Comparison of a series of laccase mediators in the electro-oxidation reactions of non-phenolic lignin model compounds

Article abstract of DOI:10.1016/j.electacta.2013.05.112

The electro-oxidations of non-phenolic lignin model compounds in an electrolytic mediator system (EMS) have been investigated with several laccase mediators, including N-hydroxyphthalimide (NHPI), 1-hydroxybenzotriazole (HBT), violuric acid (VLA), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), under the same reaction conditions. In the bulk electrolyses of the monomeric model compound [1-(4′-ethoxy-3′-methoxyphenyl)ethanol (1G)], oxidation with NHPI gave the corresponding C_α=O product (2G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO afforded 2G in moderate yields. The highest reaction selectivity for the guaiacylunits was found in the oxidation conducted in the presence of ABTS, although the yield was low. In the bulk electrolyses of the dimeric model compound [4-ethoxy-3-methoxyphenylglycerol- β-guaiacyl ether (3G)], the oxidation with NHPI gave the corresponding C_α=O product (4G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO gave 4G in low yields. In contrast, the oxidation with ABTS gave a C_α-C_β cleavage product (5G) in 5.5% yield. The selectivity of the mediators in the EMS reaction effectively reflected the mechanisms of their reactions, as reported for the laccase mediator system. NHPI was confirmed to be the best mediator in the present system for the selective C_α-carbonylation of the non-phenolic β-O-4 structures in lignin.

Full text of DOI:10.1016/j.electacta.2013.05.112

Accepted Manuscript
Title: Comparison of a series of laccase mediators in the
electro-oxidation reactions of non-phenolic lignin model
compounds
Author: Takumi Shiraishi Yumi Sannami Hiroshi
Kamitakahara Toshiyuki Takano
PII:
S0013-4686(13)01035-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.electacta.2013.05.112
EA 20600
To appear in:
Electrochimica Acta
Received date:
Revised date:
Accepted date:
12-12-2012
16-5-2013
21-5-2013
Please cite this article as: T. Shiraishi, Y. Sannami, H. Kamitakahara, T.
Takano, Comparison of a series of laccase mediators in the electro-oxidation
reactions of non-phenolic lignin model compounds, Electrochimica Acta (2013),
http://dx.doi.org/10.1016/j.electacta.2013.05.112
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2 Comparison of a series of laccase mediators in the
3 electro-oxidation reactions of non-phenolic lignin model
4 compounds
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Takumi Shiraishia, Yumi Sannamia, Hiroshi Kamitakaharaa, Toshiyuki Takanoa,*
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Division of Forest and Biomaterials Science, Graduate School of Agriculture, Kyoto
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University, Kyoto, Japan
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* Corresponding author. Tel: +81-75-753-6254; Fax: +81-75-753-6300.
E-mail address: takatmys@kais.kyoto-u.ac.jp (T. Takano)
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Highlight
The electro-oxidations of non-phenolic lignin model compounds with laccase mediators (NHPI,
HBT, VLA, TEMPO, ABTS) have been investigated under the same reaction conditions.
The reaction selectivity of the mediators in the electrolytic mediator system was basically
reflected to their reaction mechanism in the laccase mediator systems.
NHPI was the best mediator for selective Cα-carbonylation of non-phenolic β-O-4 structures
in lignin in the present system.
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ABSTRACT
The electro-oxidations of non-phenolic lignin model compounds in an electrolytic mediator
system (EMS) have been investigated with several laccase mediators, including
N-hydroxyphthalimide (NHPI), 1-hydroxybenzotriazole (HBT), violuric acid (VLA),
2,2,6,6-tetramethylpiperidine-N-oxyl
(TEMPO),
and
2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), under the same reaction
conditions. In the bulk electrolyses of the monomeric model compound
[1-(4’-ethoxy-3’-methoxyphenyl)ethanol (1G)], oxidation with NHPI gave the corresponding
Cα=O product (2G) in high yield, whereas the oxidations with HBT, VLA, and TEMPO
afforded 2G in moderate yields. The highest reaction selectivity for the guaiacyl-units was
found in the oxidation conducted in the presence of ABTS, although the yield was low. In the
bulk
electrolyses
of
the
dimeric
model
compound
[4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (3G)], the oxidation with NHPI gave
the corresponding Cα=O product (4G) in high yield, whereas the oxidations with HBT, VLA,
and TEMPO gave 4G in low yields. In contrast, the oxidation with ABTS gave a Cα-Cβ
cleavage product (5G) in 5.5% yield. The selectivity of the mediators in the EMS reaction
effectively reflected the mechanisms of their reactions, as reported for the laccase mediator
system. NHPI was confirmed to be the best mediator in the present system for the selective
Cα-carbonylation of the non-phenolic β-O-4 structures in lignin.
