A study of the oxidation of ethers with the enzyme laccase under mediation by two N-OH-type compounds

Article abstract of DOI:10.1039/b210978f

The oxidation of ethers of various structure by O₂ with two laccase/>N-OH systems (i.e., HBT, 1-hydroxybenzotriazole, and HPI, N-hydroxyphthalimide) is described. The process affords carbonylic products in reasonable-to-good yields. The oxidation is carried out by the intermediate >N-O^? species of the mediators, through a radical H-atom abstraction (HAT) route that represents an elaboration of the HAT route followed with benzyl alcohols. Alternative mechanisms have been considered and dismissed. A comparison with a similar oxidation of ethers by the laccase/TEMPO system, reported previously, is made. A clear-cut specialisation of the mediator vs. the substrate emerges, i.e. the laccase/HBT system is more competent for the oxidation of ethers, whereas the laccase/TEMPO system was most proficient in the oxidation of benzyl alcohols.

Full text of DOI:10.1039/b210978f

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A study of the oxidation of ethers with the enzyme laccase under
mediation by two N–OH–type compounds
Francesca d’Acunzo, Paola Baiocco and Carlo Galli*
`
Dipartimento di Chimica and Centro CNR Meccanismi di Reazione, Universita ‘La Sapienza’,
P.le A. Moro 5, 00185 Roma, Italy. E-mail: carlo.galli@uniroma1.it; Fax: +39 06 490421
Received (in London, UK) 7th November 2002, Accepted 25th November 2002
First published as an Advance Article on the web 6th December 2002
The oxidation of ethers of various structure by O2 with two laccase/QN–OH systems (i.e., HBT,
1-hydroxybenzotriazole, and HPI, N-hydroxyphthalimide) is described. The process affords carbonylic
products in reasonable-to-good yields. The oxidation is carried out by the intermediate QN–Oꢀ species
of the mediators, through a radical H-atom abstraction (HAT) route that represents an elaboration of
the HAT route followed with benzyl alcohols. Alternative mechanisms have been considered and dismissed.
A comparison with a similar oxidation of ethers by the laccase/TEMPO system, reported previously, is
made. A clear-cut specialisation of the mediator vs. the substrate emerges, i.e. the laccase/HBT system is more
competent for the oxidation of ethers, whereas the laccase/TEMPO system was most proficient in the
oxidation of benzyl alcohols.
Oxygen re-oxidises laccase, thereby closing the catalytic cycle.
Introduction
Laccase/mediator systems may be considered for various
applications, such as textile dye bleaching,10 or environmen-
tally-friendly Kraft pulp delignification in the paper indus-
try,7b,11 or also in selective organic transformations.12–15 It is
clear that a better understanding of the mechanistic role of
the mediator, and of the spectrum of substrates accessible to
laccase/mediator systems, has both practical and fundamental
relevance. We have undertaken an evaluation of the efficiency
of a number of mediators of laccase in the oxidation of
benzylic alcohols, taken as non-phenolic models of lignin:9,16
TEMPO (i.e., 2,2,6,6-tetramethylpiperidin-1-yloxy free radi-
cal) proved the most efficient mediator.9,13 However, when
we considered benzylic ethers, which are even more representa-
tive of the lignin structure, TEMPO gave poorer perfor-
mances.17 We explained this partial failure towards oxidation
of ethers on the basis of the ionic oxidation mechanism that
the Medox form of TEMPO, i.e., an oxoammonium ion,18 fol-
lows.9,17 Other mediators of laccase, notably those presenting a
N–OH structure, such as 1-hydroxybenzotriazole (HBT) and
N-hydroxyphthalimide (HPI), have been found to follow a
different, radical mechanism of oxidation of benzyl alco-
hols.5b,7c,8,9,16,19–22 We therefore deemed it worthwhile to
explore the efficiency of these laccase/N–OH-type mediator
systems towards benzyl ethers. The present study reports our
results obtained in the mediated oxidation of some ether-type
model compounds, both lignin and non lignin-related. The
foreseeable application to enzymatic methods of Kraft pulp
delignification for paper making will be discussed.
White-rot fungi in nature are able to degrade wood by secret-
ing ligninolytic enzymes.1 Laccase, one of these enzymes, is
more precisely a family of ‘blue-copper’ oxidase proteins, con-
taining four copper ions in the active site; these enzymes have a
molecular mass of about 70 000 Da, and perform the oxidation
of a broad range of substrates, including lignin, with the con-
comitant reduction of O2 to water.1,2 Laccase cooperates with
other enzymes, such as lignin peroxidase (LiP)3 and manganese
peroxidase (MnP),4 in this task. LiP and MnP are heme-based
peroxidase enzymes. Laccase has a lower redox potential (ca.
