Redox silencing of the Fenton reaction system by an alkylitaconic acid, ceriporic acid B produced by a selective lignin-degrading fungus, Ceriporiopsis subvermispora

Article abstract of DOI:10.1039/b614379b

The selective lignin-degrading fungus, Ceriporiopsis subvermispora secretes alkylitaconic acids (ceriporic acids) during wood decay. We reported that ceriporic acid B (hexadecylitaconic acid) was protective against the depolymerization of cellulose by the Fenton reaction. To understand the redox silencing effects, we analyzed the physicochemical and redox properties of itaconic, octylitaconic and hexadecylitaconic acids. The initial rate of HO production by the Fenton system with Fe³⁺, H₂O₂ and l-cysteine was suppressed by hexadecylitaconic and octylitaconic acids by 0.04 and 0.16 of the reaction rate without chelators. ESR, O₂ uptake and the assay of Fe²⁺ with BPS demonstrated that Fe³⁺ reduction by l-cysteine was suppressed by hexadecylitaconic and octylitaconic acids while the reaction of Fe²⁺ with H₂O₂ was not suppressed by the two alkylitaconic acids. Ligand exchange experiments with NTA demonstrated that Fe³⁺ chelation by two carboxyl groups of alkylitaconic acids is a critical step in iron redox modulation. In stark contrast, the production of HO and reduction of Fe³⁺ were not suppressed by itaconic acid due to HO-initiated degradation of the chelator. The strong redox silencing effects by a series of alkylitaconic acids have attracted interest in controlling microbial plant cell wall degradation and chemoprotection against cellular oxidative injury. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b614379b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Redox silencing of the Fenton reaction system by an alkylitaconic acid,
ceriporic acid B produced by a selective lignin-degrading fungus,
Ceriporiopsis subvermispora
Yasunori Ohashi,a,b Yoshihiko Kan, Takahito Watanabe, Yoichi Honda and Takashi Watanabe*
a
a
a
a
Received 3rd October 2006, Accepted 9th January 2007
First published as an Advance Article on the web 26th January 2007
DOI: 10.1039/b614379b
The selective lignin-degrading fungus, Ceriporiopsis subvermispora secretes alkylitaconic acids (ceriporic
acids) during wood decay. We reported that ceriporic acid B (hexadecylitaconic acid) was protective
against the depolymerization of cellulose by the Fenton reaction. To understand the redox silencing
effects, we analyzed the physicochemical and redox properties of itaconic, octylitaconic and
•
3+
hexadecylitaconic acids. The initial rate of HO production by the Fenton system with Fe , H
2
O
2
and
L-cysteine was suppressed by hexadecylitaconic and octylitaconic acids by 0.04 and 0.16 of the reaction
2
+
3+
rate without chelators. ESR, O
2
uptake and the assay of Fe with BPS demonstrated that Fe
reduction by L-cysteine was suppressed by hexadecylitaconic and octylitaconic acids while the reaction
2
+
of Fe with H
2
O was not suppressed by the two alkylitaconic acids. Ligand exchange experiments with
2
3
+
NTA demonstrated that Fe chelation by two carboxyl groups of alkylitaconic acids is a critical step in
iron redox modulation. In stark contrast, the production of HO and reduction of Fe were not
suppressed by itaconic acid due to HO -initiated degradation of the chelator. The strong redox silencing
•
3+
•
effects by a series of alkylitaconic acids have attracted interest in controlling microbial plant cell wall
degradation and chemoprotection against cellular oxidative injury.
Introduction
as the semiquinone radical reduce molecular oxygen to produce
3
+
superoxide, which in turn reduces Fe or disproportionate into
Iron ions play a key role in fungal metabolism and growth. Since
the bioavailability of iron is limited in nature, a number of micro-
organisms produce iron-chelating agents, such as siderophores, to
aid in the assimilation of ferric iron. On the other hand, some
microorganisms produce iron-chelating metabolites to sequester
3+
H
2
O . Fe is directly reduced by semiquinone radicals and lignin-
2
derived phenols such as guaiacol and catechol. Thus, if some
inhibition systems for the iron redox reactions were not involved in
the wood decaying systems, production of the cellulolytic oxidant,
hydroxyl radical is inevitable. Therefore, we hypothesized that
selective white rot fungi secrete low-molecular-mass chelators
which inhibit the depolymerization of cellulose by inhibiting the
redox cycling of iron ions in the Fenton system during free-radical-
1
2
3+
Fe ions to facilitate metal tolerance. Wood rot fungi secrete Fe -
reducing extracellular metabolites to promote the production of
hydroxyl radicals by the Fenton reaction to attack wood cell
3–10
•
walls. The Fenton-based OH-producing system plays a central
20
mediated lignin biodegradation. We then demonstrated that ceri-
3
–13
role in wood decay by brown rot fungi.
It was shown that
poric acid B, an alkylitaconic acid produced by C. subvermispora,
3
+
natural Fe -reductants secreted by the brown rot basidiomycete
inhibited the depolymerization of cellulose by the Fenton reaction
Gloeophyllum trabeum depolymerized cellulose in combination
with iron ions and H
brown rot decay supports that the Fenton reaction is the major
pathway in the breakdown of cellulose by these microorganisms.