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Keywords
Cα-carbonylation, Cα-Cβ cleavage, electrolytic mediator system (EMS), laccase mediator,
lignin
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1. Introduction
Kraft pulping is one of the most important manufacturing processes in the pulp and paper
industry. During the pulping treatment, strong alkali is used in the presence of a sodium
sulfide catalyst for the cleavage of the lignin. Unfortunately, the application of such forcing
conditions can lead to appreciable levels of degradation in the carbohydrates, and the
development of a pretreatment process to facilitate the delignification would therefore be
desirable for high-yield pulping. The cleavage of the non-phenolic β-O-4 linkages of lignin is
believed to be the rate-determining step during the main delignification process [1]. In
contrast, it has also been reported that the alkali cleavage of non-phenolic β-O-4 linkages can
be significantly accelerated by the presence of Cα-carbonyl groups [2, 3]. Selective
Cα-carbonylation in non-phenolic β-O-4 substructures is therefore a target of considerable
interest for the development of a pretreatment process for kraft pulping.
The oxidation reactions of lignin in the laccase- and electrolytic-mediator systems
(LMS and EMS, respectively) have been proposed as eco-friendly processes for kraft pulping
(Fig.1) [4-7]. In these systems, lignin is indirectly oxidized thorough a mediator. To date,
many papers have been published concerning the oxidation of non-phenolic lignin model
compounds in an LMS [8-11]. A variety of different compounds have been reported as laccase
mediators, including N-hydroxyphthalimide (NHPI), 1-hydroxybenzotriazole (HBT), violuric
acid
(VLA),
2,2,6,6-tetramethylpiperidine-N-oxyl
(TEMPO),
and
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) (Fig. 2). The laccase mediators
can be divided into three different groups based on their reaction mechanisms in the LMS,
which include (1) hydrogen atom transfer (HAT) mechanism-type mediators such as NHPI,
HBT, and VLA; (2) ionic mechanism-type mediators such as TEMPO; and (3) electron
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transfer (ET) mechanism-type mediators such as ABTS [12-14]. It is well known that the
Cα-carbonylation process competes with Cα-Cβ cleavage during the oxidation of non-phenolic
substrates in the LMS. Oxidations involving HAT mechanism type mediators (e.g., NHPI,
HBT, and VLA) and ionic mechanism type mediators (e.g., TEMPO) preferentially afforded
the Cα-carbonyl product in the LMS, whereas oxidations involving ET mechanism type
mediator (e.g., ABTS) gave the Cα-carbonyl product together with the Cα-Cβ cleavage
products [9].
The use of EMS has two advantages over LMS, in that (1) a wide range of pH and
temperature conditions can be selected; and (2) a mediator with a high redox potential can be
used. The mediators that have been proposed for LMS can be also used in EMS. Only a few
reports concerning the oxidation of non-phenolic lignin model compounds in EMS, however,
have been published in the literature [15-19]. The oxidation reactions of a non-phenolic β-O-4
dimer in the EMS have been reported using several different mediators, including NHPI [15],
VLA [16], HBT and ABTS [17], and tris(4-bromophenyl)amine [19]. Furthermore, the
oxidation of veratryl alcohol has been reported with HBT and ABTS [18]. The mediators that
have been proposed for use with LMS could be also used in the EMS, where they are
expected to afford similar results to those achieved in the EMS. Interestingly, however, the
EMS and LMS oxidations of a non-phenolic β-O-4 dimer with the same mediator gave
different results, in that the oxidation of non-phenolic β-O-4 dimer in LMS with HBT gave a
Cα-carbonyl product exclusively, whereas the oxidation in the EMS with HBT gave a mixture
of the Cα-carbonyl and Cα-Cβ cleavage products [17]. A satisfactory comparison of the
laccase mediators has been hampered by the different electro-oxidation conditions. A
systematic investigation of laccase mediators in both the EMS is therefore needed. Herein, a
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comparison of several representative laccase mediators, including NHPI, HBT, VLA, TEMPO,
and ABTS has been conducted with NHPI under the same electro-oxidation conditions
described in the previous paper [15].