0.7 – 0.8 V/NHE)2,5 than LiP and MnP, which are more
powerful oxidants (more than 1.3 V/NHE), and therefore is
able to catalyse mono-electronic oxidation steps only with
the easy-to-oxidise phenolic components of lignin (phenol-
oxidase activity).1,2 Lignin is a three-dimensional, insoluble
aromatic polymer that constitutes 15–30% of biomass. Its
structure encompasses a number of different types of links
between its constituents, among which ether and C–C diaryl
linkages represent more than 70% of all the functional groups
present.6 It was an important discovery, therefore, to find that
the action of laccase, a readily available and easier to manipu-
late enzyme than LiP and MnP, can be expanded towards
more difficult to oxidise non-phenolic substrates through the
use of appropriate mediators.7–9 The role of a mediator in
laccase oxidations is outlined in Scheme 1.
Once oxidised by laccase, the mediator (i.e., Medox) per-
forms a non-enzymatic oxidation of the substrate, by using
mechanisms that may be unavailable to laccase, thereby
explaining the possibility to oxidise non-phenolic substrates,
and widening the usefulness of a purely enzymatic method.9
Results and discussion
Several ether-type substrates were investigated, which were
considered representative of structural motifs present in the
lignin polymer, or also significant from a mechanistic view-
point. They include: diaryl ethers, benzyl ethers, alkyl ethers
and an epoxide. These precursors were oxidised with the lac-
case/HBT and laccase/HPI systems, at room temperature
for a 24 h reaction time. The solvent was a 0.1 M citrate buffer
Scheme 1
DOI: 10.1039/b210978f
New J. Chem., 2003, 27, 329–332
329
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Table 1 Results of the oxidation reactions of ethers with two laccase/N–OH-type mediators, compared with previous results obtained with the
laccase-TEMPO system
Reaction products (yield, %) with
a
Run
Substrate
Laccase-HPI
Laccase-HBT
Laccase-TEMPO
1
2
3
4
PhCH2OPh
PhCH2OCH2Ph
PhOPh
PhCHO (6%) PhCOOPh (4%)
PhCHO (8%) PhCOOPh (6%)
PhCHO (21%) PhCOOCH2Ph (8%)
no reaction
no reaction
b
PhCHO (16%) PhCOOCH2Ph (2%)
no reaction
—
PhCHO (16%) PhCOOCH2Ph (3%)
no reaction
no reaction
p-MeO–C6H4–C6H4–OMe-p
no reaction
5
6
7
8
9
4-MeO–C6H4CH2OMe
3,4-dimethoxy-C6H3CH2OMe
cyclo-Hex-CH2OMe
—
ArCHO (38%) ArCOOMe (15%)
—
ArCHO (32%) ArCOOMe (43%)
ArCHO (67%) ArCOOMe (25%)
no reaction
—
ArCHO (18%)
no reaction
10
—
no reaction
no reaction
c
11
PhCH2OCH2Ph
—
PhCHO (50%) PhCOOCH2Ph (16%)
—
a
b
c
From ref. 17. In this compound, part of PhCHO also originates by further oxidation of the alcohol fragment, i.e., PhCH2OH (see text). Employing a
substrate:mediator 1:1 ratio.
(pH 5) solution containing 4% MeCN, in order to ensure suffi-
cient solubility of the substrates.23 The reaction conditions
employed, and in particular the molar defect of mediator with
respect to the substrate, are consistent with Scheme 1, where
the mediator shuttles back and forth between its natural and
oxidised states (Medox), due to the intervention of laccase
and oxygen, while carrying out the non-enzymatic oxidation
of the substrate in a catalytic cycle.
Many of the investigated substrates were converted into
carbonyl products, and the oxidation yields are reported in
Table 1; a comparison is provided with the results previously
obtained with the laccase/TEMPO system.17 It must be
observed that the yields are calculated with respect to the sub-
strate, the molar amount of the mediator being only one-third
of that. Accordingly, some yields of product are greater than
100%, if evaluated with respect to the mediator, thereby sup-
porting an oxidation process with turnover. No other oxida-
tion products were found, besides those indicated; the
amount of substrate recovered in each experiment is not indi-
cated, but always complements the amount of product
detected. The structure of the products was confirmed by
GC-MS and by comparison with authentic samples. No con-
version to products was obtained in the absence of laccase,
nor of the mediator, with any of these non-phenolic substrates.
species follows
a
hydrogen atom transfer (HAT)
route.8,9,16,19–22 The HAT mechanism, in the case of benzylic
alcohols as substrates, has been supported by the determina-
tion of the effect of substituents (Hammett correlation),16,20
by kinetic isotope effect measurements,16,19 and by the product
distribution with a suitable probe molecule.16,19 In order to
account for the oxidation of ethers, particularly with respect
to the formation of ester-like products, the HAT mechanism
needs to be somewhat implemented, as reported in Scheme 2.
