Lifetime, reduction potential and base-induced fragmentation of the veratryl alcohol radical cation in aqueous solution. Pulse radiolysis studies on a ligninase "Mediator"

Article abstract of DOI:10.1021/jp9812482

The radical cation of veratryl alcohol (3,4-dimethoxybenzyl alcohol), VA^?+, was produced in aqueous solution, mainly by oxidation with the radiation chemically generated SO₄^?- or Tl²⁺. By electron-transfer equilibration with thioanisole as a redox standard, the reduction potential of VA^?+ was determined to be 1.36 ± 0.01 V/NHE. On the basis of product analysis results, the radical cation undergoes a C - H deprotonation reaction from the side chain, leading to an α-hydroxybenzyl-type radical, with k = (17 ± 1) s^-1 at pH ≤ 5, as determined by time-resolved conductance. The α-hydroxybenzyl-type radical was also produced by reduction of veratryl aldehyde with the hydrated electron, and the pK_a value of this radical was determined to be 10.0. The deprotonation of VA^?+ is enhanced by bases such as OH^-, with the rate constant being 1.3 × 10⁹ M^-1 s^-1. In contrast, the corresponding rate constant for reaction of OH with the radical cation of veratryl alcohol methyl ether, whose reduction potential is also 1.36 V/NHE, is only 2 × 10⁷ M^-1 s^-1. With the veratryl alcohol derivative, 3,4-(MeO)₂C₆H₃CH(OH)CMe₃, the radical cation undergoes both a proton loss from the benzylic position and a C_α-Cβ fragmentation with the ratio, at pH ≤ 5, of ca. 1:2. The decay of the radical cation is strongly enhanced by OH (k = 8.3 × 10⁹ M^-1 s^-1), with the base induction favoring the C-C fragmentation relative to the proton loss from the benzylic position. The possible bearing of these results with respect to the role of VA in the lignin peroxidase-catalyzed decomposition of lignin is discussed.

Full text of DOI:10.1021/jp9812482

J. Phys. Chem. A 1998, 102, 7337-7342
7337
ARTICLES
Lifetime, Reduction Potential and Base-Induced Fragmentation of the Veratryl Alcohol
Radical Cation in Aqueous Solution. Pulse Radiolysis Studies on a Ligninase “Mediator”
Massimo Bietti,1a Enrico Baciocchi,*,1b and Steen Steenken*,1c
Dipartimento di Scienze e Tecnologie Chimiche, UniVersita´ “Tor Vergata”, Via della Ricerca Scientifica,
I-00133 Roma, Italy, Dipartimento di Chimica, UniVersita´ “La Sapienza”, P.le A. Moro 5, I-00185 Roma,
Italy, and Max-Planck-Institut fu¨r Strahlenchemie, D-45413 Mu¨lheim, Germany
ReceiVed: February 24, 1998; In Final Form: June 18, 1998
The radical cation of veratryl alcohol (3,4-dimethoxybenzyl alcohol), VA•+, was produced in aqueous solution,
mainly by oxidation with the radiation chemically generated SO4•- or Tl2+. By electron-transfer equilibration
with thioanisole as a redox standard, the reduction potential of VA•+ was determined to be 1.36 ( 0.01
V/NHE. On the basis of product analysis results, the radical cation undergoes a C-H deprotonation reaction
from the side chain, leading to an R-hydroxybenzyl-type radical, with k ) (17 ( 1) s-1 at pH e 5, as determined
by time-resolved conductance. The R-hydroxybenzyl-type radical was also produced by reduction of veratryl
aldehyde with the hydrated electron, and the pKa value of this radical was determined to be 10.0. The
deprotonation of VA•+ is enhanced by bases such as OH-, with the rate constant being 1.3 × 109 M-1 s-1.
In contrast, the corresponding rate constant for reaction of OH- with the radical cation of veratryl alcohol
-1
methyl ether, whose reduction potential is also 1.36 V/NHE, is only 2 × 107 M
s-1. With the veratryl
alcohol derivative, 3,4-(MeO)2C6H3CH(OH)CMe3, the radical cation undergoes both a proton loss from the
benzylic position and a CR-Câ fragmentation with the ratio, at pH e 5, of ca. 1:2. The decay of the radical
cation is strongly enhanced by OH- (k ) 8.3 × 109 M-1 s-1), with the base induction favoring the C-C
fragmentation relative to the proton loss from the benzylic position. The possible bearing of these results
with respect to the role of VA in the lignin peroxidase-catalyzed decomposition of lignin is discussed.