2. Experimental
The
substrates
[1-(4’-ethoxy-3’-methoxyphenyl)ethanol
(guaiacyl
type,
1G),
1-(4’-ethoxy-3’,5’-dimethoxyphenyl)ethanol (syringyl type, 1S), 1-(4’-ethoxyphenyl)ethanol
(p-hydroxyphenyl type, 1H), and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (3G)]
and the authentic samples (Fig. 3) were prepared as described by Shiraishi et al. [20]. The
laccase mediators and all of the other chemicals used in the study were obtained from
commercial sources and used as received (Nacalai Tesque Inc., Kyoto, Japan). In contrast to
our previous publications [15], where a 0.1 M LiClO4 solution in CH3CN was predominantly
used as the electrolyte for the cyclic voltammetry (CV) measurements and bulk electrolyses, a
0.1 M LiClO4 solution in a mixture of CH3CN and H2O (7/3 - v/v) was used in the current
study because VLA and ABTS were insoluble in CH3CN.
The CV measurements were performed with an ALS electrochemical analyzer (ALS
650B; BAS, Tokyo, Japan) in an undivided cell (5 mL) [working electrode: 1.6 mm diameter
platinum disk; reference electrode: Ag/Ag+ reference electrode (0.1 M LiClO4, 0.01 M AgNO3
in CH3CN), counter electrode: platinum wire electrolyte: 0.1 M LiClO4 in CH3CN/H2O = 7/3
(v/v), concentration of the mediator: 5 mM, 2,6-lutidine: 25mM, compound 1G: 25 mM].
Bulk electrolyses of the non-phenolic monomer 1G and the dimer 3G with the
laccase mediator at fixed potentials (Tables 1 and 3) were carried out with the ALS
electrochemical analyzer equipped with a power booster (ALS 680) in the anode chamber of a
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divided cell, until the current value dropped to about 1 mA, unless otherwise noted. [The
divided cell: in the anode chamber, a carbon felt working electrode (2.4 × 3.0 cm2 for 1G or
1.5 × 2.0 cm2 for 3G), the Ag/Ag+ reference electrode, a substrate (5 mM), a mediator (1 mM),
2,6-lutidine (25 mM), and 0.1 M LiClO4 in CH3CN/H2O = 7/3 (v/v) (20 mL for 1G or 10 mL
for 3G); in the cathode chamber, the platinum plate electrode, 0.1 M tetra-n-butyl ammonium
perchlorate (TBAP) in CH3CN/H2O = 7/3 (v/v) (5 mL)]. Upon completion of the electrolysis,
CH3CN (0.5 ml) containing an internal standard (36 μmol benzhydrol for 1G or 18 μmol
benzophenone for 3G) was added to the anode chamber. The anolyte was extracted three
times with EtOAc, and the combined organic layers were washed with distilled water, dried
over Na2SO4, and concentrated to dryness to give the products as colorless oil. The products
were subjected to gas chromatography for quantification of the different components as
previously described in the literature for the oxidations of 1G [20] and 3G [15].
The bulk electrolyses of a mixture of non-phenolic monomers 1G, 1S and 1H (2.5
mM in each case) were performed in accordance with the procedure described above for the
electrolyses of 1G. The relative reactivity ratios (kG/kS and kG/kH, see Eq. 1) of the mediators
were calculated from the yields of the oxidation products [21].
kG
1G + MedOX ⎯⎯→ 2G + Med
kS
141
1S + MedOX ⎯⎯→ 2S + Med
(1)
kH
1H + MedOX ⎯⎯→ 2H + Med
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3. Results and discussion
3.1. Cyclic voltammetry (CV) studies
Cyclic voltammetry (CV) measurements of the laccase mediators were performed to allow for
the determination of the applied potential in the bulk electrolysis and for simple qualitative
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analysis of the electrolysis. Fig. 4A–F show the CVs of the laccase mediators in the presence
and in the absence of the base catalyst (2,6-lutidine) in a 0.1 M LiClO4 solution in
CH3CN/H2O = 7/3 (v/v). In the CVs of NHPI (Fig.4A), the oxidation peak potential (0.90 V
of peak (a)) of NHPI in the absence of the catalyst in CH3CN/H2O was different from that in
CH3CN (1.78 V, data in the previous paper [15]), indicating that the oxidation potential was
significantly influenced by the reaction conditions in the EMS. The oxidation peak potentials
of N-hydroxylamine derivatives are known to be shifted in a negative direction in the
presence of a base [22, 23]. A negative shift in the oxidation peak potential of NHPI was also
observed in the CVs in CH3CN/H2O (from 0.90 V of the shoulder peak (a) to 0.54 V of peak
(c)), as well as in the CVs conducted in CH3CN [15]. These shifts could be attributed to the
deprotonation of NHPI by 2,6-lutidine to give the NHPI-anion. The anion could then be
oxidized on the electrode at 0.54 V to give the phthalimide-N-oxyl (PINO) radical (Fig. 5). A
reduction peak was not observed in the CV of NHPI conducted in the presence of 2,6-lutidine,
suggesting that the PINO radical was unstable in the system. In the CVs of HBT (Fig. 4B), a
negative shift was observed in the oxidation peak potential (from 0.72 V of peak (d) to 0.50 V
of peak (e)) in the presence of 2,6-lutidine. The reduction peak was not found in the CV with
2,6-lutidine, suggesting that the benzotriazole-N-oxyl (BTNO) radical was unstable in the
system. The poor stability of the BTNO radical was also reported by Bourbonnais et al. [18].
