Alkoxyl- and carbon-centered radicals as primary agents for degrading non-phenolic lignin-substructure model compounds

Article abstract of DOI:10.1039/c0ob00797h

Lignin degradation by white-rot fungi proceeds via free radical reaction catalyzed by oxidative enzymes and metabolites. Basidiomycetes called selective white-rot fungi degrade both phenolic and non-phenolic lignin substructures without penetration of extracellular enzymes into the cell wall. Extracellular lipid peroxidation has been proposed as a possible ligninolytic mechanism, and radical species degrading the recalcitrant non-phenolic lignin substructures have been discussed. Reactions between the non-phenolic lignin model compounds and radicals produced from azo compounds in air have previously been analysed, and peroxyl radical (PR) is postulated to be responsible for lignin degradation (Kapich et al., FEBS Lett., 1999, 461, 115-119). However, because the thermolysis of azo compounds in air generates both a carbon-centred radical (CR) and a peroxyl radical (PR), we re-examined the reactivity of the three radicals alkoxyl radical (AR), CR and PR towards non-phenolic monomeric and dimeric lignin model compounds. The dimeric lignin model compound is degraded by CR produced by reaction of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), which under N₂ atmosphere cleaves the α-β bond in 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol to yield 4-ethoxy-3-methoxybenzaldehyde. However, it is not degraded by the PR produced by reaction of Ce⁴⁺/tert-BuOOH. In addition, it is degraded by AR produced by reaction of Ti³⁺/tert-BuOOH. PR and AR are generated in the presence and absence of veratryl alcohol, respectively. Rapid-flow ESR analysis of the radical species demonstrates that AR but not PR reacts with the lignin model compound. Thus, AR and CR are primary agents for the degradation of non-phenolic lignin substructures.

Full text of DOI:10.1039/c0ob00797h

View Article Online / Journal Homepage / Table of Contents for this issue
Organic &
Biomolecular
Chemistry
Dynamic Article Links
Cite this: Org. Biomol. Chem., 2011, 9, 2481
www.rsc.org/obc
PAPER
Alkoxyl- and carbon-centered radicals as primary agents for degrading
non-phenolic lignin-substructure model compounds
Yasunori Ohashi, Yukiko Uno, Rudianto Amirta, Takahito Watanabe, Yoichi Honda and Takashi Watanabe*
Received 29th September 2010, Accepted 22nd December 2010
DOI: 10.1039/c0ob00797h
Lignin degradation by white-rot fungi proceeds via free radical reaction catalyzed by oxidative enzymes
and metabolites. Basidiomycetes called selective white-rot fungi degrade both phenolic and
non-phenolic lignin substructures without penetration of extracellular enzymes into the cell wall.
Extracellular lipid peroxidation has been proposed as a possible ligninolytic mechanism, and radical
species degrading the recalcitrant non-phenolic lignin substructures have been discussed. Reactions
between the non-phenolic lignin model compounds and radicals produced from azo compounds in air
have previously been analysed, and peroxyl radical (PR) is postulated to be responsible for lignin
degradation (Kapich et al., FEBS Lett., 1999, 461, 115–119). However, because the thermolysis of azo
compounds in air generates both a carbon-centred radical (CR) and a peroxyl radical (PR), we
re-examined the reactivity of the three radicals alkoxyl radical (AR), CR and PR towards non-phenolic
monomeric and dimeric lignin model compounds. The dimeric lignin model compound is degraded by
CR produced by reaction of 2,2¢-azobis(2-amidinopropane) dihydrochloride (AAPH), which under N2
atmosphere cleaves the a–b bond in 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-
1,3-propanediol to yield 4-ethoxy-3-methoxybenzaldehyde. However, it is not degraded by the PR
produced by reaction of Ce4+/tert-BuOOH. In addition, it is degraded by AR produced by reaction of
Ti3+/tert-BuOOH. PR and AR are generated in the presence and absence of veratryl alcohol,
respectively. Rapid-flow ESR analysis of the radical species demonstrates that AR but not PR reacts
with the lignin model compound. Thus, AR and CR are primary agents for the degradation of
non-phenolic lignin substructures.
The lipid peroxidation process catalyzed by MnP degrades non-
phenolic b-O-4 lignin model compounds to release benzylic
Introduction
fragments.5,6 This reaction is likely relevant to ligninolysis in sound
wood, where enzymes cannot penetrate, because manganic ions
produced by MnPs are diffusible oxidants in the presence of
chelators, and initiate radical chain reactions from unsaturated
fatty acids or hydroperoxide intermediates.7 With respect to
the radical species responsible for degradation of non-phenolic
lignin model compounds by lipid peroxidation, Kapich et al.
hypothesized that peroxyl radicals (PRs) are principal agents
for the degradation of non-phenolic lignin model compounds,
based on their experiments showing that radicals derived from
azo compounds 2,2¢-azobis(2-amidinopropane) dihydrochloride
(AAPH) and 4,4¢-azobis(4-cyanovaleric acid) (ACVA) decom-
posed a non-phenolic lignin model compound.8 However, their
report did not address the possibility that initial carbon-centred
radicals (CRs) from the azo compounds react directly with the
lignin model compounds before reacting with molecular oxygen as
follows:9
Lignin, a heterogeneous phenolic polymer built from phenyl-
propane units linked together by C–C and C–O–C bonds, is
amongst the most abundant biopolymers on earth. Lignin in
plant cell walls, associated with hemicelluloses, creates a naturally
occurring composite material that imparts strength and rigidity
to the plant. Growing demand exists to produce bioethanol and
chemicals from lignocellulosics in response to problems of global
warming and fossil-fuel depletion. For conversion of lignocellu-
losics to biofuels and chemicals by enzymatic saccharification and
fermentation, degradation of the lignin network is necessary.
The selective white-rot fungus Ceriporiopsis subvermispora can
potentially degrade the lignin network without significant loss of
cellulose.1–3 C. subvermispora degrades lignin without penetration
of its extracellular enzymes into the wood cell wall. Past work
has shown that this fungus secretes manganese peroxidase (MnP)
and fatty acids to oxidize lipids to hydroperoxides and aldehydes.4
R–N N–R → 2R∑ + N2
Laboratory of Biomass Conversion, Research Institute for Sustainable
Humanosphere, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
E-mail: twatanab@rish.kyoto-u.ac.jp; Fax: +81-774-38-3681; Tel: +81-
774-38-3640
(1)
R∑ + O2 → RO2
∑
(2)
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2481–2491 | 2481
©

View Article Online
Fig. 1 Chemical structures of authentic compounds (3, 5–11), the dimeric lignin model compound (4) and veratryl alcohol (12). 1 and 2 are intermediates
of 3 and 4, respectively.