Introduction
The oxidative degradation of lignins to lower molecular
weight aromatic compounds has been intensively studied,2,3 due
Second only to cellulose, lignin is the most abundant
biopolymer on earth. It is estimated that the planet currently
contains 3 × 1011 metric tons of lignin with an annual
biosynthetic rate of ∼2 × 1010 tons. Lignin constitutes ∼30%
of the dry weight of softwoods and about 20% of the weight of
hardwoods. The presence of lignin within the cellulosic fiber
wall, mixed with hemicelluloses, creates a composite material,
which imparts strength and rigidity to trees and plants.
Chemically, lignin is built from phenylpropane units linked
together by different bonds to give an amorphous, 3D-cross-
linked network polymer, formed via a random free-radical
polymerization process and characterized by the presence of
several different bonding patterns. Among these, the most
abundant (50-60% of the linkages) are the aryl ether â-O-4
interunit linkages, connecting a phenoxy oxygen with the â
carbon of the side chain. Benzyl alcohol groups are also
common structures in lignin, accounting for more than 30% of
the phenylpropane units.2
to the mechanistic interest and commercial importance. In this
respect, several oxidizing systems have been employed, such
as chlorine dioxide,4 air in alkaline solution at high temperature,5
or nitroaromatics.6 However, chemical pulping processes are
low yield and require significant waste treatment and chemical
recycling operations. Moreover, the effluents from these
processes cannot be recycled and contain large amounts of
compounds that are known to have toxic, mutagenic, and
carcinogenic effects. Thus, increased public concern about the
environmental effects of the pulp and paper industry is rapidly
moving it toward alternative technologies.
In this respect, biopulping, defined as the treatment of wood
chips with lignin-degrading fungi prior to pulping, is attracting
growing attention thanks to the reduction in environmental
impact and energy consumption it allows.2 Among these fungi,
particularly interesting is the white rot fungus Phanerochaete
chrysosporium, which secretes a ferric hemoprotein, lignin
peroxidase (LiP), that is able to cleave oxidatively the side chain
CR-Câ bond in both lignin and lignin model compounds.7 For
a mechanistic explanation, one-electron oxidation of lignin by
LiP to form the corresponding radical cation, which subsequently
undergoes C-C bond cleavage, was initially proposed.7c-d,8
However, it was observed that lignin degradation by LiP requires
the presence of veratryl alcohol (VA, 3,4-dimethoxybenzyl
The chemical separation of lignin from cellulose has been
termed “delignification” and is one of the complex processes
of the pulp and paper industry. The process of delignification
results in the production of vast amounts of lignin whose
properties may vary depending on which delignification process
was employed and at which stage of delignification the lignin
was isolated.
S1089-5639(98)01248-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/02/1998

7338 J. Phys. Chem. A, Vol. 102, No. 38, 1998
Bietti et al.
alcohol),9 a secondary metabolite of Phanerochaete chrysos-
porium, and moreover that the geometry of the active site of
the enzyme prevents direct interaction with the lignin polymer
but not with VA.10
[TA][VA•+]
[TA•+][VA]
(7)
kf
TA•+ + VA y
\
kz TA + VA•+
K ) kf/kr )
r
This is visible in the insets of Figure 1, where the (first-order)
buildup of VA•+ at 430 nm and the decay of TA•+ at 530 nm
are presented. The equilibrium constant for reaction 7 can be
obtained by measuring the concentration ratio [VA•+]/[TA•+],
via the absorbances at equilibrium, as a function of [TA]/[VA].