The oxidation peak potentials in the absence (peak (f)) and presence (peak (h)) of 2,6-lutidine
were 0.58 and 0.37 V, respectively, in the CVs of VLA (Fig. 4C), clearly indicating the
occurrence of a negative shift. A reduction peak (peaks (i)) was clearly observed in the CV
conducted in the presence of 2,6-lutidine, suggesting that the VLA radical was stable even in
the presence of 2,6-lutidine, and this observation was consistent with data reported elsewhere
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[16,24]. A relatively low oxidation peak potential (peak (j) 0.32 V) was found for TEMPO in
the CV conducted in the absence of 2,6-lutidine (Fig. 4D), whereas no shift was observed in
the CV conducted in the presence of 2,6-lutidine. A reduction peak (peak (k)) was clearly
observed in this particular case. Furthermore, peak (j) was attributed to the single-electron
oxidation of TEMPO to the corresponding oxoammonium ion (TEMPO+), whereas the
reduction peak (k) was attributed to the reduction from TEMPO+ to TEMPO, suggesting that
TEMPO+ was stable in the system. It was also reported in the CV of TEMPO-poly (acrylic
acid) modified glassy carbon disk electrode [25].
In the CV of ABTS conducted in the absence of a catalyst at the standard scan rate
(0.05 Vs-1), two oxidation peaks were observed at (l) at 0.28 V and (m) at 0.73 V (Fig. 4E),
corresponding to the single-electron oxidations from ABTS to ABTS+• and from ABTS+• to
ABTS2+, respectively. No negative shift was observed in this particular case. Two reduction
peaks (n) at 0.35 V and (o) at 0.24 V were also found. The peak (n) was not considered the
reduction peak from ABTS2+ to ABTS+• because of the big difference between the oxidation
peak (m) and reduction peak (n), whereas peak (o) was attributed to the reduction of ABTS+•
to ABTS. Bourbonnais et al. [18] reported that the reduction peak of ABTS2+ was not
observed in the CV conducted in the presence of a catalyst at a slow scan rate (0.20 Vs-1).
They also explained the lack of a reduction peak on the basis of the enhanced intensity of the
reduction peak of ABTS+• from the comproportionation reaction between ABTS and ABTS2+.
Peak (n) might be considered the peak corresponding to the comproportionation reaction, but
further investigation is required.
Fig. 4F shows the CV of ABTS in the absence of the catalyst at an enhanced scan
rate (0.5 Vs-1). The CV possessed a profile consistent with those previously reported in the
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literature [18]. The CV contained a peak (p) corresponding to the reduction of ABTS2+ to
ABTS+• and there was also a reduction in the intensity of peak (n), suggesting that ABTS2+
was relatively unstable. The current data therefore suggested that ABTS2+ was spontaneously
converted to an unknown ABTS intermediate, and that the intermediate was reduced to
ABTS+• at the potential of peak (n) in this particular CV at the standard scan rate.
It is well known that the catalytic efficiency of a mediator can be evaluated based on
the CVs of the mediator conducted in the presence and in the absence of a substrate [18]. The
catalytic efficiency of a mediator can be expressed by the ratio ik/ic, where ik is the anodic
peak current of the mediator in the presence of a substrate and ic is the anodic peak current of
the mediator in isolation [26, 27]. Fig. 4G-K show the CVs of the laccase mediators with
2,6-lutidine in the absence and presence of a substrate (compound 1G) in a 0.1 M LiClO4
solution of CH3CN/H2O = 7/3 (v/v). The anodic current was enhanced in the CVs of NHPI
and HBT (Fig. 4G and H), suggesting that NHPI and HBT were reactive towards compound
1G. The catalytic efficiencies of NHPI and HBT were 3.6 and 1.4, respectively. In contrast,
the CVs of VLA and TEMPO showed no significant change in the presence of compound 1G
(Fig. 4I and J), suggesting that VLA and TEMPO were not useful mediators in the current
system. No enhancement was observed in the anodic current in the CVs of ABTS (Fig. K)
following the addition of compound 1G. Interestingly, Bourbonnais et al. [18] reported that
the anodic current was enhanced in the presence of veratryl alcohol in a 0.05 M sodium citrate
buffer solution (pH 4) [18]. These results confirmed that the catalytic efficiency of the
mediators was significantly influenced by the reaction conditions in the EMS. Consequently,
the results of the CV studies revealed that NHPI was the most suitable mediator in the current
system, when used in conjunction with 2,6-lutidine in a 0.1 M LiClO4 solution of
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CH3CN/H2O = 7/3 (v/v).