Therefore, re-examination is necessary to determine the reac-
tivity of PRs when reaction of CRs with molecular oxygen is
excluded. PRs are relatively stable radicals and serve as chain-
carrying radicals in lipid peroxidation.10 This suggests that PRs
function as diffusible oxidants, abstracting hydrogen from C–H
bonds with small bond dissociation energies (BDEs), including
the bis-allylic C–H bond, but do not play a central role in
hydrogen abstraction from the recalcitrant non-phenolic lignin
model compounds. To clarify the reactivity of each radical species,
we generated AR, CR and PR, and analysed their reactivity
towards lignin model compounds by using GC–MS and ESR,
including a rapid-flow technique. Herein, we discuss these results
in relation to the BDEs and energy gaps before and after reaction
obtained by molecular orbital calculations.
Fig. 1 shows the chemical structures of the lignin
model compounds and authentic compounds used in
this study. 3-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-(2-
methoxyphenoxy)-1-propanone (1) was synthesized from
1-(4-hydroxy-3-methoxyphenyl)ethanone
via
bromination,
Williamson ether synthesis with sodium 2-methoxyphenolate
and aldol condensation with methanol, as described by
Hosoya et al. (1980).11 1-(4-Hydroxy-3-methoxyphenyl)-2-
(2-methoxyphenoxy)-1,3-propanediol (2) was synthesized by
reduction of 1 with NaBH4. 1 and 2 were ethylated with
diazoethane to give 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxy-
2-(2-methoxyphenoxy)-1-propanone (3, [MS (-TMS) m/z (%):
418 (M+, 7.1), 268 (18.8), 179 (100), 151 (20.7), 150 (22.5),
123 (28.2), 73 (84.4)]) and 1-(4-ethoxy-3-methoxyphenyl)-2-
(2-methoxyphenoxy)-1,3-propanediol (4, [MS (-Ac) m/z (%):
432 (M+, 2.5), 207 (9.7), 206 (10.7), 181 (30.1), 149 (6.3), 124
(9.4), 43 (100)]), respectively. 1-(4-Ethoxy-3-methoxyphenyl)-
2-(2-methoxyphenoxy)-2-propen-1-one (5, [MS m/z (%): 328
(M+, 12.5), 179 (100), 151 (99.5), 149 (68.4), 135 (37.1),
123 (77.4), 77 (99.6)]) was obtained by degradation of 4 in
Experimental
Materials
Acetic acid, sodium acetate, pyridine, toluene, hydrogen per-
oxide, 2,2¢-azobis(2-amidinopropane) dihydrochloride (AAPH),
2-methoxyphenol and H218O were obtained from Wako
Pure Chemical Industries (Osaka, Japan). 18O2 was pur-
chased from Shoko Company (Tokyo, Japan). Acetic anhy-
dride and chloroform were obtained from Nacalai Tesque,
Inc. (Kyoto, Japan). tert-Butyl hydroperoxide (tert-BuOOH)
was a gift from NOF Corporation (Tokyo, Japan). Fluoran-
thene, 2,4,6-tri-tert-butylnitrosobenzene (BNB) and 4-ethoxy-
3-methoxybenzaldehyde were obtained from Tokyo Chemical
Industry Company (Tokyo, Japan). 5,5-Dimethyl-1-pyrroline-
N-oxide (DMPO), a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-
POBN) and 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium
salt (DBNBS) were obtained from Labotec (Tokyo, Japan). All
other chemicals used were of analytical reagent grade. Water
was purified with a compact ultrapure water system (EASYpure,
Barnstead, IA, USA).
a
laccase/1-hydroxybenzotriazole system.12 1-(4-Ethoxy-3-
methoxyphenyl)-1,2,3-propanetriol (6, [MS (-Ac) m/z (%):
368 (M+, 1.9), 206 (8.3), 181 (24.7), 151 (4.9), 43 (100)]) was
synthesized from 3-(4-hydroxy-3-methoxyphenyl)-2-propen-1-ol
via ethylation by diazoethane and oxidation by OsO4.13 6 was
oxidized by 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) to
give 1-(4-ethoxy-3-methoxyphenyl)-2,3-dihydroxy-1-propanone
(7, [MS (-Ac) m/z (%): 324 (M+, 4.3), 265 (2.0), 207 (8.4),
179 (29.6), 151 (21.7), 123 (9.8), 43 (100)]).14,15 1-(4-Ethoxy-3-
methoxyphenyl)-3-hydroxy-1-propanone (8, [MS (-TMS) m/z
(%): 296 (M+, 28.4), 253 (19.5), 206 (50.5), 179 (20.2), 151
(16.8), 75 (100), 73 (85.8)]) was synthesized from 3-(4-hydroxy-
3-methoxyphenyl)-2-propen-1-ol by ethylation with diazoethane,
hydrogenation to a double bond by Pd–C/NaBH4/HCl and
oxidation with DDQ.16,17 4-Ethoxy-3-methoxybenzoic acid (10,
[MS (-TMS) m/z (%): 268 (M+, 40.6), 253 (92.5), 225 (37.7), 209
2482 | Org. Biomol. Chem., 2011, 9, 2481–2491
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
(92.0), 181 (51.5), 73 (100)]) was produced from vanillic acid by
reaction with ethyl iodide.18
of the lignin model compound 4 solution (20 mM in acetone)
and 56 mL of tert-BuOOH (69%) were added to a test tube
and diluted with 794 mL of H2O. Then, 100 mL of TiCl3 or
Ce(NH4)2(NO3)6 (10 mM each) was added and the mixture was
stirred for 5 or 10 min to produce ARs or PRs, respectively. After
the reaction, 100 mL of fluoranthene (1 mM in dichloromethane)
was added, chloroform extraction was carried out and the extract
was evaporated. The residue was acetylated with 100 mL of acetic
anhydride in 100 mL of pyridine at room temperature for 12 h.
After evaporation, 100 mL of chloroform–MeOH (1 : 1, v/v) was
added to the vial and the sample was subjected to GC–MS analysis.
Experiments were carried out in triplicate.
Instrumental analysis
GC–MS analysis was performed using a gas chromatograph–mass
spectrometer (GCMS-QP5050A, Shimadzu, Kyoto, Japan) on a
DB-1 column (length = 30 m; i.d = 0.25 mm; thickness = 1 mm; J&W
Scientific Inc., CA, USA). Electron-impact mass spectra (EI-MS)
were recorded at an ionization energy of 70 eV. The column oven
temperature was maintained at 50 ◦C for 1 min, raised to 150 ◦C at
a rate of 30 ◦C min-1, maintained there for 1 min, raised to 280 ◦C
at a rate of 5 ◦C min-1, and maintained there.