A second way to obtain K is from a kinetic analysis, that is, via
the rate of approach to equilibrium. The result of this analysis
is shown in the first (from the left) inset in Figure 1; the numbers
obtained were: K ) 1.3 × 102, kf ) 3.3 × 109 M-1 s-1, kr )
2.6 × 107 M-1 s-1, to be compared with K ) 1.8 × 102 obtained
from the absorbances at equilibrium.24 The constant K reflects
the difference between the reduction potentials of VA•+ and
TA•+, and with the Nernst equation, it can be converted into a
potential. When 1.49 V/NHE is taken as the absolute potential
for TA•+,23 the reduction potentials E(VA•+ + e- f VA)
determined with these two methods, absorbances at equilibrium
and kinetic analysis, result as 1.356 and 1.365 V, respectively,
leading to an averaged value of 1.36 ( 0.01 V/NHE.25 This
number, which refers to aqueous solution, is the same as that
(1.36 V)19 measured in acetone solution using cyclic voltam-
metry. On this basis, a number can be attached to the oxidizing
power of LiP. Obviously, the oxidizing power has to be >1.36
V/NHE for the reaction to be exothermic. This is within the
range of a recently published estimate (g1.2 V/NHE),26 which
is based on the ability of LiP to oxidize a series of methoxy-
benzenes with known oxidation potentials.27
Different mechanistic roles have thus been proposed for the
action of VA on lignin degradation catalyzed by LiP, forex-
ample, it could act as a redox mediator, get oxidized by LiP to
yield VA•+ as a diffusible oxidant able to abstract one electron
from lignin,11,12 protect the enzyme from inactivation by H2O2,13
or donate electrons to LiP in order to complete the catalytic
cycle.14 More recently, it has been proposed that VA•+ reacts
with a nucleophile in the active-site channel and is subsequently
regenerated outside the channel where it can interact with the
lignin polymer.15
Thus, VA, although it does not contain a Câ in the side chain
(that is, C-C cleavage is not possible), has been intensively
studied with respect to the properties and reactions of its radical
cation.16-19 However, there are still unsolved questions, relat-
ing, in particular, to the spectroscopic properties and to the
lifetime of this radical cation. For example, whereas Candeias
reports k(decay) ) 17 s-1 16
, the number measured by Aust (with
a different method of VA•+ production) is 1.2 × 103 s-1 19
. This
difference is important in conjunction with the question of
whether VA•+ is able to act as a diffusible oxidant in the enzyme
system.20 Also, there is so far no information on the suscep-
tibility of VA•+ toward catalytic decomposition, especially with
respect to bases. However, this is a likely reaction, as judged
by the behavior of model systems.21 Thus, to obtain a more
complete and consistent picture, we have performed the experi-
ments described in the following.
2. Decay of the Radical Cation. a. In the Absence of
Bases. In the absence of electron-transfer partners, VA•+ can
have a very long lifetime. In practice, the decay by (bimo-
lecular) radical-radical reactions is an important path limiting
the lifetime of VA•+. However, at VA•+ concentrations of e1
µM, the radical cation is seen to disappear in a reaction that is
predominantly first-order and that is assigned, on the basis of
steady-state γ-radiolysis studies showing the formation of
veratryl aldehyde as the main reaction product, to C-H
deprotonation from the benzylic carbon of the side chain,28
Results and Discussion
1. Reduction Potential and Absorption Spectrum of VA•+.
In Figure 1 is presented the absorption spectrum (triangles) of
the radical cation of veratryl alcohol (VA) as measured in an
aqueous solution containing 0.05 mM VA, 1 mM thioanisole
(TA), 0.1 M 2-methyl-2-propanol (to scavenge •OH, eq 2), and
5 mM K2S2O8 (to scavenge e-aq to produce the oxidant SO4
eq 3).
,
•-
The generation of VA•+, whose absorption spectrum (Figure
1, triangles) is essentially the same as that described by Candeias
and Harvey16 but not as that of Khindaria et al.,17 occurs via
the following reactions (eqs 1-4):
which is shown in eq 8, with k ) (17 ( 1) s-1
.
H2O ' H+, •OH, e-
(1)
aq
•OH + CH3C(CH3)2OH f H2O + •CH2C(CH3)2OH (2)
The R-hydroxybenzyl-type radical resulting from the depro-
tonation, eq 8, was also produced in an alternative way, that is,
by reaction of veratrylaldehyde (VCHO) with e-aq, as shown
in eq 9
e-aq + S2O82- f SO42- + SO4
(3)
(4)
•-
SO4•- + VA (TA) f SO42- + VA•+ (TA•+)
Alternatively, it was produced via eqs 5 and 6:
•OH + Tl+ + H+ f Tl2+ + H2O
(5)
(6)
Tl2+ + VA (TA) f Tl+ + VA•+ (TA•+)
.
By variation of the concentration of veratrylaldehyde, k(e-
aq
TA was added to the VA solutions as a “redox standard” (the
reduction potential of TA•+, whose maximum absorption values
are at 310 and 530 nm,22 has been determined23 to be 1.49
V/NHE), and it was found that a reversible electron transfer
takes place between VA and TA•+, according to the equation
+ VCHO) was determined to be 1.6 × 1010 M-1 s-1. The
absorption spectrum of the radical is shown in Figure 2 at two
pH values: at pH 12 (triangles), where the ketyl radical anion
predominates, and at pH 6 (circles), where the neutral form (the
conjugate acid) is present. By measurement of the absorption

Veratryl Alcohol Radical Cation
J. Phys. Chem. A, Vol. 102, No. 38, 1998 7339
Figure 3. (a) Conductance increase initiated by a 1 Gy pulse in an
N2O-saturated aqueous solution at pH 3.4 containing 0.5 mM Tl2SO4
and 0.1 mM VA at 25 °C. (b) 8.3 mM 2-methyl-2-propanol added to
the solution in (a). (c) The solution in (a) saturated with the mixture
N2O/O2 ) 1:1.