The production of hydroxyl radicals is also proposed as a possible
low molecular mass agent that increases the porosity of wood
cell walls in non-selective white rot.
rot fungi with high cellulolytic activity, a selective white rot
fungus, Ceriporiopsis subvermispora is able to decompose lignin
3+
21
even in the presence of Fe -reductants such as hydroquinone.
14
2
O
2
.
Structural analysis of cellulose after
The suppressive effects of ceriporic acids in the Fenton system
are in contrast to the effects of itaconic acid, a metabolite of
1
5
22
23
fungi such as Aspergillus itaconicus, Helicobasidium mompa,
24
25
Ustilago zeae, U. maydis and some yeasts belonging to the
genus Candida. To assess redox modulation by (alkyl)itaconic
26
8
,16–19
In contrast to wood
acids, we synthesized octyl (1-undecene-2,3-dicarboxylic acid) and
hexadecyl (ceriporic acid B; 1-nonadecene-2,3-dicarboxylic acid)
itaconic acids, and comparatively analyzed the physicochemical
and redox properties of itaconic acid derivatives with a long,
medium and no alkyl side chain.
4
in wood cell walls without excessive cellulose damage. Lignin
biodegradation proceeds by a free radical process in the presence
of molecular oxygen and transition metals. Reductive radicals such
a
Materials and methods
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-
Materials
7
74-38-3640
b
Itaconic acid, 1-bromohexadecane, 1-bromooctane, FeCl , FeSO ,
bathophenanthrolinedisulfonic acid disodium salt (BPS), H
Research Fellow of the Japan Society for the Promotion of Science (JSPS),
Japan
3
4
2
O
2
,
8
40 | Org. Biomol. Chem., 2007, 5, 840–847
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L-cysteine and L(+)-ascorbic acid were obtained from Wako
Pure Chemical Industries (Osaka, Japan). Magnesium turnings,
nitrilotriacetic acid (NTA) and hydroquinone were obtained from
Nacalai Tesque (Kyoto, Japan). A spin trapping reagent, a-
(
4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was obtained
from Labotec (Tokyo, Japan). All other chemicals used were of
analytical reagent grade. Water was purified using an EASYpureꢀC
compact ultrapure water system (Barnstead, IA) throughout this
study.
Instrumental analysis
GC-MS analysis was carried out with a Shimadzu GCMS-
QP5050A gas chromatograph mass spectrometer (Kyoto, Japan)
equipped with a DB-5HT column (length, 30 m; i.d., 0.25 mm;
thickness, 1 lm; J & W Scientific Inc., CA). The electron impact
mass spectrum (EI-MS) was recorded at an ionization energy of
◦
Fig. 1 Synthetic route for 1-nonadecene-2,3-dicarboxylic acid and 1-
undecene-2,3-dicarboxylic acid. 1, itaconic acid; 2, dimethyl itaconate;
3, dimethyl (a-bromomethyl)fumarate; 4, dimethyl hexadecylitaconate
(dimethyl 1-nonadecene-2,3-dicarboxylate); 5, hexadecylitaconic acid
7
3
0 eV. The column oven temperature was maintained at 50 C for
◦
◦
◦
−1
min and subsequently raised from 50 C to 100 C at 40 C min ,
◦
◦
◦
−1
◦
◦
from 100 C to 210 C at 3 C min , and from 210 C to 300 C
at 12 C min and maintained for 15 min. H- and C-NMR
◦
−1
1
13
(
ceriporic acid B; 1-nonadecene-2,3-dicarboxylic acid); 6, dimethyl
spectra were measured with a JEOL k-400 NMR spectrometer
octylitaconate (dimethyl 1-undecene-2,3-dicarboxylate); 7, octylitaconic
acid (1-undecene-2,3-dicarboxylic acid).
◦
(
Tokyo, Japan) at 400 MHz, 20 C in CDCl
3
. Surface tension was
¨
measured using a KRUSS Digital Tensiometer K9 (Hamburg,
Germany). pH titration was carried out using a HORIBA pH
meter D21 (Kyoto, Japan). UV–vis absorption spectra were
measured with a HITACHI U-3310 Spectrophotometer (Tokyo,
1
1); 22.6 (C-10); 27.4 (C-5); 29.3–29.6 (C-6–C-9); 31.8 (C-4); 47.8
C-3); 127.4 (C-2); 137.6 (C-1); 171.1 (C(2)–COOH); 179.8 (C(3)
COOH), m/z 224(1%), 196 (3), 179 (1), 163 (1), 150 (4), 139 (7),
26 (100), 111 (8), 98(35), 81 (8), 70 (10), 57 (28), 43 (60).
(
–
◦
Japan) at 25 C. ESR spectra were measured with a JEOL JES-
1
FR30 Free Radical Monitor (Tokyo, Japan) at room temperature
under the following conditions: frequency, 9.425 GHz; center field,
Determination of surface tension activity of octylitaconic acid
3
35.6 mT; sweep width, 5.0 mT; modulation width, 0.10 mT;
receiver gain, 320; data points, 4096; time constant, 0.10 s; sweep
time, 1.0 min; power, 4.0 mW.
The surface tension of octylitaconic acid was compared with that
of Tween 20 and Tween 80. Each compound was dissolved in water
−
1
−1
at a concentration range of 0.1 mg L to 100 mg L . Each sample
5 mL) was placed in a vessel and surface tension was measured
(
Synthesis of ceriporic acid B and its analogues
in triplicate.