The QN–OH mediator is converted into the Med-Oꢀ form by
the enzyme (Scheme 1),5b,7c,9 and subsequently it abstracts a
H-atom from reactive C–H bonds, to give a radical intermedi-
ate (A). This explains why benzylic substrates (ArCH2OR) can
react (runs 1, 2, 5–8), whereas diaryl ethers (runs 3 and 4),
lacking weak C–H bonds, cannot. In the case of an aliphatic
Reactivity of substrate
A first glimpse at Table 1 shows that mediators HBT and HPI
behave similarly, as far as reactivity with substrate and extent
of conversion is concerned, and clearly differ with respect to
the behaviour of mediator TEMPO.17 In general, conversion
into oxidised products is higher with HBT and HPI. In
particular, both HBT and HPI enable the laccase-mediated
oxidation of PhCH2OPh, which did not react with laccase-
TEMPO.17 These results are reasonably accounted for if we
consider that the oxidised forms of the mediators (i.e. the
oxammonium ion form of TEMPO on the one hand, and the
QN–Oꢀ radical form of HBT and HPI on the other) oxidise
the substrates through different mechanisms. Whereas an
ionic mechanism of oxidation is followed by the TEMPO-
oxoammonium,9,13,17,18 where the nucleophilic character
of the substrate plays an important role,17 the QN–Oꢀ
Scheme 2
330
New J. Chem., 2003, 27, 329–332

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Scheme 4
Scheme 3
(5%) conversion into the lactone was observed. It was accom-
panied by a Ca–Cb bond cleavage product (ca. 10%, by GC-
MS), which is a typical outcome of radical cations of
ethers.26,27 The difference with the efficient oxidation of iso-
chroman to 3,4-dihydroxycoumarin under mild condition,
with the laccase-HBT and laccase-HPI systems, is striking
and confirms the operation of a different, radical in nature,
mechanism of oxidation by the Med-Oꢀ species, at least with
substrates endowed with a redox potential equal or higher than
1.4–1.5 V/NHE.
Further along this line, the anodic oxidation of trans-stil-
bene oxide to the radical cation intermediate has been found
to give rise to benzophenone and diphenylacetic acid;26 neither
of these two products was found in our case, because trans-
stilbene oxide was recovered unreacted from incubation with
laccase-HBT. Once again, no ET route appears to be pursued
by the Med-Oꢀ species.
ether (run 9), the stronger C–H bond makes the abstraction
step endothermic (Scheme 3)24 to such extent that the HAT
route slows down substantially.
The strength of vinyl-like C–H bonds (BDE, ca. 110 kcal
molÀ1 25
in precursor epoxide (run 10), is such that the HAT
)
route is no longer accessible, and no reaction takes place (vide
infra).
Radical intermediate A may be further oxidised to a carbo-
cation,26 either by the Med-Oꢀ or by laccase itself. Addition
of water to the carbocation would give a hemiacetal, which
gives rise to the observed aldehyde through loss of an alcohol
molecule. The latter can also be oxidised: this is the case with
(PhCH2)2O, where benzyl alcohol is initially produced along
with benzaldehyde; benzyl alcohol is oxidised in turn to provide
additional benzaldehyde. On the other hand, further oxidation
of the hemiacetal by Med-Oꢀ yields the other observed product,
that is the ester.26,27 The second route occurs almost exclusively
with the two cyclic precursors (runs 5 and 6), most likely due to
the higher stability of the cyclic (sugar-analogue) hemiacetal
intermediate, to afford the corresponding lactones in syntheti-
cally interesting yields. In contrast, oxidation to the ester-level
was only a minor path in the laccase-TEMPO case.13,17,18
Finally, an increase of the molar amount of HBT, from a
mediator:substrate ratio of 1:3 to 1:1, with an equal reaction
Conclusion
The oxidation of ethers of various structure by two laccase/
QN–OH systems is described. The oxidation is carried out by
the intermediate QN–Oꢀ species according to a radical HAT
route, which represents an elaboration of the HAT route fol-
lowed with the benzyl alcohols;8,9,16,19–22 alternative mechan-
isms have been considered and dismissed. Synthetically
interesting results have been obtained in the oxidation of two
cyclic ethers to the corresponding lactones. The present oxida-
tion procedure appears more efficient than one recently
described, which relies on ruminal microbes.33
time, causes
a sizeable increase in the conversion of
(PhCH2)2O to products (run 11 vs. 2), as expected for a process
where the oxidation efficiency depends on the nature and
amount of the mediator (Scheme 1).