3.2. Bulk electrolyses of the non-phenolic monomers 1G, 1S and 1H
The laccase mediators were submitted to the bulk electrolyses of non-phenolic monomer 1G
with 2,6-lutidine in a 0.1 M LiClO4 solution of CH3CN/H2O = 7/3 (v/v). Several different
applied potentials were used, including 0.70 V for NHPI, 0.50 V for HBT, 0.40 V for VLA,
0.40 V for TEMPO, and 0.30 and 0.75 V for ABTS. The values for NHPI and HBT were
decided on the basis of the potentials with the highest catalytic efficiencies, whereas the other
three were decided on the basis of their oxidation potentials. The quantitative results are
summarized in Table 1. The HBT-mediated electro-oxidation of compound 1G afforded
compound 2G in 50.5% yield, whereas the NHPI-mediated electro-oxidation gave compound
2G in 94.1% yield. The moderate yield in the former case was attributed to the short lifetime
of the BTNO radical. The behavior observed in the current VLA-mediated electro-oxidation
was particularly unusual. Although a significant reduction was observed in the current within
the first seconds to ca. 2 mA, with a further gradual reduction to 1 mA over the following 15
min, compound 2G was not detected. Based on the CV studies of the VLA described above,
the VLA radical was expected to be stable. When the reaction time was increased from 15 min
(i.e. until the current dropped to 1 mA) to 3 days (i.e. until the current dropped to 0.02 mA),
the yield of compound 2G increased to 47.6%, suggesting that the VLA radical was stable, but
less reactive towards compound 1G. TEMPO-mediated electro-oxidation of compound 1G
afforded compound 2G in 60.2% yield, suggesting that the benzylic alcohol was easily
oxidized in the TEMPO-mediated electro-oxidation as well as in the TEMPO-mediated
oxidation with Laccase [28]. The ABTS-mediated electro-oxidation of compound 1G with
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2,6-lutidine was initially conducted at 0.30 V (corresponded to the oxidation peak (l) for the
conversion of ABTS to ABTS+•). The current fell to 1 mA within several seconds, suggesting
that all of the electricity was consumed in the formation of ABTS+•, although no further
reactions were detected. This result suggested that the ABTS+• provided a similar level of
reactivity to the veratryl alcohol [18]. The ABTS-mediated electro-oxidation was then
conducted both in the absence and in the presence of 2,6-lutidine at 0.75 V (corresponding to
the oxidation peak (m) for the conversion of ABTS+• to ABTS2+), affording compound 2G in
3.0 and 16.2% yields, respectively. 2,6-Lutidine effectively improved the yield of compound
2G, likely by accelerating the deprotonation of the radical cation intermediate formed via the
single electron transfer of compound 1G according to the ET mechanism, as well as by the
direct electro-oxidation of compound 1G [20]. The yield of compound 2G, however, was low,
even when 2,6-lutidine was used, and this was attributed to the rapid conversion of ABTS2+ to
the unknown ABTS intermediate, as described in the discussion of the CV studies involving
ABTS.
The substrate selectivity in the EMS was also an important variable for the laccase
mediators. The relative reactivity ratios were determined from the yields of compounds 2G,
2S and 2H upon competition of the electrolyses of the mixtures of 1G, 1S and 1H [21]. The
results are shown in Table 2. In all of the electro-oxidations, the kG/kS and kG/kH values were
greater than 1, indicating that the preferential oxidation of the guaiacyl-type compound 1G
had proceeded. Although the substrate selectivity for the guaiacyl-type compound in the
ABTS-mediated electro-oxidation was very high, the yields of the corresponding Cα-carbonyl
compounds were low. The preference for the oxidation of the guaiacyl-type compound over
the syringyl-type and p-hydroxyphenyl-type compounds could be attributable to the higher
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ionization potential of the non-phenolic syringyl-type compound [29, 30] and the higher
oxidation potential of compound 1H (1.4 V for 1H and 1.0 V for 1G [20]), respectively. Other
factors potentially affecting the preference of the laccase mediators for oxidation should also
be considered, such as the polar effects of the radical transition state in the NHPI-mediated
electro-oxidation [31, 32]. Further investigations would be required to develop a clearer
understanding of the observed substrate selectivity.