Hexadecylitaconic acid (ceriporic acid B; 1-nonadecene-2,3-
dicarboxylic acid, 5) and dimethyl hexadecylitaconate (dimethyl
Acid dissociation constants of ceriporic acid B and its analogues
1
-nonadecene-2,3-dicarboxylate, 4) were synthesized according to
27
Fig. 1 as described.
To determine the acid dissociation constant of hexadecylitaconic
acid, octylitaconic acid, and itaconic acid, pH titration was
carried out at room temperature. The electrode was calibrated
using standard aqueous buffers (pH 4.0 and 7.0). Solutions were
prepared with 50% (v/v) EtOH(aq) due to the low solubility of
hexadecylitaconic acid in water, and ionic strength was adjusted
Dimethyl octylitaconate (dimethyl 1-undecene-2,3-dicar-
boxylate, 6) was synthesized using the Grignard reaction from
dimethyl (a-bromomethyl)fumarate, 3. Procedures for the reaction
27
and purification were as described for hexadecylitaconic acid
except for the preparation of the Grignard reagent. The tem-
◦
perature for the Grignard reaction was lowered from 50–55 C
to 100 mM by adding NaNO . Ten millilitres of hexadecylitaconic
3
◦
to 30–35 C due to the higher reactivity of 1-bromooctane with
acid, octylitaconic acid, and itaconic acid solution (1 mM each)
magnesium compared to that of 1-bromohexadecane. Distilled
tetrahydrofuran (74.8 mL), compound 3 (3.94 g, 16.6 mM) and 1-
bromooctane (4.24 g, 22.0 mM) were prepared to give compound
containing HNO (4 mM) were titrated with 10 mM NaOH in 50%
(v/v) EtOH(aq). The pH was measured after every incremental
addition of NaOH solution (20 lL) with stirring until the pH
3
6
(1.54 g, 5.70 mM: 34.3% from 3). Compound 6 was then
value reached 12.0.
demethylated by acidolysis. A mixture of 6 (0.88 g, 3.3 mM),
formic acid (22.9 mL), hydroquinone (4.6 mg) as a polymerization
inhibitor, and sulfuric acid (0.323 g) was heated at 100 C for
•
Effects of ceriporic acid B and its analogues on HO production
◦
3
hours with stirring to give octylitaconic acid (1-undecene-2,3-
dicarboxylic acid, 7, 0.40 g, 1.7 mM: 51% from 6): d (400 MHz,
CDCl ) 0.85 (3H, s, CH CH ); 1.23 (12H, m, CH ); 1.64 (1H, m,
H-4); 1.86 (1H, m, H-4); 3.37 (1H, t, H-3); 5.78 (1H, s, CH
=,
trans); 6.44 (1H, s, CH (400 MHz, CDCl ) 14.1 (C-
=, cis), d
To investigate the effects of itaconic acid derivatives on HO•
production in the Fenton system, the spin trapping method was
carried out as described. With an excess of ethanol versus 4-
POBN, HO produced by the Fenton reaction reacts with ethanol
to yield the a-hydroxyethyl radical according to (eqn (1)). The
H
28
3
2
3
2
•
2
2
C
3
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a-hydroxyethyl radical is trapped with 4-POBN (eqn (2)), and the
spin adduct produced is quantified by ESR:
1.0 mL and the experiments were carried out in triplicate. The final
concentrations of each reagent at the starting point of the reaction
are described in the legends of Fig. 8 and Table 3.
•
•
CH
3
CH
2
OH + HO → CH
3
C HOH + H
2
O
(1)
(2)
Theoretical calculation of HOMO energy of ceriporic acid B and
its analogues
•
CH
3
C HOH + 4-POBN → 4-POBN-CH
2
(CH )OH
3
Concentrated stock solutions of hexadecylitaconic acid, octylita-
conic acid, dimethyl hexadecylitaconate, dimethyl octylitaconate
and itaconic acid (30 mM each) were prepared in ethanol. Those
Differences in HOMO (highest occupied molecular orbital) ener-
gies of geometrically optimized itaconic acid derivatives including
itaconic acid, octylitaconic acid, and hexadecylitaconic acid were
estimated by the MOS-F module in FUJITSU WinMOPAC
Version 3.5 (Tokyo, Japan, 2002), semi-empirical molecular orbital
calculation software examining the effect of alkyl chain lengths of
of FeSO
(
(
4
and FeCl
0.5 mM each) and H
25 mM, pH 4.0).
The required amounts of succinate buffer and FeCl
3
(1 mM each), 4-POBN (1 M), reductants
2
O
2
(2 mM) were prepared in succinate buffer
stock
3+
3
itaconic acid derivatives on the inhibitory effect on Fe reduction
solution were poured into a 10 mL vial immediately after prepa-
ration, and then the stock solution of itaconic acid derivatives
and ethanol were added to the solution. After pre-incubation for
•
and HO production.
