with the other
laccase/ Q N–OH-type mediators.8 The determination has been
extended here to the laccase/NHA system on a suitable mono-
deuteriated benzyl alcohol, such as 5-D. In the HAT route,
either Hꢀ or Dꢀ ought to be intramolecularly abstracted from
precursor 5-D, with the concomitant formation of either
Ar–CDO or Ar–CHO, respectively (Scheme 8).
Mass spectrometric determination of the relative amount of
the two aldehydes (6 and 6-D) allowed to determine the intra-
molecular kH/kD selectivity (Table 3).14c The obtained value,
i.e. 5.2, although being significantly larger than 1, and there-
fore consistent with a rate-determining C–H or C–D bond
cleavage, is slightly smaller (close to the limits of the experi-
mental errors) than the intramolecular kH/kD values obtained
with the other HAT oxidants, ranging from 6.2 to 6.5.8,14c The
determination was therefore repeated in an intermolecular
mode, by competition of un-deuterated (5) and bis-deuterated
(i.e., 5-D2) precursors, and provided the slightly larger kH/kD
value of 6.4, fully consistent with those of the other HAT med-
iators.8,14c For comparison, the intramolecular kH/kD value
determined for the laccase/TEMPO system with substrate 5-
D, i.e. 2.6, is sizably different from that of laccase/NHA, well
beyond the experimental errors. This smaller kH/kD value is
Table 3 Kinetic isotope effect determinationsa
b
Oxidant
kH/kD
Laccase þ HBT
Laccase þ HPI
6.4c
6.2c
5.2d
2.6
Laccase þ NHA
Laccase þ TEMPO
a
Approximate errors in the triplicated determinations: ꢃ8%. See
b
Experimental for conditions. See Scheme 8. Intramolecular values,
obtained with substrate 5-D. c Data from ref. 8. The intermolecular
value, obtained from a mixture of 5 and 5-D2 , is 6.4
d
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C e n t r e N a t i o n a l d e l a R e c h e r c h e S c i e n t i f i q u e 2 0 0 4
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N e w . J . C h e m . , 2 0 0 4 , 2 8 , 3 6 6 – 3 7 2

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determined by GC analysis with respect to an internal stan-
laccase/TEMPO system oxidises not only the non-phenolic
subunits of lignin but also the primary C6 alcohol functþional-
cies,35 thereby reducing the strength of the paper and resulting
less valuable as a biotechnological alternative.
dard (acetophenone or p-methoxyacetophenone), suitable
response factors being determined from authentic products,
and calculated with respect to the substrate molar amount.
With less volatile compounds, a HPLC analysis was employed,
using benzophenone as the internal standard.33a The competi-
tion experiments for the Hammett correlation were similarly
run on a 60 mmol amount of each of the substrates, dissolved
in 0.25 mL MeCN, and added to 6 mL of citrate buffer (over-
all, 4% MeCN) along with 20 mmol of NHA and 10 units of
laccase, and the yields of products determined after 3–6 h.
Relative reactivity values were calculated by the use of the
standard integrated equation for competitive reactions.8 Unex-
pectedly, 4-NO2-benzyl alcohol gave analytical problems with
the laccase/NHA system, so that it was necessary to resort to
another electron-withdrawing substituted benzyl alcohol, such
as 4-CF3-C6H4CH2OH, for the Hammett treatment. In order
to reckon the kH/kD ratios for the kinetic isotope effect deter-
minations, both intra- and intermolecular ones, the relative
amounts of the Ar-CHO and Ar-CDO oxidation products
(Scheme 8) were determined by GC-MS analyses in the SIM
mode, after a 5 h reaction time either from 5-D or from an
equimolar mixture of 5 and 5-D2 .
ity of the polysaccharide cellulose,34 through the QN O spe-
=
Experimental section
General remarks
NMR spectra were taken on a AC 200 Bruker instrument. A
VARIAN CP 3800 instrument, fitted with a 30 m ꢄ 0.25 mm
methyl silicone gum capillary column (CPSil5CB), was
employed in the GC analyses. The identity of the products
was confirmed by GC-MS analyses, run on a HP 5892 GC,
equipped with a 30 m ꢄ 0.2 mm methyl silicone gum capillary
column, and coupled to a HP 5972 MSD instrument, operat-
ing at 70 eV. A Hewlett-Packard 1050 HPLC system (pump,
detector, and solvent delivery system), equipped with a Supel-
cosil LC-18-DB 25 cm ꢄ 4.6 mm column and a HP 3395B
integrator, was employed in the HPLC analyses. These were
carried out at 0.5–1 mL minꢁ1 flow rate with
water:MeOH mixed solvent as the eluent.