3.3. Bulk electrolyses of the non-phenolic dimer 3G
The laccase mediators were subjected to bulk electrolyses of the non-phenolic β-O-4 dimer
3G in the presence of 2,6-lutidine in a 0.1 M LiClO4 solution in CH3CN/H2O = 7/3 (v/v). The
gas chromatograms of the products in the bulk electrolyses are shown in Fig. 6A-E, and the
quantitative results are summarized in Table 3. In the NHPI-mediated electro-oxidation of
compound 3G, nearly all of the starting material 3G was consumed (Fig. 6A) and a Cα=O
product (compound 4G) was obtained in 92.3% yield [15]. In the HBT- and VLA-mediated
electro-oxidations of compound 3G, compound 4G was obtained in 14.1 and 11.5% yields,
respectively, with most of the starting material 3G being recovered in both cases (Fig. 6B and
C). These results indicated that the PINO radical was reactive towards compound 3G, and that
the BTNO and VLA radicals were not as reactive, as expected from the CV studies of the
mediators. Although the peak (q) observed at 4.4 min did not correspond to the peak derived
from compound 5G in the gas chromatogram of the products in the electro-oxidation with
HBT (Fig. 6B), it appeared to correspond to the peak from compound 5G. The formation of a
Cα-Cβ cleavage product (compound 5G) was not detected in the electro-oxidation reactions
involving the N-hydroxylamine compounds (i.e. NHPI, HBT and VLA). The reaction
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selectivity observed for the N-hydroxylamine compounds was consistent with the results
expected on the basis of their reaction mechanism (HAT mechanism) in the LMS. The
TEMPO-mediated electro-oxidation of compound 3G also preferentially afforded compound
4G in 2.0% yield, with most of the starting material 3G also being recovered (Fig. 6D),
suggesting that TEMPO was less reactive towards compound 3G. Further, a much lower yield
was observed for the electro-oxidation of dimer 3G in this particular case than in the
oxidation of monomer 1G. These results were in agreement with the data observed in the LMS
conducted with TEMPO [28]. The low yield in this case could be attributed to the steric
hindrance afforded by the Cα-OH moiety of the substrate. The electron donating effect of the
B-ring in intermediate 6G (Fig. 7) could also have an impact on the yield of the reaction,
because electron donating groups have been reported to oppose the deprotonation of
TEMPO-adducts [28]. Although the use of TEMPO as a selective oxidation catalyst for the
oxidation of primary alcohols has been well documented in the literature [33], no Cγ-oxidation
products were observed in the current electro-oxidation reactions using TEMPO. The
ABTS-mediated electro-oxidation of compound 3G gave compounds 4 and 5 in 2.8 and 5.5%
yields, respectively, suggesting that the Cα-Cβ cleavage reaction proceeded preferentially. The
results suggested that ABTS operated via an ET mechanism in the LMS. Overall, the results
demonstrated that the reaction selectivities of the laccase mediators in the EMS were in
agreement with those observed in the LMS using the same mediators [9], and that NHPI was
the best mediator for the Cα-carbonylation in the present system.
4. Conclusions
The oxidations of non-phenolic lignin model compounds were investigated under the same
14
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308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
conditions in the EMS with a variety of different laccase mediators, including NHPI, HBT,
VLA, TEMPO, and ABTS.
The CV studies of the mediators showed that NHPI and HBT demonstrated a high
level of catalytic efficiency towards the non-phenolic monomer 1G in the system with
2,6-lutidine, and that the VLA radical and TEMPO+ were stable. In the bulk electrolyses of
the non-phenolic monomer 1G, the NHPI-mediated electro-oxidation afforded a Cα=O
product (compound 2G) in high yield, whereas the HBT-, VLA-, TEMPO- and
ABTS-mediated electro-oxidations afforded 2G in only moderate or low yields. The
pronounced lifetime of the VLA radical was also confirmed by the extended reaction time of
the VLA-mediated electro-oxidation reaction. The guaiacyl-units were found to be more
reactive than the syringyl- and p-hydroxyphenyl-type units in the current system. Although
the highest reaction selectivity for the guaiacyl-units was found in the ABTS-mediated
electro-oxidation reaction, the conversion efficiency was low. In contrast, in the bulk
electrolyses of the non-phenolic dimer 3G, the NHPI-, HBT-, VLA-, TEMPO-mediated
electro-oxidation reactions afforded a Cα=O compound (compound 4G) exclusively, but the
yield was extremely low in the TEMPO-mediated electro-oxidation. The ABTS-mediated
electro-oxidation reaction gave a Cα-Cβ cleavage product (compound 5G) preferentially in a
low yield. The reaction selectivity of the mediators in the EMS effectively reflected their
reaction mechanisms, as reported in the LMS. Consequently, the current study confirmed that
NHPI was the best of the mediators tested in the current system [0.1 M
LiClO4/2.6-lutidine/(CH3CN/H2O=7/3 by vol.)] for the selective Cα-carbonylation of
non-phenolic β-O-4 structures in lignin, although optimum conditions for the HBT-, VLA-,
TEMPO-, and ABTS-mediated electro-oxidation reactions should be investigated in greater
15
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331
332
detail.