•-
three different ways: (i) with SO4 (eq 4); (ii) with Tl2+ (eq
6); (iii) via •OH, by reaction of H+ with the OH adduct of VA
(eq 11), analogous to reactions previously studied.31 The latter
method provides an internal standard for the concentration
changes of H+, since the production of the radical cation is
accompanied by the loss of H+ (eq 11) and the decay of the
radical cation (eq 8) by the same amount of gain of H+
Figure 1. Time-resolved absorption spectra for the reaction of
thioanisole radical cation (TA•+) with veratryl alcohol (VA) obtained
by pulse radiolysis in an Ar-saturated aqueous solution, recorded 1 µs
(circles), 6 µs (filled squares), and 20 µs (triangles) after the 3 MeV,
300 ns pulse. The insets on the right show the decay of TA•+ monitored
at 530 nm and the corresponding buildup of VA•+ at 430 nm. The left
inset shows the kinetic analysis of the electron-transfer equilibrium (see
text).
.
It was found that the rate of conductance increase resulting
from the disappearance of VA•+ depended on the method of its
generation; with methods i and iii, the measured rate was (1-
2) × 102 s-1, whereas with method ii it was 17 ( 1 s-1. This
value is the same as that of Candeias and Harvey,16 and we
suggest that this is the correct one on the basis of the following
•
considerations. In system iii, OH is likely to react with VA
not only with the nucleus but also with the side chain, in which
case neutral radicals are formed,32 which may react rapidly with
VA•+ and thus limit its lifetime. Along this line, in system i,
the lifetime of VA•+ would be limited by the tert-butyl alcohol
radicals produced according to eq 2. To check this idea, in
system ii tert-butyl alcohol was added in a concentration
sufficient to scavenge 50% of the initially produced OH radicals,
Figure 2. Absorption spectra recorded at 8 µs after the pulse on
irradiating deoxygenated 0.2 mM solutions of veratrylaldehyde contain-
ing 0.1 M 2-methyl-2-propanol at pH 6.0 (circles) and 12.1 (triangles).
For obtaining the ꢀ-values, it is assumed that G(radical) ) G(e-aq) )
2.7.
•
leading to the production of Me2C(OH)CH2 , thus preventing
at 340 nm as a function of pH, the pKa value of the neutral
radical was determined (see inset in Figure 2) to be 10.0, which
is higher (as expected) than that (pKa ) 9.3, measured in an
analogous way) of the corresponding radical from 4-methoxy-
benzaldehyde (whereby k(e-aq) + 4-methoxybenzaldehyde )
3.2 × 1010 M-1 s-1).29 Oxidation, for example, with oxygen,
of the R-hydroxybenzyl-type radical produced in eq 8 leads to
the final product, veratrylaldehyde. By observation of the decay
rate of the radical as a function of [O2], the rate constant for
reaction 10,
•OH from producing Tl2+ and thus VA•+ (via eqs 5 and 6). In
fact, as seen in Figure 3b, addition of tert-butyl alcohol led to
a drastic increase in the rate of proton formation, indicating the
enhanced disappearance of VA•+. This would be understandable
if by radical-radical decay of a radical cation and a neutral
radical a carbocation was produced, which typically has a much
higher reactivity with water (to give H+) than a radical cation.33
Thus, we suggest that the high value (1.2 × 103 s-1 19,34
)
reported
by Aust for the decay rate of VA•+ is an artifact indicating the
presence of radicals other than VA•+ under his experimental
conditions. Aust also reports19 that there is an effect of oxygen
on the lifetime of VA•+. We have now checked this, using
method ii to generate the radical cation and monitoring its decay
by the conductance technique. From the very small enhancing
effect of O2 on the rate of conductance regeneration (compare
Figure 3 parts a and c), the value we obtain is k(O2 + VA•+) e
which probably proceeds by an addition/elimination mecha-
nism,30 was determined to be 1.6 × 109 M-1 s-1
.
8 × 103 M-1 s-1
.