ESR spin-trapping was peformed using a free-radical monitor
(JES-FR30, JEOL, Tokyo, Japan) at room temperature under
the following conditions: frequency = 9.425 GHz; centre field =
335.6 mT; sweep width = 5.0 mT; modulation width = 0.10 mT; re-
ceiver gain = 320; data points = 4096; time constant = 0.10 s; sweep
time = 1.0 min; power = 4.0 mW. Rapid-flow ESR was performed
using an ESR spectrometer (EMX, Bruker Biospin) equipped with
a dielectric mixing resonator (ER4117D-MVT, Ibaraki, Japan)
at room temperature as described in the literature19–21 under
the following conditions: frequency = 9.62 GHz; centre field =
343.0 mT; sweep width = 5.0 mT; modulation width = 0.063 mT;
receiver gain = 2 ¥ 105; data points = 512; time constant = 0.082 s;
sweep time = 42 s; power = 10 mW.
Analysis of radicals derived from AAPH and tert-BuOOH
CRs and PRs derived from AAPH under N2 or O2 atmosphere
were analysed using ESR spin-trapping. First, 40 mL of AAPH
(100 mM in 20 mM sodium acetate buffer, pH 5), 40 mL of DBNBS
(100 mM in H2O) or BNB (100 mM in methanol, ethanol or
DMSO) and 320 mL of sodium acetate buffer (20 mM, pH 5)
or solvent (methanol, ethanol or DMSO) were mi◦xed in a test
tube. The samples were shaken at 100 rpm and 60 C for 5 min
under N2 or O2 atmosphere. After reaction, the spin adducts were
analysed using ESR. Although, the ESR signals of BNB adducts
were accumulated 20 times, those from the other spin traps were
accumulated just once.
ARs derived from tert-BuOOH by the reaction with Ti3+ were
analysed using ESR spin-trapping. First, 56 mL of tert-BuOOH
(69%) and 100 mL of 4-POBN (100 mM) were diluted with 744 mL
of H2O in a test tube. Then, 100 mL of TiCl3 (10 mM) was added,
the solution was mixed vigorously and the ESR spectrum was
recorded immediately.
Detection of lignin model compound degradation products by
radicals derived from AAPH
Degradation products of lignin model compound 4 by radicals
derived from AAPH were detected by GC–MS analysis. AAPH
(2.7 mg), 950 mL of sodium acetate buffer (20 mM, pH 5.0)
and 50 mL of the lignin model compound 4 solution (20 mM
in acetone) were added to a test tube and shaken at 100 rpm and
60 ◦C for 3 h under N2 or O2 atmosphere. After the reaction,
100 mL of fluoranthene (1 mM in dichloromethane) was added
and chloroform extraction was carried out. The organic layer
was separated, divided into two equal fractions, transferred into
separate vials and evaporated. One fraction was acetylated with
100 mL of acetic anhydride in 100 mL of pyridine at room
temperature for 12 h, while the other was trimethylsilylated with
25 mL of N,O-bis(trimethylsilyl)trifluoroacetamide in 200 mL of
pyridine at 60 ◦C for 1 h. After evaporation, 100 mL of chloroform–
MeOH (1 : 1, v/v) was added to each vial and the sample was
subjected to GC–MS analysis. The same experiments were carried
out with H218O/N2 and H216O/18O2 instead of H216O/N2 and
H216O/16O2. All experiments were carried out in triplicate.
PRs derived from tert-BuOOH by the reaction with Ce4+
were analysed using ESR spin-trapping. First, 100 mL of
Ce(NH4)2(NO3)6 (10 mM in H2O), 56 mL of tert-BuOOH (69%),
100 mL of 4-POBN (100 mM) and 744 mL of H2O were mixed
in a test tube. The solution was mixed vigorously and the ESR
spectrum was recorded immediately.
Analysis of radicals derived from veratryl alcohol
AR and PR derived from the two peroxides tert-BuOOH and H2O2
were analysed directly in the presence and absence of veratryl
alcohol 12 by using rapid-flow ESR. Two aqueous solutions
were prepared: Solution A contained 10 mM TiCl3 or 6 mM
of Ce(NH4)2(NO3)6 and 190 mM H2SO4; Solution B contained
0–80 mM tert-BuOOH or 0–90 mM H2O2, 0–150 mM veratryl
alcohol 12 and 190 mM H2SO4. The H2SO4 was added to stabilize
Ti3+ and to accelerate the reaction of Ce4+ with the peroxides.
Each solution was filled into a 25 mL syringe, the syringes were
set on a syringe pump and the solutions were flowed into a mixing
chamber. Spectra were accumulated 30 times. Flow rates after
mixing are listed in the figure legend.
Reactions of lignin model compound with Ti3+/tert-BuOOH and
Ce4+/tert-BuOOH systems
AR and PR are produced from tert-BuOOH by reaction with Ti3+
(ref. 22) and Ce4+ (ref. 23) as follows:
Ti3+ + tert-BuO–OH → Ti4+ + tert-BuO∑ + OH-
Simulation of bond dissociation energy
(3)
The bond dissociation energies (BDEs) were simulated by cal-
culating the difference in the energy before and after reaction
A–H → A∑ + H∑, where A–H is the parent molecule and A∑
is the corresponding hydrogen-abstracted radical. The BDE of
Ce4+ + tert-BuOO–H → Ce3+ + tert-BuOO∑ + H+
(4)
We investigated the reactions of these unitary-radical-
production systems with lignin model compound 4. First, 50 mL
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2481–2491 | 2483
©

View Article Online
Table 1 Degradation products of non-phenolic b-O-4 lignin model
compound 4 from reaction with AAPH-derived radicals
Table 2 Degradation products of non-phenolic b-O-4 lignin model
compound 4 from reaction with metal–hydroperoxide systems
Intensity ratio (%)a
Intensity ratio (%)a
Sample
3
4
5
6 + 7b
8
9
10 11
Sample
3
4
5
7
9
11
-AAPH (N2)
+AAPH (N2)
-AAPH (O2)
+AAPH (O2)
1.7
6.9
1.5
5.7
96.6
42.0
98.2
63.7
1.6
6.6
0.3
Ti3+
100.0
47.6
92.1
97.2
3.0
1.1
41.4
18.6
Ti3+/tert-BuOOH
Ce4+
25.9
2.6
2.2
10.9
0.7
0.3
1.0
0.2
6.4
4.1
8.4
0.3
0.3
2.7
2.6
1.8 3.6
Ce4+/tert-BuOOH
Reactions were carried out in sodium acetate buffer (pH = 5.0) containing
5% (v/v) acetone. [AAPH] = 10 mM, [lignin model compound 4] =
1.0 mM.a The values of 4, 5, 6+7, 9 and 11 were calculated using the ratio
of acetylated samples with an internal standard. The values of 3, 8 and 10
were calculated using the ratio of trimethylsilylated samples. b Because 6
and 7 are difficult to separate by our GC–MS conditions, the sum of the
ratio is given.