Results
3
0 min, stock solutions of 4-POBN, reductants, and H
2
2
O were
added sequentially. The total volume of the reaction solution was
Surface tension activity of octylitaconic acid
2
1
00 lL. The amount of spin adduct was measured at 1, 5, 10, and
5 minutes after the addition of H . These experiments were
Surface tensions of Tween 20, Tween 80, and octylitaconic acid are
shown in Fig. 2. Surface activities of octylitaconic acid were lower
than those of Tween 20 and Tween 80 between the concentrations
2
O
2
carried out in triplicate. The final concentrations of each reagent
at the starting point of the reaction are described in each legend
of the figures and tables.
−
1
−1
of 0.1 mg L and 10 mg L . At concentrations higher than
5
−
1
0 mg L , surface activities increased linearly and exceeded
The experiments using FeSO
carried out in the same way.
4
instead of FeCl –reductant were
3
those of Tween 20 and Tween 80. It was difficult to measure the
surface activities of octylitaconic acid at concentrations higher
−
1
than 100 mg L due to low solubility.
Effects of ceriporic acid B and its analogues on dissolved oxygen
consumption in the process of iron redox reaction
2
+
The production of Fe was determined by oxygen uptake exper-
iments. Stock solutions of hexadecylitaconic acid, octylitaconic
acid and itaconic acid (30 mM each) were prepared in ethanol,
whereas those of FeSO
mM) and H (20 mM) were prepared in water.
Stock solutions of H , L-cysteine, and itaconic acid derivative
in ethanol were mixed in a 10 mL test tube equipped with an
oxygen electrode. Aqueous FeCl was added to start the reaction.
instead of FeCl –L-cysteine were carried
4
and FeCl
3
(10 mM each), L-cysteine (5
2
O
2
2
O
2
3
Experiments using FeSO
out in the same way.
4
3
Effects of ceriporic acid B and its analogues on the reduction of
3
+
Fe
2
+
2+
2+
BPS, a strong chelator of Fe , was used to determine Fe
3
+
Fig. 2 Surface tension activities of octylitaconic acid (ꢀ), Tween 20 (᭺)
and Tween 80 (ꢁ).
produced by the reduction of Fe . The production of [Fe(BPS) ]
3
was determined by the absorbance at 534 nm (eqn (3)) (e534 is
−
1
−1 29,30
22 140 L eq cm ).
2
+
2+
Fe + 3BPS → [Fe(BPS)
3
]
(3)
Acid dissociation constants of ceriporic acid B and its analogues
Stock solutions of hexadecylitaconic acid, octylitaconic acid
and itaconic acid (4 mM each) were prepared using ethanol. Those
The pH titration curves for (alkyl)itaconic acid are shown in Fig. 3.
The pK
a
and pK
a
values of itaconic acid in aqueous ethanol
1
2
3
+
of NTA (4 mM), BPS (10 mM) and the reductant for Fe (100
mM) were prepared using succinate buffer (25 mM, pH 4.0). FeCl
2 mM) was dissolved in succinate buffer (25 mM, pH 4.0) and
solution were 4.5 and 6.5, respectively. These values were slightly
higher than those in a previous report (pK , 3.8 and pK , 5.7)
3
a
a
2
1
31
(
measured in water. The pK
a
and pK
a
of hexadecylitaconic
1
2
3
+
immediately used for the assay of Fe reduction. A stock solution
of the itaconic acid derivative was added to FeCl solution in a
0 mL vial. Stock solutions of BPS and the reductant were finally
acid were 4.5 and 7.8, respectively. For octylitaconic acid, pK
a
1
3
and pK
a
were 4.6 and 7.6, respectively. Differences between the
2
1
pK
a
values of these three itaconic acid derivatives were within
1
3
+
added after incubating Fe with NTA for 30 min. Absorption was
measured at 534 nm. The total volume of the reaction solution was
0.1; however, the pK
a
of itaconic acid was lower than those of
2
hexadecylitaconic acid and octylitaconic acid by 1.3 and 1.1,
8
42 | Org. Biomol. Chem., 2007, 5, 840–847
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•
Effects of ceriporic acid B and its analogues on HO production
The production of HO• in the Fenton system containing
alkyl)itaconic acid (octylitaconic acid, dimethyl octylitaconate
(
3
+
and itaconic acid) and a reductant for Fe , L-cysteine, was deter-
mined (Fig. 5). No suppressive effects were found for dimethyl
octylitaconate (ꢀ). Itaconic acid (᭛) slightly accelerated HO pro-
duction, whereas HO production was suppressed by octylitaconic
•
•
acid (᭺).
Fig. 3 pH titration curves of 1 mM itaconic acid (ꢀ), octylitaconic acid
᭺) and hexadecylitaconic acid (ꢁ) in the presence of 100 mM NaNO
and 4 mM HNO in aqueous EtOH (50%, v/v) at room temperature.