To resolve the discrepancy in the literature16,19 on the rate of
reaction 8, we measured this reaction by using the time-resolved
ac-conductance technique, monitoring the production of H+ at
pH ≈ 3 (see Figure 3). The radical cation was produced in
As a further model compound for lignin, an alcohol was
studied that is derived from VA by substitution of one hydrogen
at the benzylic position by a tert-butyl group, that is, 3,4-
•-
(MeO)2C6H3CH(OH)CMe3 (VAtBu). On reaction with SO4

7340 J. Phys. Chem. A, Vol. 102, No. 38, 1998
Bietti et al.
SCHEME 2
this reaction, the OH function of VA was methylated, thus
producing veratryl alcohol methyl ether (VAME), and this
Figure 4. Dependence on [OH-] of kobs for decay of VA•+ (circles)
and VAME•+ (squares). The O2-saturated aqueous solutions contained
compound was oxidized with SO4 or Tl2+, analogous to
•-
•
0.1 M 2-methyl-2-propanol (to scavenge OH), 10 mM K2S2O8 (to
reaction 4 or 6. The absorption spectrum of the resulting radical
cation, VAME•+, was very similar to that of VA•+, showing
absorption bands at 295 and 425 nm, and at low dose (e1 Gy/
pulse) the radical cation (as produced with Tl2+) decayed at
pH e 5 predominantly by first-order kinetics with36 k ) 20 (
1 s-1, a value very similar to that of VA•+. The reduction
potential of VAME•+ was measured by a method analogous to
that described above for VA•+, and the value 1.36 V/NHE was
obtained. In contrast to the situation with VA•+, the decay of
VAME•+ increased only slightly in the presence of OH-. From
the slope of the kobs(decay) vs [OH-] plot (see Figure 4), the
rate constant for decay of the radical cation by reaction with
OH- is only 2 × 107 M-1 s-1, that is, 65 times lower than that
for the case of the alcohol, VA, and not much higher than that
(2.1 × 106 M-1 s-1) we measured for the reaction of the radical
cation of 3,4-dimethoxytoluene with OH-, where deprotonation
involves necessarily the C-H bond of the methyl group.41 This
indicates that the interaction of OH- with VAME•+ should also
involve base attack at the C-H bond,42 in contrast to the case
of VA•+ where it is the O-H bond that is involved.
scavenge e-aq), 1 mM Na2B4O7, and 0.5 mM of VA (VAME).
SCHEME 1
We found that also the rate of fragmentation of the radical
cation of 3,4-(MeO)2C6H3CH(OH)CMe3 increases strongly with
increasing [OH-]. From the slope of the corresponding plot,
the second-order rate constant for the OH--induced fragmenta-
tion (which now almost exclusively involves the C-C bond)38
is 8.3 × 109 M-1 s-1, a value somewhat larger than that for
or Tl2+, this compound gave a spectrum very similar to that of
VA•+, which, at pH e 5 and low dose, decayed in a first-order
reaction with the averaged35 rate constant36 k ) (24 ( 1) s-1
.
On the basis of product analysis studies performed after
γ-irradiation, the decay at pH ) 4-5 leads to two products:
veratrylaldehyde and the ketone 3,4-(MeO)2C6H3C(O)CMe3,
with the ratio aldehyde/ketone ) 2. The production of
veratrylaldehyde clearly indicates that the radical cation under-
goes a C-C fragmentation of the side chain (Scheme 1, path
a),37 whereas the ketone is the product of a C-H deprotonation
reaction (path b)38 followed by oxidation of the resulting
R-hydroxybenzyl-type radical.39
VA•+ (see above)43 and close to that ((1-2) × 1010 M-1 s-1
)
for diffusion-controlled OH- reactions with protons bonded to
hetero atoms (the rate constants for reaction of OH- with C-H
protons are typically up to several orders of magnitude
lower).44,45 The similar rate constants for reactions of VA•+
and VAtBu•+, with OH- despite the different bonds cleaved in
the two processes, make it therefore reasonable to assume that
OH- deprotonates in both cases the OH function in the side
chain of the radical cations.46 The result would be a radical
zwitterion (Scheme 2). However, the radical zwitterion, being
less electron deficient than the radical cation, should be less
prone to undergo C-C or C-H fragmentation reactions. Thus,
the experimentally observed positive “catalysis” of OH- cannot
be explained by the formation of the radical zwitterion as such.