a Reactions were carried out in water containing 5% (v/v) acetone. [lignin
model compound 4] = 1.0 mM, [tert-BuOOH] = 0.40 M, [Ti3+] = [Ce4+] =
1 mM. All values were calculated using the ratio of acetylated samples
with an internal standard.
3, dehydrated dimeric ketone 5, triol 6, monomeric dihydroxyl
ketone 7, monomeric monohydroxyl ketone 8, benzylic acid 10
and guaiacol 11. These results indicate that the radicals derived
from AAPH can selectively break the a–b bond in lignin model
compound 4, and a-oxidation and b-O-4 bond cleavage occur. A
major difference between the reactions under N2 and O2 atmo-
spheres is that benzylic acid 10 is produced from benzaldehyde
9 under O2 atmosphere. The products formed by reaction under
O2 atmosphere are consistent with products reported to form by
reaction in air.8 Fig. 3 shows a comparison between H216O and
H218O under N2 and 18O2 atmospheres. This result clearly indicates
that the oxygen atom in water is incorporated into a carbonyl
group of benzaldehyde 9 even in the presence of dissolved oxygen.
radicals such as CR and PR derived from AAPH and tert-
BuO∑ and tert-BuOO∑ were calculated to compare their capability
to abstract hydrogen. In this case, A–H is the hydrogen-added
product from the radical. This simulation is based on a concept
described by Wright et al. (2001).24 The geometries of the closed-
shell species were optimized using the AM1 method. Theoretical
molecular orbital (MO) calculations for the optimized structure
were performed at the B3LYP/6-311G** level of theory. For
radicals, an unrestricted approach was applied. The energy of the
hydrogen atom was set to its exact value, -0.50000 au (-313.75
kcal mol-1). All calculations were performed using Spartan ’06
software (Wavefunction Inc., Irvine, CA, USA).
Reactions of lignin model compound with Ti3+/tert-BuOOH and
Ce4+/tert-BuOOH systems
Results
The lignin model compound 4 was reacted with the Ti3+/tert-
BuOOH and Ce4+/tert-BuOOH systems.
Analyisis of lignin model compound degradation products by
radicals derived from AAPH
Reaction with the Ti3+/tert-BuOOH system degrades the lignin
model compound 4 to give dimeric ketone 3, dehydrated dimeric
ketone 5, monomeric dihydroxyl ketone 7, benzaldehyde 9 and
guaiacol 11 (Table 2). However, without tert-BuOOH, the lignin
model compound 4 is not decomposed. Thus, non-phenolic b-O-4
lignin model compound 4 is decomposed by the alkoxyl radical
(tert-BuO∑).
Non-phenolic b-O-4 lignin model compound 4 was reacted with
radicals produced by the thermolysis of AAPH under N2 and
O2 atmospheres. Yields of the degradation products of lignin
model compound 4 are shown in Table 1. The GC–MS total
ion chromatogram of the degradation products from the lignin
model compound 4 after reaction with AAPH-derived radicals
under N2 atmosphere is exemplified in Fig. 2. Under both N2 and
O2 atmospheres, a major degradation product is benzaldehyde
9. Under N2 atmosphere, a small amount of the following
additional products is produced: dimeric ketone 3, monomeric
monohydroxyl ketone 8 and guaiacol 11. Under O2 atmosphere,
the following additional products are produced: dimeric ketone
tert-BuOOH is known to react with Ce4+ to give tert-BuOO∑.
Reaction with the Ce4+/tert-BuOOH system degrades the lignin
model compound 4 to give dimeric ketone 3, dehydrated dimeric
ketone 5 and guaiacol 11. However, without tert-BuOOH, the
lignin model compound 4 produces dimeric ketone 3, dehydrated
dimeric ketone 5 and guaiacol 11 at intensities almost the same
Fig. 2 GC–MS total ion chromatogram of the degradation products from the lignin model compound (4) after reaction with AAPH-derived radicals
under N2 atmosphere.
2484 | Org. Biomol. Chem., 2011, 9, 2481–2491
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 3 Mass spectra of 9 produced from lignin model compound 4 by the AAPH-derived radical under various reaction conditions: (a) H216O solvent
under N2 atmosphere; (b) H218O solvent under N2 atmosphere; (c) H216O solvent under 18O2 atmosphere. Other reaction conditions were as listed in
Table 1.
as for the complete reaction system with tert-BuOOH. A minor
difference between the two reaction systems is that, without tert-
BuOOH, monomeric dihydroxyl ketone 7 and benzaldehyde 9
are produced. Thus, although Ce4+ reacts directly with the lignin
model compound 4, it reacts more readily with tert-BuOOH,
producing PRs, which are unreactive towards the lignin model
compound 4.
5 is thought to be produced by deacetylation of the acetyl
derivative of benzyl ketone 3 at elevated temperature based on
GC–MS analysis.17 In the present study, 3 is produced in the
reactions of AAPH under both O2 and N2 atmospheres, but 5
is produced only in the reaction under O2 atmosphere (Table 1),
suggesting that 5 is produced during radical reaction of the lignin
model compound 4.
Analysis of radicals derived from AAPH and peroxides
Fig. 4 shows the ESR spectra of DBNBS spin adducts produced
by reaction with AAPH-derived radicals under N2 and O2
atmospheres. Under the N2 atmosphere, only a triplet signal
(1 : 1 : 1) is evident. However, under the O2 atmosphere, both a
triplet signal and a sextet signal (1 : 3 : 3 : 3 : 3 : 1) are evident,
and the triplet signal is lower in intensity than that under N2
atmosphere. Therefore, we conclude that the triplet signals with
hfsc a(N) = 1.28 mT originate from the CR adduct.