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
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18
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426
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428
Table 1 Bulk electrolysis of 1-(4’-ethoxy-3’-methoxyphenyl)ethanol (1G) with different mediators
Mediator
(eq.)
Potential
2,6-Lutidine Yield of 2Ga
Electricity
(F mol-1)
CEb
(%)
(eq.)
(%)
(vs Ag/Ag / V)
NHPIc
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.2
0.70
0.50
0.40
0.40d
0.40
0.30
0.75
0.75
5
5
5
5
5
5
5
0
94.1
50.5
-
2.23
1.38
0.22
2.86
1.56
0.16
1.23
0.55
79.8
73.0
-
HBT
VLA
VLA
47.6
60.2
-
33.3
59.7
-
TEMPO
ABTS
ABTS
ABTS
16.2
26.3
3.0
10.9
429
430
431
432
433
434
435
436
aYields (based on 1G) were determined by GC using benzhydrol as an IS; bcurrent efficiency;
cdata in the previous paper [15]; duntil current dropped to 0.02 mA (for 3 days)
Table 2 Reactivity ratio of the mediators in the bulk electrolyses of a mixture of 1-(4’-ethoxyphenyl)ethanols
(1G, 1S and 1H)
Mediator
Potential
Reactivity ratioa
Yieldb (%)
2S
Electricity
(F mol-1)
(vs Ag/Ag+ / V)
kG/kS
2.6
kG/kH
3.1
kS/kH
1.2
2G
2H
22.3
13.1
33.0
29.9
4.7
NHPI
HBT
0.70
0.50
0.40
0.40
0.75
53.5
32.7
40.5
36.5
19.1
25.3
0.67
0.60
0.88
0.34
0.46
1.7
2.9
1.7
21.6
VLA
1.9
1.3
0.7
24.4
TEMPO
3.8
1.3
0.3
10.9
ABTS
13.0
4.2
0.3
2.3
437
438
439
440
441
442
aReactivity ratio was determined by the method reported by d’Acunzo et al. [21];
bYields (based on 1G, 1S and 1H, respectively) were determined by GC using benzhydrol as an IS.
Table 3 Bulk electrolyses of 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (3G)
Mediator
(eq.)
Potential
2,6-Lutidine
(eq.)
Yielda (%)
Electricity
(F mol-1)
CEb
(%)
(vsAg/Ag+ / V)
4G
5G
NHPIc
0.2
0.2
0.2
0.2
0.2
0.70
0.50
0.40c
0.40
0.75
5
5
5
5
5
92.3
14.1
11.5
2.0
n.d.e
n.d.e
n.d.e
n.d.e
5.5
2.23
0.75
0.79
0.41
1.03
82.7
37.4
29.2
1.9
HBT
VLAd
TEMPO
ABTS
2.8
1.3
443
444
445
aYields (based on 3G) were determined by GC using benzhydrol as an IS; bcurrent efficiency;
cdata in the previous paper [15]; d until current dropped to 0.02 mA (for 12 h); enot detected
19
Page 19 of 25

446
447
448
449
450
451
Figure 1 Laccase mediator system (LMS) and electrolytic mediator system (EMS)
Figure 2 Laccase mediators
Figure 3 Non-phenolic lignin model compounds (substrates and authentic samples) used in the current study
Figure 4 Cyclic voltammograms of NHPI (A, G), HBT (B, H), VLA (C, I) TEMPO (D, J) and
ABTS (E, F, K) reactions in the presence (solid line) or absence (dashed line, pH 6) of 2,6-lutidine
(A-F, pH 8), as well as in the presence (solid line) or absence (dashed line) of compound 1 (G-K); 0.1
452
M LiClO4 in CH3CN/H2O = 7/3 by (v/v); scan rate 0.05 V s-1 (A-E), 0.5 V s-1 (F), 0.01 V s-1 (G-K) at
453
454
455
456
457
458
459
28°C
Figure 5 Reaction mechanism for the NHPI-mediated electro-oxidation
Figure 6 Gas chromatograms of the products observed in the electro-oxidation reactions
conducted in the presence of NHPI (A), HBT (B), VLA (C) , TEMPO (D) and ABTS (E).