However, if it is assumed that in the radical zwitterion an
intramolecular electron transfer occurs from the oxyanion in
the side chain to the electron “hole” in the aromatic π-system,
b. In the Presence of Bases. Monitoring the absorption of
VA•+ at 430 nm, we found that its decay rate can be
considerably increased by adding OH- to the solution.40 In fact,
the rate of decay of VA•+ increases linearly with [OH-] (see
Figure 4), and, from the slope of this dependence, the second-
order rate constant for reaction of OH- with VA•+, which leads
to the formation of the R-hydroxybenzyl-type radical,28 results
as (1.3 ( 0.3) × 109 M-1 s-1. To elucidate the mechanism of

Veratryl Alcohol Radical Cation
J. Phys. Chem. A, Vol. 102, No. 38, 1998 7341
References and Notes
an oxyl radical is the result, and radicals of this type are known
to undergo â-C-C fragmentation (Scheme 2, step d) and, also,
in aqueous solution, 1,2-H-atom shift reactions (step e),47 thus,
the mechanism accounts for all the experimental observations.
Formation of the benzyloxyl radical may also be concerted48
with base-induced deprotonation of the radical cation (Scheme
2, path b). The same type of mechanism has recently been
proposed21 for the case of 4-(mono)methoxybenzyl alcohol
radical cations.49 This indicates that this mechanism may be a
general one for radical cations with an OH-function at the
R-position50 of the side chain.
(1) (a) Universita` “Tor Vergata”. (b) Universita` “La Sapienza” (c) Max-
Planck-Institut.
(2) Biotechnology in the Pulp and Paper Industry; Eriksson, K.-E. L.,
Ed.; Springer-Verlag: Berlin Heidelberg, 1997.
(3) Pardini, V. L.; Smith, C. Z.; Utley, J. H. P.; Vargas, R. R.; Viertler,
H. J. Org. Chem. 1991, 56, 7305.
(4) Gunnarsson, P.-I.; Ljunggren, S. Acta Chem. Scand. 1996, 50, 422.
(5) Chum, H. L.; Baizer, M. M. The Electrochemistry of Biomass and
DeriVed Materials; ACS Monograph 183; American Chemical Society:
Washington, DC, 1985; p 244.
(6) Othmer, K. Encyclopedia of Chemical Technology, 3rd ed.; J.
Wiley: New York, 1983; vol. 23, p 704.
(7) (a) Tien, M.; Kirk, T. K. Science 1983, 221, 661. (b) Glenn, J. K.;
Morgan, M. A.; Mayfield, M. B.; Kuwahara, M.; Gold, M. H. Biochem.
Biophys. Res. Commun. 1983, 114, 1077. (c) Schoemaker, H. E.; Harvey,
P. J.; Bowen, R. M.; Palmer, J. M. FEBS Lett. 1985, 183, 7. (d) Harvey, P.
J.; Schoemaker, H. E.; Bowen, R. M.; Palmer, J. M. FEBS Lett. 1985, 183,
13. (e) Hammel, K. E.; Kalyanaraman, B.; Kirk, T. K. Proc. Natl. Acad.
Sci. U.S.A. 1986, 83, 3708. (f) Hammel, K. E.; Jensen, K. A.; Mozuch, M.
D.; Landucci, L. L.; Tien, M.; Pease, E. A. J. Biol. Chem. 1993, 268, 12274.
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Summary and Conclusions
The reduction potential of the radical cation of veratryl
•-
alcohol, VA•+, as produced in aqueous solution with SO4
,
was determined by electron-transfer equilibration with thioani-
sole as a redox standard to be 1.36 V/NHE. The radical cation
undergoes a C-H deprotonation reaction from the side chain
with k ) (17 ( 1) s-1 at pH e 5. This value confirms a
previous one16 and indicates that VA•+ should in fact be able
to act as a diffusible mediator11,12,16 in the degradation of lignin
by LiP. The deprotonation reaction of VA•+ is induced by OH-,
with the “catalytic” rate constant being 1.3 × 109 M-1 s-1. In
contrast, the corresponding rate constant for reaction of OH-
with the radical cation of veratryl alcohol methyl ether,
VAME•+, whose reduction potential is also 1.36 V/NHE, is only
2 × 107 M-1 s-1. From this large difference in reactivity, which
cannot (as derived from the redox potentials) be due to a
difference in acidity, it is concluded that in the former case (the
alcohol) the reaction of OH- involves the deprotonation from
the alcohol function to yield (in a stepwise or concerted fashion)
a benzyloxyl radical followed by a 1,2 H-atom shift, which
constitutes the loss of H from the carbon. In the case of the
ether, however, interaction of OH- is with the hydrogen on the
benzylic carbon. In the case of VA, an important consequence
of the “catalysis” by OH- is that the lifetime of the radical cation
at pH g 7 is limited by this reaction (for example, k(pseudo
first order) at pH ) 8 is 1.6 × 103 s-1), rather than by the
uncatalyzed decay. With the veratryl alcohol derivative, 3,4-
(MeO)2C6H3CH(OH)CMe3, the radical cation undergoes both
a proton loss from the benzylic position and a CR-Câ
fragmentation with the ratio, at pH e 5, of ca. 1:2. The decay
of the radical cation is strongly enhanced by OH- (k ) 8.3 ×
109 M-1 s-1), with the base induction favoring the C-C
fragmentation relative to the proton loss from the benzylic
position.43 The conclusion to be drawn from this for the
biological decomposition of lignin is that in view of an optimal
functioning of VA as a diffusible mediator in the oxidation of
lignin by LiP it is clearly advantageous for the biological system
to avoid the enhancement of the VA•+ decay by OH-, that is,
it is important to keep the pH of the enzyme environment as
low as possible. On the other hand, to take advantage of the
OH- effect of radical cation decomposition, the (local) pH in
the vicinity of the lignin macromolecule should be high. A
conclusion of relevance to practical or industrial mechanistic
applications is that for all processes of lignin degradation based
on one-electron oxidation, basic media should be superior to
acidic media.