Fig. 5 shows the ESR accumulation spectra of BNB spin adducts
produced by the AAPH-derived radicals under N2 atmosphere in
90% (v/v) of various solvents. Fig. 5a, for methanol solvent, shows
triple quintet and quartet signals from 0.5 to 20.5 min. The quartet
signals disappear from 21 to 41 min. For the triple quintet signals,
Fig. 4 ESR spectra of DBNBS spin adducts produced by reaction with
AAPH-derived radicals in 18 mM sodium acetate buffer (pH 5) under
various atmospheres: (a) N2 atmosphere; (b) O2 atmosphere. [AAPH] =
[DBNBS] = 10 mM.
hfsc a(N) = a(Hb) = 1.38 mT; a(Hmeta) = 0.08 mT originates from the
hydroxymethyl radical adduct produced by hydrogen abstraction
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2481–2491 | 2485
©

View Article Online
Fig. 6 ESR spectra of 4-POBN spin adducts produced in various
tert-BuOOH systems: (a) Ti3+/tert-BuOOH system; (b) Ce4+/tert-BuOOH
system. [tert-BuOOH] = 0.40 M; [4-POBN] = 10 mM; [Ti3+] = [Ce4+] = 1 mM.
Fig. 5 ESR spectra of BNB spin adducts produced by reaction with
AAPH-derived radicals under N2 atmosphere in 90% (v/v) of various
solvents: (a) methanol; (b) ethanol; (c) DMSO. [AAPH] = [BNB] = 10 mM.
Reaction of Ti3+ with H2O2 gives hydroxyl radicals (HO∑)
according to eqn (5):22
Ti3+ + HO–OH → Ti4+ + HO∑ + OH-
(5)
from methanol by CR derived from AAPH. Fig. 5b, for ethanol
solvent, shows the presence of a similar a-hydroxyethyl radical
adduct, with hfsc a(N) = 1.41 mT and a(Hb) = 1.34 mT. Fig. 5c,
for DMSO solvent, shows triple triplet signals, with hfsc a(N) =
1.01 mT and a(Hmeta) = 0.20 mT. This radical is assigned to a CR
adduct due to the small a(N) value because BNB reportedly traps
bulky radicals such as tertiary CR to give anilino (not nitroxide)
spin adducts with small a(N) values.25,26 Under O2 atmosphere, no
ESR signals are observed for all three solvents. BNB is known to
be a strong spin-trapping agent for CR,27 which explains why ESR
spin adducts are detected under N2 but not O2 atmosphere.
Fig. 6 shows the ESR spectra of 4-POBN spin adducts produced
by the reaction of tert-BuOOH with Ti3+ and Ce4+ in the presence
of 4-POBN. Reaction of the Ti3+/tert-BuOOH system gives an
AR adduct (a(N) = 1.63, a(H) = 0.29); while the reaction of the
Ce4+/tert-BuOOH system gives a PR adduct (a(N) = 1.51 mT,
a(H) = 0.23 mT). The hfsc values are identical to those previously
reported.28,29
Fig. 7 shows the ESR spectra of various Ti3+ reaction systems. In
Fig. 7a, reaction with H2O2 causes signals from hydroxyl radicals
(HO∑) to appear. In Fig. 7b, reaction with H2O2 in the presence
of veratryl alcohol 12 causes signals from HO∑ to disappear and
a set of new complex signals to appear; the new signals are
assigned to radicals derived from hydroxylated veratryl alcohol.
The spectral pattern differs from those previously reported for
the same reaction.30 In Fig. 7c, reaction with tert-BuOOH causes
signals from tert-BuO∑ and H3C∑ to appear. Reaction of Ti3+ with
tert-BuOOH is known to give tert-BuO∑, according to eqn (3).
However, the alkoxyl radical readily decomposes by b-scission to
give a ketone and a CR—that is, tert-BuO∑ decomposes by b-
scission to give a methyl radical, according to eqn (6):22
tert-BuO∑ → CH3COCH3 + H3C∑
(6)
In Fig. 7d, reaction with tert-BuOOH in the presence of veratryl
alcohol 12 causes signals from tert-BuO∑ to disappear and signals
from H3C∑ to grow slightly smaller, indicating that tert-BuO∑
preferentially reacts with veratryl alcohol 12. Unlike for the
Ti3+/H2O2 system, radicals derived from the substrate are not
directly detected.
Radicals derived from veratryl alcohol
Radicals generated by the reaction of Ti3+ with H2O2 or tert-
BuOOH in the presence and absence of veratryl alcohol 12 were
analysed directly using rapid-flow ESR.
Fig. 8 shows the ESR spectra of various Ce4+ reaction systems.
In Fig. 8a, reaction with H2O2 gives a broad ESR signal assigned
2486 | Org. Biomol. Chem., 2011, 9, 2481–2491
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 7 ESR spectra of various Ti3+ reaction systems (VA = veratryl alcohol 12): (a) Ti3+/H2O2 system; (b) Ti3+/H2O2/VA system; (c) Ti3+/tert-BuOOH
system; (d) Ti3+/tert-BuOOH/VA system. The concentration of each reagent after mixing was as follows: [Ti3+] = 5 mM; [H2O2] = 45 mM; [tert-BuOOH] =
40 mM; [VA] = 75 mM; [H2SO4] = 190 mM. The flow rates after mixing were as follows: (a) and (b), 20 mL min-1; (c) and (d), 0.1 mL min-1. The assigned
radical structure is shown in (b).
to the hydroperoxyl radical (HOO∑), which is produced according
to eqn (7):31
12. This result is in accordance with degradation of lignin model
compound 4 by the radical systems (Table 2).
Ce4+ + HOO–H → Ce3+ + HOO∑ + H+
(7)
Calculation of the bond dissociation energy
The BDEs of 4 were calculated by the MO method (Table 3).
We first calculated the BDEs of the C–H bonds in linoleic acid
to verify the accuracy of the simulation. Values for the bonds
at the bis-allyl and allyl positions were calculated to be 76.1
and 87.5 kcal mol-1, respectively, in good agreement with values
previously reported.33 We then simulated the BDEs of the C–H
bonds in four stereoisomers of the lignin model compound 4. For
each isomer, the value for the bond at the benzyl position is the
smallest. Values are smaller for the erythro isomer than for the
threo isomer. Energy differences for the molecules before and after
hydrogen abstraction at the benzyl position by the three radicals,
AR, CR and PR, were calculated to be in the order AR > CR >
PR (Table 4).