(
3
3
respectively. The chain lengths of the two alkylitaconic acids,
hexadecylitaconic acid and octylitaconic acid, did not affect the
pKa1 or pKa2 values significantly.
pH dependence on the molar fraction of each acid was
calculated using the pK
a
values, as shown in Fig. 4(a)–(c). It
−
was found that the pH dependence of [HA ] of itaconic acid is
different from that of octylitaconic acid and hexadecylitaconic
Fig. 5 Effects of octylitaconic acid (᭺), dimethyl octylitaconate (ꢁ),
•
3+
2 2
and itaconic acid (᭛) on HO production by Fe –L-cysteine and H O
−
acid; the maximum [HA ] of itaconic acid was lower than that of
in succinate buffer (pH = 4.0) containing 20% (v/v) EtOH at room
−
octylitaconic acid and hexadecylitaconic acid. The highest [HA ]
3+
temperature. [Fe ] = 0.1 mM, [itaconate derivative] = 3 mM, [H
2
O
2
] =
molar fraction of itaconic acid was 81.6% at pH 5.5 although those
0
.5 mM, [L-cysteine] = 0.05 mM, [POBN] = 100 mM. In a control
of hexadecylitaconic acid and octylitaconic acid were 94.1% and
experiment (ꢀ), no itaconate derivatives were added.
−
9
5.7% at pH 6.1, respectively. The [HA ] of itaconic acid was the
primary fraction at pH 4.6–6.3 although with hexadecylitaconic
acid and octylitaconic acid the primary fraction was at pH 4.7–7.5
and pH 4.6–7.7, respectively. Around 80% of the three itaconic
2
+
Fig. 6 shows the results of the direct reactions of Fe with
octylitaconic acid, dimethyl octylitaconate or itaconic acid. In the
2
+
Fe system, the amount of spin adduct rapidly increased within
a minute and reached a plateau. Among the three (alkyl)itaconic
acid derivatives exist as “H A” at pH 4, used in the experiments
2
3
+
for the determination of their inhibitory effects on Fe reduction
•
and HO production.
Fig. 6 Effects of octylitaconic acid (᭺), dimethyl octylitaconate (ꢁ), and
•
2+
Fig. 4 The pH dependencies of molar fraction diagrams of itaconic
acid derivatives (1 mM) in aqueous EtOH (50%, v/v) at 100 mM ionic
strength and room temperature. (a) Itaconic acid; (b) octylitaconic acid;
2 2
itaconic acid (᭛) on HO production by Fe and H O in succinate buffer
2+
(pH = 4.0) containing 20% (v/v) EtOH at room temperature. [Fe ] =
0.1 mM, [itaconate derivative] = 3 mM, [H
2
O
2
] = 0.5 mM, [POBN] =
(
(
c) hexadecylitaconic acid. Each line shows the molar fraction of [H
2
A]
100 mM. In control experiment (ꢀ), no itaconic acid derivatives were
), [HA ] (---) and [A2−] (· · ·).
−
added.
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•
3+
Table 1 Initial rate and cumulative amount of HO production by the reaction of Fe –reductant–itaconate derivative
a
a,b
Reductant
L-Cysteine
Itaconate derivative
Initial rate
Cumulative amount
—
1.00
0.04
0.94
0.16
0.89
0.96
1.00
0.11
0.84
0.38
0.98
1.04
1.00
0.12
0.97
0.16
0.81
0.80
1.00
0.04
0.96
0.05
1.03
1.16
1.00
0.13
1.03
0.11
1.02
1.21
1.00
0.13
1.03
0.04
1.02
1.06
Hexadecylitaconic acid
Dimethyl hexadecylitaconate
Octylitaconic acid
Dimethyl octylitaconate
Itaconic acid
L(+)-Ascorbic acid
Hydroquinone
—
Hexadecylitaconic acid
Dimethyl hexadecylitaconate
Octylitaconic acid
Dimethyl octylitaconate
Itaconic acid
—
Hexadecylitaconic acid
Dimethyl hexadecylitaconate
Octylitaconic acid
Dimethyl octylitaconate
Itaconic acid
3
+
Experiments were carried out in succinate buffer (pH = 4.0) containing 20% (v/v) EtOH at room temperature. [Fe ] = 0.1 mM, [H
2
O
2
] = 0.5 mM,
a
[
itaconate derivative] = 3 mM, [reductant] = 0.05 mM, [4-POBN] = 100 mM. Experimental procedure is described in Materials and Methods. Values are
b
•
expressed as a ratio of the two reaction systems with and without itaconic acid derivatives. Cumulative amount of HO produced during the first 15 min.
•
acid derivatives, suppression of HO production was found only for
Effects of ceriporic acid B and its analogues on dissolved oxygen
consumption in the process of iron redox reaction
3
+
octylitaconic acid as in the Fe –reductant system. The production
of HO in the presence of octylitaconic acid was less than 3% of
•
The effects of hexadecylitaconic acid and its analogues on
dissolved oxygen consumption in the iron redox reaction are
the control.
3
+
All results in the reactions for the Fe –reductant system
and those for Fe are shown in Table 1 and 2, respectively.
When hexadecylitaconic acid or octylitaconic acid was added
to the Fe –reduction and Fe systems, HO production was
inhibited 15 minutes after starting the reaction. In the com-
parison between the system of Fe –reductant and Fe , the
2
+
2
+
shown in Fig. 7. Dissolved oxygen was consumed by Fe in the
presence of hexadecylitaconic acid, octylitaconic acid and itaconic
3
+
3
+
2+
•
acid according to eqn (1). Mixing Fe , itaconic acid and L-cysteine
decreased dissolved oxygen; however, dissolved oxygen was not
3
+
3
+
2+
consumed when Fe was premixed with hexadecylitaconic acid or
octylitaconic acid followed by addition of L-cysteine as a reductant
•
inhibition effect of HO production by hexadecylitaconic acid
3
+
3
+
for Fe .
and octylitaconic acid in the Fe –reductant system was larger
2
+
than that in the Fe system. Furthermore, the initial rates of
HO production in the Fe system containing hexadecylitaconic
acid and octylitaconic acid were bigger than in the Fe –reductant
system.