(9) Hammel, K. E.; Moen, M. A. Enzyme Microb. Technol. 1991, 13,
5.
(10) (a) Piontek, K.; Glumoff, T.; Winterhalter, K. FEBS Lett. 1993,
315, 119. (b) Poulos, T. L.; Edwards, S. L.; Wariishi, H.; Gold, M. H. J.
Biol. Chem. 1993, 268, 4429.
(11) Harvey, P. J.; Schoemaker, H. E.; Palmer, J. M. FEBS Lett. 1986,
195, 242.
(12) Goodwin, D. C.; Aust, S. D.; Grover, T. A. Biochemistry 1995,
34, 5060.
(13) Valli, K.; Wariishi, H.; Gold, M. H. Biochemistry 1990, 29, 8535.
(14) Koduri, R. S.; Tien, M. Biochemistry 1994, 33, 4225.
(15) Schoemaker, H. E.; Piontek, K. Pure Appl. Chem. 1996, 68, 2089.
(16) Candeias, L. P.; Harvey, P. J. J. Biol. Chem. 1995, 270, 16745.
(17) Khindaria, A.; Grover, T. A.; Aust, S. D. Biochemistry 1995, 34,
6020.
(18) Khindaria, A.; Yamazaki, I.; Aust, S. D. Biochemistry 1995, 34,
16860.
(19) Khindaria, A.; Yamazaki, I.; Aust, S. D. Biochemistry 1996, 35,
6418.
(20) There is also the problem of the “stabilization” of VA•+ by LiP;
k(decay) of VA•+, as generated by LiP, was reported19 to be 1.8 s-1
.
(21) Baciocchi, E.; Bietti, M.; Steenken, S. J. Am. Chem. Soc. 1997,
119, 4078.
(22) Ioele, M.; Steenken, S.; Baciocchi, E. J. Phys. Chem. A 1997, 101,
2979.
(23) This, our number, is from electron-transfer equilibration between
TA•+ and 3-(2-methoxyphenoxyl)-1,2-propanediol, for which the potential
was reported to be 1.40 V/NHE (a) Jonsson, M.; Lind, J.; Merenyi, G.;
Eriksen, T. E. J. Chem. Soc., Perkin Trans. 2, 1995, 67.
(24) For details on this technique, see the following. Wardman, P. J.
Phys. Chem. Ref. Data 1989, 18, 1637. Steenken, S.; Neta, P. J. Phys. Chem.
1982, 86, 3661. Reference 23a. Typically, the K from the absorbances is
more reliable than that from the rates.
(25) To check this number, we have equilibrated VA•+ also with
guanosine, whose potential relative to that (1.13 V)23a of 1,2,4-trimethoxy-
benzene has been measured to be 1.26 V/NHE (Steenken, S.; Jovanovic,
S. V. J. Am. Chem. Soc. 1997, 119, 617). To improve the reliability of the
potential for 1,2,4-trimethoxybenzene, we have now measured this value
by cyclic voltammetry in aqueous solution, pH 3-5, with 0.1 M KCl as
the electrolyte, to be 1.14 V/NHE, which is in excellent agreement with
the pulse radiolysis value from ref 23a. Then, from the measured difference
in the potential between guanosine and VA, E(VA•+) results as 1.37 V/NHE,
a number which is practically the same as that from the comparison with
TA•+ (see text).