In Fig. 8b, reaction with H2O2 in the presence of veratryl alcohol
12 causes no spectral change, indicating that the hydroperoxyl
radical is unreactive towards veratryl alcohol 12. However, when
peroxides are omitted from the reaction system, Ce4+ reacts directly
with veratryl alcohol 12 to give veratryl alcohol cation radical
(eqn (8), where VA = veratryl alcohol 12), whose ESR spectrum
is identical to that previously reported for veratryl alcohol cation
radical produced by the same reaction.32
∑
Ce4+ + VA → Ce3+ + VA+
(8)
In Fig. 8c, reaction with tert-BuOOH gives a broad signal at-
tributed to tert-BuOO∑. When reactions are performed at different
concentrations of tert-BuOOH and fixed concentrations of Ce4+
and veratryl alcohol 12, the spectra change with the concentration
of tert-BuOOH. Without tert-BuOOH, the spectra are identical to
that for the veratryl cation radical. Signals from the veratryl cation
radical decrease and new signals from tert-BuOO∑ increase with
increasing concentration of tert-BuOOH. Thus, in the presence
of tert-BuOOH and veratryl alcohol 12, Ce4+ preferentially reacts
with tert-BuOOH to give tert-BuOO∑ (eqn (4)), but signals from
tert-BuOO∑ increase due to low reactivity towards veratryl alcohol
Discussion
In studies of white rot, oxidative enzymes capable of decomposing
non-phenolic b-O-4 lignin model compounds are of major con-
cern. Lignin peroxidases (LiPs) and versatile peroxidases (VPs)
are target enzymes that decompose the recalcitrant structure.
However, a majority of white-rot fungi do not secrete LiPs and
VPs, and most laccases (Lccs) and manganese peroxidases (MnPs)
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2481–2491 | 2487
©

View Article Online
Table 3 Bond dissociation energies of the C–H bond in non-phenolic
b-O-4 lignin model compound 4
from enzymes, if chelators for manganese ions are involved because
chelated Mn3+ initiates radical chain reactions in both unsaturated
fatty acids and lipid hydroperoxides.7 A major concern in these
radical reactions is what type of radical species initially oxidizes
the recalcitrant structure of lignin. Kapich et al. analysed radicals
by means of thermolysis of azo compounds and concluded that
PR is responsible for the degradation of non-phenolic lignin.8
However, CR involvement in the degradation cannot be excluded
because the researchers carried out the thermolysis reactions only
under aerobic atmosphere, where CR competitively reacts with
molecular oxygen and the lignin model compounds.
BDE/kcal mol-1
erythro
threo
Hydrogen type a-R, b-S
a-S, b-R
a-R, b-R
a-S, b-S
a-H
85.1
105.5
101.7
85.3
105.5
101.9
92.3
106.5
101.3
92.5
106.6
101.5
b-H
g-H
–OCH3
–OCH2CH3
–OCH2CH3
ring-H
103.5, 104.5 104.4, 105.0 103.5, 104.4 103.6, 104.5
103.5
108.8
102.2
108.0
103.6
108.7
103.4
108.8
To determine the relative reactivities of the radical species, we
generated ARs, CRs and PRs and analysed the reactivity of each
towards non-phenolic lignin model compounds by using ESR and
GC–MS.
117.2–121.1 117.3–119.5 117.3–119.5 117.2–120.0
Table 4 Changes in potential of radicals after hydrogen abstraction
To determine the relative reactivity of CR, we derived CR from
AAPH (by thermolysis under N2 atmosphere). We found that the
radical cleaves the a–b bond of the lignin model compound 4
to produce 3-ethoxy-4-methoxybenzaldehyde 9 (Table 1). Baiocco
et al. reported that, in reactions of laccase with benzyl alcohol in
the presence of mediator compounds, both the hydrogen abstrac-
tion at the benzyl position [radical hydrogen-atom-transfer (HAT)
route] and electron abstraction from a benzene ring [electron-
transfer (ET) route] occur, producing benzaldehyde.36 Our ESR
spin-trapping experiments show that CR derived from AAPH
also reacts with methanol and ethanol to abstract a hydrogen
atom from the a-carbon (Fig. 5). Because CR is known to serve
as a hydrogen acceptor rather than an electron acceptor,37–39 it is
reasonable that the CR derived from AAPH abstracts a hydrogen
atom from the benzyl position of the lignin model compound
4 to produce a benzyl radical. Experiments of the lignin model
compound 4 degradation by thermolysis of AAPH under O2
atmosphere give a fragmentation pattern that closely resembles
that under anaerobic atmosphere, except for the generation of
benzoic acid 10 under O2 atmosphere, which can be ascribed to
Radical
–BDE/kcal mol-1
AAPH (-C∑)
AAPH (-COO∑)
tert-BuO∑
-93.8
-87.5
-105.1
-86.5
tert-BuOO∑
cannot directly oxidize the non-phenolic b-O-4 lignin model
compounds. Rather, co-oxidation of mediators and lipids by these
enzymes decomposes the lignin model compounds.
We previously demonstrated that radical reactions of copper
complexes with organic hydroperoxides produce free radicals and
depolymerize a synthetic non-phenolic lignin polymer at ambient
temperature in aqueous media.34 The radical reactions delignify
both hardwood and softwood, resulting in fibre separation without
apparent morphological changes in the cell wall thickness, as
observed in selective white rot.35 This suggests the potential role
of free radicals as a strong lignin oxidizing agent in selective white
rot, where lignin decomposes at sites far from enzymes. Lipid
peroxidation by MnP also generates free radicals even at sites far
Fig. 8 ESR spectra of various Ce4+ reaction systems (VA = veratryl alcohol 12): (a) Ce4+/H2O2 system; (b) Ce4+/H2O2/VA system; (c)
Ce4+/tert-BuOOH/VA system. The concentration of each reagent after mixing was as follows: [Ce4+] = 3 mM; [H2O2] = 45 mM; [tert-BuOOH] =
40 mM; [VA] = 75 mM; [H2SO4] = 190 mM. The flow rates after mixing were 0.1 mL min-1.
2488 | Org. Biomol. Chem., 2011, 9, 2481–2491
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Fig. 9 Proposed pathway for degradation of lignin model compound 4 by free radicals.
autoxidation of benzaldehyde 9 (Table 1). Thus, CR derived from
AAPH initially reacts with the lignin model compound 4.