a 3:2
Chemical oxidations
Materials
In the Hammett treatment with ‘preformed’ ABTS2þ, 20 mmol
of ABTS were dissolved in 1.5 mL of 2 M H2SO4 ; 40 mmol of
Many of the substrates and products were commercially avail-
able (Aldrich) and used without further purification. Other
precursors and products were available from previous investi-
gations,7e,8,13,25,33a including the mono- and bis-deuteriated
alcohols (5-D and 5-D2).36 A multi-step literature procedure
was followed for the synthesis of NHA,37 which requires the
preliminary reduction of nitrobenzene to phenylhydroxyl-
amine over Zn powder, followed by acetylation of the latter.
The final compound was obtained in a 55% overall yield as a
yellow solid, mp 63–65 ꢂC (lit.37 65–66 ꢂC), with the following
1H-NMR spectrum [d (ppm) in CDCl3 : 2.1 (s, 3H, PhN(OH)-
COCH3), 7.3–7.4 (m, 5H, PhN(OH)COMe), 8.9 (broad, 1H,
ArN(OH)COMe).
IV
(NH4)2Ce (NO3)6 (viz. CAN) dissolved in 1.5 mL of 2 M
H2SO4were added, and the red colour of ABTS2þ developed
immediately.14c A 60 mmol amount of each of the two compet-
ing substrates was added very quickly at this point, and the
resulting solution stirred at room temperature for 3 min, or
until the red colour had turned blue (i.e., ABTSꢀþ). Conven-
tional work-up followed.
Electrochemical measurements
Cyclic voltammetry (CV) experiments were carried out at
25 ꢂC in a three-electrode circuit, using an electrochemical
computerized system (Amel System 5000, version 2.1) and a
glassy-carbon working electrode (planar disk, diameter 3
mm). A saturated calomel electrode (SCE) was used as refer-
ence and a platinum cylinder electrode (surface 1 cm2) was
employed as an auxiliary electrode. The E values are referred
to NHE. The scanning rate range was from 0.005 V sꢁ1 to 5
V sꢁ1. In the CV experiments, NHA was 1 mM in a 0.01 M
sodium acetate buffer solution containing 0.1 M LiClO4 .
Enzyme purification
Laccase from a strain of Trametes villosa (viz. Poliporus pinsi-
tus) (Novo Nordisk Biotech) was employed. It was purified by
ion-exchange chromatography on Q-Sepharose by elution with
phosphate buffer, and laccase fractions having an absorption
ratio A280/A610 of 20–30 were considered sufficiently pure.6
The collected fractions were concentrated by dialysis in cellu-
lose membrane tubing (Sigma) against poly(ethylene glycol)
to a final activity of 9000 U/mL, as determined spectrophoto-
metrically by the standard reaction with ABTS.38
Acknowledgements
Thanks are due to Novo Nordisk Biotech (Denmark) for a
generous gift of laccase, and the EU for financial support
(grant QLK5-CT-1999-01277) to the OXYDELIGN project.
Enzymatic reactions
The oxidation reactions were performed at room tempera-
ture.7e,8 In order to ensure solubility of both substrate (60
mmol) and NHA (20 mmol), they were weighted in the same
vial, dissolved in 120 mL of MeCN and added to 3 mL of
the buffered water solution (pH 5; 0.1 M in sodium citrate),
so that a final 4% MeCN mixed solvent resulted. The proper
amount of a diluted solution of purified laccase was then
added. The buffer solution had been purged with O2 for 30
min prior to the addition of the reagents. Analogously, the
50% dioxane mixed solvent was prepared by employing 1.5
mL of dioxane to dissolve substrate and NHA, and then add-
ing it to 1.5 mL of the buffer solution. The concentration of the
reagents was: [substrate] 20 mM, [NHA] 6 mM, with 15 units
of laccase. The reaction time was 24 h, in general, an atmo-
sphere of oxygen being kept in the reaction vessel by means
of a hemi-inflated latex balloon. The yields of oxidation were
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