Figure 7 Proposed reaction mechanism of the TEMPO-mediated electro-oxidation of compound 3G
20
Page 20 of 25

Graphical Abstract (for review)
Graphical Abstract
HO
HO
HO
O
H
O
O
O
Electro-oxidation
OMe
OMe
+
OMe
OMe
OMe
NHPI
HBT
VLA
TEMPO
ABTS
OEt
OEt
Cα=O product (4G)
OEt
Lignin dimer (3G)
Cα-Cβ Cleavage
product (5G)
Page 21 of 25

Figure(s)
(a) Laccase mediator system (LMS)
O2
+
Mediator-OX
Mediator
4H
Lignin
laccase
2H2O
Lignin-
OX
laccase-OX
(b) Electrolytic mediator system (EMS)
-
e
Mediator-OX
Mediator
Lignin
Lignin-OX
(Anode)
Figure 1 Laccase mediator system (LMS) and electrolytic mediator
system (EMS)
OH
N
O
O
O
N
N
N
OH
N
HN
NH
O
OH
O
HBT
NHPI
VLA
O S
3
S
N
S
N
N
N
N
SO
3
O
ABTS
TEMPO
Figure 2 Laccase mediators
HO
O
G: R1=-OCH3, R2= -H
S: R1=R2= -OCH3
H: R1=R2= -H
R
2
R
1
R
2
R
1
OEt
OEt
1G, 1S, 1H
2G, 2S, 2H
HO
O
HO
HO
O
H
O
O
OMe
OMe
OMe
OMe
OMe
OEt
5G
OEt
4G
OEt
3G
Figure 3 Non-phenolic lignin model compounds (substrates and
authentic samples) used in the current study
Page 22 of 25

NHPI
o
(A)
(G)
a
c
ik
10 μA
10 μA
c
ic
b
HBT
(B)
(H)
d
e
10 μA
p
10 μA
ik
ic
VLA
(C)
(I)
h
f
10 μA
10 μA
g
i
TEMPO
j
(D)
(J)
10 μA
10 μA
k
ABTS
(K)
(E)
m
l
10 μA
10 μA
o
n
1.5
0
0.5
1.0
(F)
m
p
E vs Ag/Ag+ / V
l
20 μA
Figure 4 Cyclic voltammograms of NHPI (A, G), HBT (B, H), VLA (C,
I) TEMPO (D, J) and ABTS (E, F, K) reactions in the presence (solid
line) or absence (dashed line, pH 6) of 2,6-lutidine (A-F, pH 8), as well as
in the presence (solid line) or absence (dashed line) of compound 1 (G-K);
0.1 M LiClO4 in CH3CN/H2O = 7/3 by (v/v); scan rate 0.05 V s-1 (A-E),
n
o
0
0.5
1.0
1.5
E vs Ag/Ag+ / V
0.5 V s-1 (F), 0.01 V s-1 (G-K) at 28°C
Page 23 of 25

N
O
O
2,6-lutidine
(Anode)
N
O
N
O
H
O
O
O
NHPI
NHPI-anion
Substrate-OX
e-
N
O
Substrate
O
PINO-radical
Figure 5 Reaction mechanism for the NHPI-mediated electro-oxidation
HO
HO
HO
N
O
N
B
O
HO
O
O
O
O
H
O
H
OMe
OMe
OMe
N
A
OMe
OMe
OMe
OEt
4G
OEt
6G
OEt
3G
Figure 7 Proposed reaction mechanism of the TEMPO-mediated electro-
oxidation of compound 3G
Page 24 of 25

(A) NHPI
I.S.
4G
(B) HBT
3G
I.S.
͌
4G
4G
(q)
(C) VLA
3G
͌
I.S.
(D) TEMPO
3G
I.S.
͌
4G
4G
(E) ABTS
3G
I.S.
͌
5G
4
12
20
28
retention time / min
Figure 6 Gas chromatograms of the products observed in
the electro-oxidation reactions conducted in the presence of
NHPI (A), HBT (B), VLA (C) , TEMPO (D) and ABTS (E).
Page 25 of 25