To determine the relative reactivity of PR, we derived PR from
the Ce4+/tert-BuOOH reaction system in the presence of the lignin
model compound 4. The lignin model compound 4 remains stable
under this condition (Table 2). PR is known to generate di-tert-
butyl peroxide and tert-BuO∑ or singlet oxygen, according to the
following equations:40
product of the Russel reaction, tert-BuO∑ (eqn (10)), and its b-
scission product H3C∑, are not detected by ESR. Therefore, the
contributions of secondary AR and methyl radical are negligible;
the first reaction governs the overall radical species accessible
to the lignin model compounds, and PR is unreactive towards
non-phenolic lignin model compounds. Also, rapid-flow ESR
experiments clearly demonstrate that tert-BuOO∑ and HOO∑ do
not oxidize the non-phenolic lignin monomer, veratryl alcohol 12
(Fig. 8), therefore, PR derived from tert-BuOOH is unreactive
towards non-phenolic lignin model compounds.
2 tert-BuOO∑ → tert-BuOO(tert-Bu) + O2
(9)
To determine the relative reactivity of AR, we derived AR from
the Ti3+/tert-BuOOH reaction system and found that it degrades
non-phenolic lignin model compound 4 (Table 2). We also found
that AR is consumed by reaction with veratryl alcohol 12 (Fig. 7).
H3C∑ is also produced in this reaction, but the decrease in its signal
intensity is small. Thus, AR derived from tert-BuOOH reacts with
non-phenolic monomeric and dimeric lignin model compounds.
2 tert-BuOO∑ → 2 tert-BuO∑ + O2
(10)
In addition, tert-BuO∑ is known to decay by b-scission into
H3C∑ and acetone (eqn (6)) in accordance with the rate constant
for b-scission in the solvent system used.41 This means that three
radicals—tert-BuOO∑, tert-BuO∑ and H3C∑—can be produced if
the chain reactions proceed. Fig. 8 shows, however, that the
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2481–2491 | 2489
©

View Article Online
These results are in agreement with earlier findings that the
oxidation reactivity of the three radicals is in the order AR > CR ꢀ
PR. For example, Koppenol reported that the values of reduction
potential E0 for RO∑/ROH, ROO∑/ROOH, C∑H2OH/CH3OH
and CH3C∑HOH/CH3CH2OH are +1.6, +1.0, +1.2 and +1.0 V,
respectively.42 Our experiments show that AR derived from tert-
BuOOH and CR derived from AAPH react with the lignin model
compound 4, but PR derived from tert-BuOOH is unreactive
towards the dimer and veratryl alcohol 12 (E0 = 1.4 V).43 The
differential reactivity of radicals in abstracting hydrogen at the
benzyl position of lignin model compound 4 is supported by BDE
calculations (Table 4), which show that the oxidation reactivity is
in the order AR > CR ꢀ PR.
the highest reactivity towards veratryl alcohol, as determined
by rapid-flow ESR, and by the finding that energy differences
between the lignin model compound before and after hydrogen
abstraction at the benzyl position by AR, PR and CR are in the
order AR > CR > PR. In addition, PR derived from tert-BuOOH
is not reactive towards the lignin model compound, as expected
from bond dissociation energy (BDE) simulation. Incorporation
of the 18O in H218O into the benzaldehyde derivative occurs even in
the presence of dissolved oxygen. This result can be explained by
the following pathway sequence: (1) formation of an aryl cation
radical, (2) Ca–Cb cleavage, (3) formation of a benzyl radical and
cation intermediate and (4) nucleophilic attack of water. Thus, in
free-radical-mediated lignin degradation, CR- and AR-producing
systems are important for initiating hydrogen abstraction from
lignin model compounds, although the role of PR as a chain-
carrying radical should also be considered.
Finally, we propose a pathway for degradation of lignin model
compound 4 by radicals initiated by hydrogen abstraction at
the benzyl position (Fig. 9). Two reaction routes have been
proposed for the initial reaction of the lignin model compound
4 with AR and CR: (1) electron abstraction from the aromatic
ring and (2) hydrogen abstraction at the benzyl position.8 The
major reaction products are benzaldehyde 9 produced by Ca–
Cb cleavage from the first route, and benzyl ketone 3 produced
by reaction of the benzyl radical intermediate with molecular
oxygen and subsequent release of hydroperoxy radical from the
second route. The benzyl radical, an initial product of the lignin
model compound 4 by hydrogen abstraction, can be transformed
to the aryl cation radical intermediate upon elimination of a
proton. This complicates our understanding of the initial reaction.
However, because CR and AR react not as electron acceptors but
as hydrogen acceptors,37–39 it is plausible that the initial reaction
starts with hydrogen abstraction.
Acknowledgements
This work was supported by the Advanced Research Program
of the Research Institute of Innovative Technology for the Earth
(RITE), a Grant-in-Aid for Scientific Research (B, to T. W.) and a
JSPS fellowship (to Y. O.). We are indebted to M. Shigematsu
of Fukuoka University for help with MO calculations and to
K. Okuno and Y. Oya of Shizuoka University for help with rapid-
flow ESR measurements.
Notes and References
By comparing H216O and H218O under N2 and 18O2 atmospheres
(Fig. 3), it is clearly evident that 18O in water is incorporated
into a carbonyl group of benzaldehyde 9 even in the presence of
dissolved oxygen. Fig. 9 shows the following possible pathway
sequence that explains this finding: (1) formation of an aryl cation
radical, (2) Ca–Cb cleavage, (3) formation of a benzyl radical
and cation intermediate and (4) nucleophilic attack of water. This
sequence is consistent with reported reactions of the aryl cation
radical.44,45 Thus, we correctly propose that the major radical
species responsible for the degradation of non-phenolic lignin
model compound 4 in lipid peroxidation are AR and CR, both of
which abstract hydrogen from the benzyl position. Although AR
and CR are important for lignin degradation, we point out that PR
and the aryl radical may also play important roles in propagating
radical chain reactions in lipid peroxidation.
1 K. Messner and E. Srebotnik, FEMS Microbiol. Rev., 1994, 13, 351–
364.
2 R. A. Blanchette, E. W. Krueger, J. E. Haight, M. Akhtar and D. E.
Akin, J. Biotechnol., 1997, 53, 203–213.
3 E. Srebotnik and K. Messner, Appl. Environ. Microbiol., 1994, 60,
1383–1386.
4 M. Enoki, T. Watanabe, S. Nakagame, K. Koller, K. Messner, Y. Honda
and M. Kuwahara, FEMS Microbiol. Lett., 1999, 180, 205–211.
5 W. Bao, Y. Fukushima, K. A. Jensen Jr, M. A. Moen and K. E. Hammel,
FEBS Lett., 1994, 354, 297–300.
6 K. A. Jensen Jr, W. Bao, S. Kawai, E. Srebotnik and K. E Hammel,
Appl. Environ. Microbiol., 1996, 62, 3679–3686.
7 T. Watanabe, S. Katayama, M. Enoki, Y. Honda and M. Kuwahara,
Eur. J. Biochem., 2000, 13, 4222–4231.