•
2+
Effects of ceriporic acid B and its analogues on the reduction of
3
+
3
+
Fe
Effects of hexadecylitaconic acid, octylitaconic acid and itaconic
3
+
acid on Fe reduction by a reductant, L-cysteine, were investigated
in the presence and absence of a chelating reagent, NTA. When
NTA was not added (hexadecylitaconic acid +, NTA −; ᭺), the
•
2+
3
Table 2 Initial rates of HO production and cumulative amount of it at
production rate of [Fe(BPS) ] decreased compared with a control
2
+
1
5 min after reaction started in the Fe –itaconate derivative system
experiment (hexadecylitaconic acid −, NTA −; ꢁ) as shown in
Fig. 8. On the other hand, when hexadecylitaconic acid and NTA
were added (hexadecylitaconic acid +, NTA +; ꢀ), the initial rate
a
a,b
Itaconate derivative
Initial rate
Cumulative amount
2
+
—
1.00
0.45
0.91
0.36
0.92
0.89
1.00
0.26
1.05
0.21
1.03
1.02
of [Fe(BPS)
3
]
production increased about five times compared
Hexadecylitaconic acid
Dimethyl hexadecylitaconate
Octylitaconic acid
Dimethyl octylitaconate
Itaconic acid
with when only hexadecylitaconic acid was added (᭺).
All results are shown in Table 3. When hexadecylitaconic acid
3
+
2+
or octylitaconic acid was added to the Fe –reduction and the Fe
•
systems, HO production was inhibited 15 minutes after starting
3
+
the reaction. In a comparison of the system of Fe –reductant with
Experiments were carried out in succinate buffer (pH = 4.0) containing
2
+
•
2+
Fe , the inhibition effect of HO production by hexadecylitaconic
2
[
0% (v/v) EtOH at room temperature. [Fe ] = 0.1 mM, [H
2
O
2
] = 0.5 mM,
3
+
itaconate derivative] = 3 mM, [4-POBN] = 100 mM. Experimental
acid and octylitaconic acid in the Fe –reductant system was
greater than that with Fe . Furthermore, the initial rate of HO
production in the Fe system containing hexadecylitaconic acid or
octylitaconic acid was greater than in the Fe –reductant system.
a
procedure is described in Materials and Methods. Values are expressed
as a ratio of the two reaction systems with and without itaconic acid
2+
•
2
+
b
•
derivatives. Cumulative amount of HO produced during the first 15 min.
3
+
8
44 | Org. Biomol. Chem., 2007, 5, 840–847
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2+
3
Fig. 8 Effects of hexadecylitaconic acid and NTA on [Fe(BPS) ]
3+
production by Fe –L-cysteine–BPS in succinate buffer (pH = 4.0)
containing 10% (v/v) EtOH at room temperature. [Fe ] = 0.02 mM,
hexadecylitaconic acid] = 0.04 mM, [L-cysteine] = 10 mM, [NTA] =
.04 mM, [BPS] = 1.0 mM. The complete system (hexadecylitaconic
acid +, NTA+; ꢁ) contained all reagents. The reaction system of
3+
[
0
(
hexadecylitaconic acid +, NTA −, ᭺) did not contain NTA. In the control
experiment (hexadecylitaconic acid −, NTA −, ꢀ), hexadecylitaconic acid
and NTA were omitted.
2+
3+
Table 3 Initial rate of [Fe(BPS)
reductant–itaconic acid derivatives
3
]
production in reactions of Fe –
a
Reductant
L-Cysteine
Itaconate derivative
Chelator
Initial rate
—
—
—
1.00
0.41
2.06
0.69
1.72
0.53
1.16
1.00
0.33
>10
0.56
2.00
1.11
1.06
1.00
0.34
1.47
0.62
2.20
0.44
1.66
Hexadecylitaconic acid
Hexadecylitaconic acid NTA
Octylitaconic acid
Octylitaconic acid
Itaconic acid
Itaconic acid
—
Hexadecylitaconic acid
Hexadecylitaconic acid NTA
Octylitaconic acid
Octylitaconic acid
Itaconic acid
Itaconic acid
—
Hexadecylitaconic acid
Hexadecylitaconic acid NTA
Octylitaconic acid
Octylitaconic acid
Itaconic acid
2
+
Fig. 7 Time course of dissolved oxygen consumption in Fe –H
and Fe –L-cysteine–H O
2 2
2
O
2
(a)
(b) systems containing 20% (v/v) EtOH in the
presence of hexadecylitaconic acid (᭺), itaconic acid (ꢁ) and octylitaconic
—
NTA
—
NTA
—
—
3+
2
+
acid (᭛). [itaconate derivatives] = 3 mM, [H ] = 5 mM. (a): [Fe ] =
mM. (b): [Fe ] = 1 mM, [L-cysteine] = 0.5 mM. In the control experiment
ꢀ), itaconaic acid derivatives were not added.