A clear-cut specialisation of the mediator vs. the substrate
emerges from the present study, because the laccase-HBT sys-
tem is more competent for the oxidation of ethers than the lac-
case-TEMPO system. The latter, conversely, was the most
proficient in the oxidation of benzyl alcohols, as we showed
in a previous study.9 Because the ether functional group is a
widely recurring structural feature of lignin,6 the development
of mediator systems capable of cleaving the ether group effi-
ciently and under mild conditions is promising for mediated
enzymatic methods of Kraft pulp delignification for paper
making.34
HAT vs. ET mechanism?
The possibility that the Med-Oꢀ species carries out the oxida-
tion of the substrate to intermediate A by an electron transfer
(ET) route,28 and not by a HAT route, has been considered
(Scheme 4).
Both HBT and HPI, in the QN–Oꢀ form, have redox poten-
tials in the order of 1.0–1.1 V/NHE,9,29–31 that would be
perhaps sufficient to remove an electron, at least from those
substrates9 endowed with the lower redox potential, which
are easier to oxidise. The feasibility of this alternative hypo-
thesis has been tested independently, through the use of a bona
fide ET agent. Oxidation of isochroman with (NH4)2-
CeIV(NO3)6 (viz, CAN; Eꢁ 1.5 V),32 a strong mono-electronic
oxidant, has been performed in H2O:MeCN 2:1 solution at
room temperature: no oxidation product (i.e., the lactone
3,4-dihydroxycoumarin) was obtained, and the ether was
quantitatively recovered, even though CAN is certainly a
stronger monoelectronic oxidant than the Med-Oꢀ species.
Only at the reflux temperature of the mixed solvent, and
employing twice the stoichiometric amount of CAN, a modest
Experimental
General techniques
Characterisation of the structure of the reaction products was
done by 1H-NMR at 200 MHz on a Bruker AC200 NMR
instrument; chemical shifts are reported in the d scale (in
ppm) relative to residual nondeuterated solvent signals
(CDCl3). A VARIAN 3400 Star instrument, fitted with a 20
m  0.25 mm methyl silicone gum capillary column, was
New J. Chem., 2003, 27, 329–332
331

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confirmed by GC-MS analyses, run on a HP 5892 GC,
equipped with a 12 m  0.2 mm methyl silicone gum capillary
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or available from a previous investigation.9,13,16,17 Phthalan
(i.e., o-xylylene oxide; Aldrich) was oxidised to phthalide with
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Enzyme preparation
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tus) (Novo Nordisk Biotech) was employed. It was purified by
ion-exchange chromatography on Q-Sepharose by elution with
phosphate buffer,9 and an activity of 9000 U mLÀ1 was deter-
mined spectrophotometrically by the standard method with
ABTS.36
10 H.-J. Deussen and C. Johansen, PCT International patent WO
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The oxidation reactions were performed at room temperature
in stirred water solution (3 mL), buffered at pH 5 (0.1 M in
sodium citrate), containing 4% MeCN to improve the solubi-
lity of some of the substrates, and saturated by bubbling O2
for 30 min prior to the addition of the reagents.9 The concen-
tration of the reagents was: [substrate], 20 mM; [mediator], 6
mM, with 15 units of laccase. The reaction time was 24 h,
an atmosphere of O2 being maintained over the reaction vessel.
Following a conventional workup with ethyl acetate, the molar
amount of the oxidation products was determined by GC
analysis with respect to an internal standard (acetophenone
or p-methoxyacetophenone), suitable response factors being
determined from authentic compounds; the yields were calcu-
lated with respect to the molar amount of substrate.
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17 F. d’Acunzo, P. Baiocco, M. Fabbrini, C. Galli and P. Gentili,
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25 K. M. Ervin, S. Gronert, S. E. Barlow, M. K. Gilles, A. G.
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Reaction of 0.5 mmol of isochroman with 0.5 mmol of
IV
(NH4)2Ce (NO3)6 (viz, CAN) in 7.5 mL of H2O:MeCN 2:1
(v/v) at room temperature for 7 h did not afford any pro-
duct,27 and the ether was quantitatively recovered (GC). The
reaction was repeated in the same mixed solvent, by doubling
the molar amount of CAN (i.e., 1 mmol) and refluxing the
reagents for 7 h.27 The lactone (i.e., 3,4-dihydroxycoumarin)
was obtained in a 5% GC-yield, and a Ca–Cb bond cleavage
product also appeared at the GC-MS, in approximately 10%
yield; the structure of this product was not further investi-
gated. Unreacted isochroman was also recovered (75%).
27 E. Baciocchi, A. Piermattei, C. Rol, R. Ruzziconi and G. V.
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Acknowledgements
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Thanks are due to Novo Nordisk Biotech (Denmark) for a
generous gift of laccase. We also thank the EU for financial
support (grant QLK5-CT-1999-01277) to the OXYDELIGN
project.
31 K. Krikstopaitis and J. Kulys, Electrochem. Commun., 2000,
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