8 A. N. Kapich, K. A. Jensen and K. E. Hammel, FEBS Lett., 1999, 461,
115–119.
9 E. Niki, Chem. Phys. Lipids, 1987, 44, 227–253.
10 B. Halliwell and J. M. C. Gutteridge, in Free Radicals in Biology and
Medicine, Oxford University Press, New York, 3rd. edn, 1985, p. 297.
11 S. Hosoya, K. Kanazawa, H. Kaneko and J. Nakano, Mokuzai
Gakkaishi, 1980, 26, 118–121.
12 S. Kawai, M. Nakagawa and H. Ohashi, Enzyme Microb. Technol.,
2002, 30, 482–489.
Conclusion
13 R. Amirta, K. Fujimori, N. Shirai, Y. Honda and T. Watanabe, Chem.
Phys. Lipids, 2003, 126, 121–131.
14 D. Walker and J. D. Hiebert, Chem. Rev., 1967, 67, 153–195.
15 H.-D. Becker, A. Bjo¨rk and E. Adler, J. Org. Chem., 1980, 45, 1596–
1600.
16 H. C. Brown and C. A. Brown, J. Am. Chem. Soc., 1962, 84, 1495.
17 S. Kawai, S. Shoji, K. Nabeta, H. Okuyama and T. Higuchi, Mokuzai
Gakkaishi, 1990, 36, 126–132.
18 O. A. Mamer, J. A. Montgomery, R. J. Deckelbaum and E. Granot,
Biol. Mass Spectrom., 1985, 12, 163–169.
19 Y. Ohashi, H. Yoshioka and H. Yoshioka, Biosci., Biotechnol.,
Biochem., 2002, 66, 847–852.
20 Y. Ohashi, Y. Takeuchi, M. Hirama, H. Yoshioka and H. Yoshioka,
Bull. Chem. Soc. Jpn., 2005, 78, 1757–1762.
We studied lignin biodegradation using white-rot fungi by investi-
gating the reactivities of free radicals towards non-phenolic lignin
model compounds. Spin-trapping ESR and GC–MS analysis show
that carbon-centred radicals (CRs) derived from 2,2¢-azobis(2-
amidinopropane) dihydrochloride (AAPH) decompose the b-
O-4 dimeric lignin model compound with accompanying a–b
bond cleavage. Reactions producing alkoxyl radical (AR) from
tert-BuOOH oxidize the a-position of the dimeric lignin model
compound and cleave the b-O-4 bond, but reactivity is less than for
reactions producing CR. The lower reactivity of the AR-producing
system can be explained by b-scission of AR because AR exhibits
2490 | Org. Biomol. Chem., 2011, 9, 2481–2491
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
21 H. Yoshioka, Y. Ohashi, H. Fukuda, Y. Senba and H. Yoshioka, J. Phys.
Chem. A, 2003, 107, 1127–1132.
22 W. T. Dixon and R. O. C. Norman, J. Chem. Soc., 1963, 3119–3124.
23 J. E. Bennett, J. Chem. Soc., Faraday Trans., 1990, 86, 3247–3252.
24 J. S. Wright, E. R. Johnson and G. A. DiLabio, J. Am. Chem. Soc.,
2001, 123, 1173–1183.
25 A. Yoshioka, T. Seino, M. Tabata and M. Takai, Holzforschung, 2000,
54, 357–364.
26 T. Seino, A. Yoshioka, M. Fujiwara, K-L. Chen, T. Erata, M. Tabata
and M. Takai, Wood Sci. Technol., 2001, 35, 97–106.
27 P. G. Mekarbane and B. J. Tabner, Magn. Reson. Chem., 2000, 38,
845–852.
34 T. Watanabe, K. Koller and K. Messner, J. Biotechnol., 1998, 62, 221–
230.
35 K. Messner, K. K. Fackler, P. Lamaipis, W. Gindl, E. Srebotnik
and T. Watanabe, ACS Symposium Series 845 Wood deterioration and
preservation, ed. B. Goodel, D. D. Nicholas and T. P. Schultz, American
Chemical Society, Washington, DC, 2003, pp. 73–96.
36 P. Baiocco, A. M. Barreca, M. Fabbrini, C. Galli and P. Gentili, Org.
Biomol. Chem., 2003, 1, 191–197.
37 K. D. Beare and M. L. Coote, J. Phys. Chem. A, 2004, 108, 7211–
7221.
38 Y. M. Riyad, R. Hermann and O. Brede, Radiat. Phys. Chem., 2005,
72, 437–445.
28 O. M. Panasenko, A. N. Osipov, A. V. Chekanov, J. Arnhold and V. I.
Sergienko, Biochemistry (Moscow), 2002, 67, 1061–1070.
29 O. M. Panasenko, A. V. Chekanov, J. Arnhold, V. I. Sergienko, A. N.
Osipov and Yu. A. Vladimirov, Biochemistry (Moscow), 2005, 70,
1209–1217.
39 J. Pe´rez-Prieto, R. E. Galian, P. O. Burgos, M. D. M. Min˜ana, M. A.
Miranda and F. Lo´pez-Ortiz, Org. Lett., 2005, 7, 3869–3872.
40 C. M. Jones and M. J. Burkitt, J. Am. Chem. Soc., 2003, 125, 6946–
6954.
41 Y. P. Tsentalovich, L. V. Kulik, N. P. Gritsan and A. V. Yurkovskaya,
J. Phys. Chem. A, 1998, 102, 7975–7980.
42 W. H. Koppenol, Free Radic. Toxicol., 1997, 3–14.
43 M. Bietti, E. Baciocchi and S. Steenken, J. Phys. Chem. A, 1998, 102,
7337–7342.
30 A. Valavanidis, B. C. Gilbert and A. C. Whitwood, Chimika chronika,
New Ser., 1995, 24, 217–232.
31 G. Czapski, H. Levanon and A. Samuni, Israel J. Chem., 1969, 7, 375–
386.
32 A. Khindaria, T. A. Grover and S. D. Aust, Biochemistry, 1995, 34,
44 M. Schmittel and A. Burghart, Angew. Chem., Int. Ed. Engl., 1997, 36,
6020–6025.
2550–2589.
33 B. A. Wagner, G. R. Buettner and C. P. Burns, Biochemistry, 1994, 33,
4449–4453.
45 E. Baciocchi, M. Bietti, M. F. Gerini and O. Lanzalunga, Biochem.
Biophys. Res. Commun., 2002, 293, 832–835.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2481–2491 | 2491
©