2
O
2
L(+)-Ascorbic acid
Hydroquinone
3+
1
(
—
NTA
—
NTA
—
—
Theoretical calculation of HOMO energy of ceriporic acid B and
its analogues
The HOMO energy of itaconic acid derivatives with different chain
length is shown in Fig. 9. Alkylitaconic acids with a long alkyl side
chain have higher HOMO energies. Differences in HOMO energy
were marked between itaconic acid and the two alkyl itaconic
acids.
—
NTA
—
Itaconic acid
NTA
Experiments were carried out in succinate buffer (pH = 4.0) containing
Discussion
3+
1
1
0% (v/v) EtOH at room temperature. [Fe ] = 0.02 mM, [BPS] =
.0 mM, [itaconate derivative] = 0.04 mM, [reductant] = 10 mM,
The biodegradation of lignin by wood rot fungi is an extracellular
free radical event that proceeds in concert with the activation of
molecular oxygen and redox cycling of transition metals. Hydroxyl
[NTA] = 0.1 mM. Experimental procedure is described in Materials and
a
Methods. Values are expressed as a ratio of two reactions systems with and
2
+
without itaconic acid derivatives. The rate of [Fe(BPS)
3
]
production in
3+
the reduction of Fe with L-cysteine, L(+)-ascorbic acid and hydroquinone
was 1.2 × 10 M s , 7.2 × 10 M s and 4.6 × 10 M s , respectively.
•
radicals (HO ), a radical species highly destructive to cellulose
−8
−1
−8
−1
−9
−1
8,9
and lignin, are proposed as principal low molecular mass
oxidants that erode wood cell walls to enhance the accessibility
of extracellular enzymes to wood cell wall components. Wood
rot fungi have versatile enzymatic and non-enzymatic systems to
3
+
inhibitors for Fe reduction is a viable target for antifungal agents
37
in wood preservation.
3
+
2+ 9,20,21,27,32–36
accelerate the reductive half cycle from Fe to Fe .
reduction of Fe promotes the overall production rate of hydroxyl
radicals in the Fenton system. Therefore, the development of
The
In contrast to the wood decay accompanied by the erosion
of wood cell walls and cellulose depolymerization, a selective
white rot fungus, C. subvermispora, decomposes lignin in wood
3
+
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alkylitaconic acids have oxygen atoms as the sole coordination site,
2
+
3+
coordination to Fe is much weaker than that to Fe as explained
38
by the HSAB theory. For example, stability constants (logK) of
iron-dicarboxylic acid complexes are as follows: Fe(II)-malonic
acid, 2.8 (k
1
); Fe(III)-malonic acid, 15.7(k
1
k
2
k ); Fe(II)-oxalic acid,
3
9
3
5
.2 (k ); Fe(III)-oxalic acid, 18 (k
1
k
2
k
3
1
k
2
k ). Therefore stability
3
constants of Fe(II)-alkylitaconic acid must be smaller than those
of Fe(III)-alkylitaconic acid.
3
+
2+
Suppression of the reductive half cycle from Fe to Fe by
alkylitaconic acids was clearly demonstrated by oxygen uptake
experiments. In general, iron chelators affected iron-mediated
2
+
2+
Haber–Weiss reactions involving the oxidation of Fe by O
2
(Fe
in
•
3
+
−
40
2+
+
O
2
→ Fe + O
2
). In the experiments of Fe with H
2
O
2
the presence of three itaconic acid derivatives, no differences were
found in the initial reaction rate of O consumption. In contrast,
uptake was strongly inhibited by hexadecylitaconic acid and
octylitaconic acid when reactions were started by mixing Fe ,
and a reductant, cysteine. No inhibitory effects were found
for itaconic acid in the reaction starting from Fe as well as in the
reaction from Fe .
The effects of (alkyl)itaconic acids on Fe reduction were
2
O
2
3
+
H
2
O
2
Fig. 9 The highest occupied molecular orbital (HOMO) energies of
itaconate derivatives with various lengths of side chain.
3
+
2
+
3
+
without erosion of the wood cell walls at a site far from enzymes.
Wood decay proceeds without extensive damage to cellulose
in an environment where free radicals, lignin-derived phenols,
2
+
3+
analyzed using a ligand for Fe , BPS. At a ligand to Fe
3
+
molar ratio of 30 : 1, Fe reduction was completely inhibited
by hexadecylitaconic acid (data not shown). No ligand exchange
reactions with NTA and EGTA occurred under this condition due
are produced in the presence of O and Fe ions. Therefore we
2
hypothesized that this fungus produces inhibitors for Fenton-
•
3+
based HO production, and isolated three novel alkylitaconic
to steric crowding. When the molar ratio of the ligand to Fe was
acids: 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A), 1-
nonadecene-2,3-dicarboxylic acid (ceriporic acid B) and (Z)-
2 : 1, hexadecylitaconic acid and octylitaconic acid suppressed
3
+
Fe reduction, but the addition of a strong iron chelator, NTA,
abolished these suppressive effects due to a ligand exchange
1
,10-nonadecadiene-2,3-dicarboxylic acid (ceriporic acid C) from
20,27,35,36
3+
extract-free wood cultures of C. subvermispora.
In vitro
reaction and the high oxidation potential of the resultant Fe -
41
experiments showed that ceriporic acid B (hexadecylitaconic
acid) inhibited the depolymerization of cellulose by the Fenton
NTA complex. These observations illustrate that alkylitaconic
acids suppressed the reductive half cycle in the Fenton system
21
3+
reaction at the physiological pH of wood decay. In this study, we
comparatively analyzed the physicochemical and redox properties
of itaconic acid derivatives with long, medium and no alkyl side
chains to discuss the structure–function relationship of this series
of metabolites.
by coordinating Fe as discussed in the ESR and oxygen uptake
experiments.
Itaconic acid is oxidized by HO• to form the b-hydroxy
42
itaconate radical, which in turn polymerizes (Fig. 10). Under
aerobic conditions, part of the radical intermediate reacts with
molecular oxygen to form the b-hydroxy peroxyl radical. The
differential profiles between itaconic and alkylitaconic acids in
oxygen consumption are ascribed to the susceptibility to oxidation
•
Production of HO was strongly suppressed by octylitaconic
acid and hexadecylitaconic acid in the Fenton reaction starting
3
+
from Fe in the presence of a reductant, cysteine, ascorbic
acid or hydroquinone. Octylitaconic acid has strong surfactant
activity comparable to that of Tween 20 and Tween 80 (Fig. 2).
•
by HO as expected from the differences in steric effects of the
side chain rather than HOMO energy. GCMS analyses after the
reactions showed that itaconic acid was decomposed due to the
radical reaction but hexadecylitaconic acid and octylitaconic acid
remained intact after the Fenton reaction in the presence and
absence of NTA (data not shown).
•
Hexadecylitaconic acid decreased the initial rate of HO produc-
tion more intensively than octylitaconic acid did, but differences
•
between the two alkylitaconic acids in the total amount of HO
produced during 15 min were negligible. When the carboxyl groups
of hexadecylitaconic acid and octylitaconic acid were esterified,
•
inhibitory effects on HO production disappeared, indicating
that free carboxyl groups and the alkyl side chain are essential
•
components for the suppression of HO production.
2
+
The reactions of Fe
with
H
2
•
O
2
in the presence of
(
alkyl)itaconic acids exhibited an HO production profile distinc-
tively different from the reactions initiated by mixing Fe , H
and a reductant. In reactions starting from Fe , the production
of HO increased rapidly in one minute, regardless of the presence
3
+
2
O
2
2
+
•
•
Fig. 10 Proposed pathway for the oxidation of itaconic acid by HO .
•
of alkylitaconic acids; however, the rate of HO production
decreased to zero within one minute after the reaction. This is
explained by the mechanism by which alkylitaconic acids inhibited
a reductive half cycle from Fe to Fe by coordinating Fe . As
In conclusion, the strong redox silencing ability of alkylitaconic
acids—hexadecylitaconic acid and its analogue octylitaconic
acid—driven by the suppression of Fe reduction has been
3
+
2+
3+
3+
8
46 | Org. Biomol. Chem., 2007, 5, 840–847
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established. Iron is an essential element for living organisms
but iron can damage tissues by catalyzing the production of
reactive oxygen species (ROS). A number of iron chelators
like desferrioxamine (DFO), 1,10-phenanthroline and pyridoxal
isonicotinoyl hydrazone (PIH) have been studied to suppress iron-
mediated oxidative injury. In view of anitioxidative therapy, it is
of considerable interest to control ROS in vivo using a series of
lipophilic and amphipathic alkylitaconic acids with different chain
lengths.
11 T. L. Highly and B. L. Illman, Biodeterior. Abstr., 1991, 5, 231–244.
1
2 A. Paszczynski, R. Crawford, D. Funk and B. Goodell, Appl. Environ.
Microbiol., 1999, 65, 674–679.
3 Z. Kerem, K. A. Jensen and K. E. Hammel, FEBS Lett., 1999, 446,
49–54.
1
14 G. Xu and B. Goodell, J. Biotechnol., 2001, 87, 43–57.
1
5 T. K. Kirk, R. Ibach, M. D. Mozuch, A. H. Conner and T. L. Highley,
Holzforschung, 1991, 45, 239–244.
6 S. Backa, J. Gierer, T. Reitberger and T. Nilsson, Holzforschung, 1993,
47, 181–187.
1
1
1
7 H. Tanaka, S. Itakura and A. Enoki, J. Biotechnol., 1999, 75, 57–70.
8 D. P. Barr, M. M. Shah, T. A. Grover and S. D. Aust, Arch. Biochem.
Biophys., 1992, 298, 480–485.
Conclusion
2
The reactivity of iron varies greatly depending on its ligand envi-
ronment. A selective white rot fungus, C. subvermispora secretes
alkylitaconic acids to suppress the production of hydroxyl radicals
by the Fenton reaction even in the presence of reductants for Fe .
The alkyl side chain and coordination by the two carboxylic groups
in the metabolite are essential for redox silencing and high stability
3
+
•
against oxidative degradation by HO . These findings indicate that
the alkyl side chain in metabolites functions as a switching “device”
to control antioxidant activities in addition to the hydrophobicity
and amphipathic properties of the metabolites.
Acknowledgements
The authors gratefully acknowledge support from the Advanced
Research Program of the Research Institute of Innovative Tech-
nology for the Earth (RITE) and a Grant-in-Aid for JSPS Fellows
(
to Y. O.).
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