(26) Kersten, P. J.; Kalyanaraman, B.; Hammel, K. E.; Reinhammar,
B.; Kirk, T. K. Biochem. J. 1990, 268, 475.
(27) A similar value was found for manganese peroxidase (see: Popp,
J. L.; Kirk, T. K. Arch. Biochem. Biophys. 1991, 288, 145).
(28) Since the lifetime of the VA•+ deprotonation product (which decays
by radical-radical reaction) is considerably shorter than that of its precursor,
VA•+, the spectrum of the 3,4-dimethoxy-R-hydroxybenzyl radical cannot
be seen at pH e 7. However, by speeding up the deprotonation reaction
with base (see Scheme 2, steps a-c, and e), the spectrum (which turns out
to be the same as that from eq 9; see Figure 2) can be easily measured.
(29) Baciocchi, E.; Bietti, M.; Steenken, S. Unpublished results.
(30) Steenken, S. In Free Radicals: Chemistry, Pathology and Medicine;
Rice-Evans, C., Dormandy, T., Eds.; Richelieu Press: London, 1988, p
51.
Supporting Information Available: Complete experimental
section with details on pulse radiolysis and γ-radiolysis experi-
ments (3 pages). Ordering information is given on any current
masthead page.

7342 J. Phys. Chem. A, Vol. 102, No. 38, 1998
Bietti et al.
(31) O’Neill, P.; Steenken, S.; Schulte-Frohlinde, D. J. Phys. Chem.
1975, 79, 2773.
therein) was used for the oxidation of this substrate at 25 °C. In this case,
the only observed product was veratrylaldehyde; no ketone was detected.
2-
(32) The yield of VA•+ from eq 11 compared to that (≡100%) from
the reaction with Tl2+ (eq 6) was found to be 35%. The remaining 65% is
assigned to H-abstraction from the substituents by •OH and/or to ipso-
addition at the MeO groups followed by elimination of MeOH to give
phenoxyl-type radicals. See the following. Steenken, S.; O’Neill, P. J. Phys.
Chem. 1977, 81, 505. O’Neill, P.; Steenken, S. Ber. Bunsen-Ges. Phys.
Chem. 1977, 81, 550. The yield of VA•+ from eq 4 was the same as that
from eq 6, which indicates that H-abstraction by SO4•- from the side chain
is not important.
(39) S2O8 oxidizes the R-hydroxybenzyl-type radical yielding the
•-
ketone, H+, and SO4 which continues the chain.
(40) This dependence of lifetime on pH constitutes a correction of the
report of Aust19 in which it is stated that the lifetime of the radical cation
is independent of pH.
(41) At pH 4-5, the rate constant for the deprotonation of the radical
cation of 3,4-dimethoxytoluene was measured (by conductance) to be 1.1
s-1
.
(42) Less likely is a change of mechanism, for example, toward OH-
addition at the ring.
(33) Reaction of a radical with the radical cation could proceed via
dimerization to yield a (delocalized) cyclohexadienyl-type carbocation or
via (electron transfer) disproportionation giving, again, a carbocation.
(34) This value was determined in air or oxygen saturated solution,
whereas that of ref 16 was measured in the absence of O2.
(43) The mechanistic implications of these findings are presently under
investigation: Baciocchi, E.; Bietti, M.; Steenken, S. To be published.
(44) Eigen, M. Angew. Chem. 1963, 75, 489; Angew. Chem., Int. Ed.
Engl. 1964, 3, 1.
(35) In the conductance traces, there were two first-order components
visible, with the ratio of the rate constants being 2:1. On the basis of the
product analysis results (see text), the higher value is assigned to the C-C
fragmentation process (Scheme 1, path a) and the lower one to the C-H
deprotonation reaction (Scheme 1, path b). The “averaged” rate constant,
(45) In acetonitrile solvent, however, rate constants for base-induced
radical cation deprotonations of up to 1010 M-1 s-1 have been measured
(Anne, A.; Hapiot, P.; Moiroux, J.; Neta, P.; Saveant, J.-M. J. Am. Chem.
Soc. 1992, 114, 4694 and references therein. See also Sehested, K.; Holcman,
J. J. Phys. Chem. 1978, 82, 651 for rate constants in aqueous solution of
up to 1010 M-1 s-1).
24 ( 1 s-1, is similar to that of VA•+
.
(36) Value obtained by the conductance technique in the dose range
0.5-2 Gy/pulse.
(46) In comparison with the strong base OH-, the efficiency of the
weaker base HPO42- is much lower, that is, k(HPO42- + radical cation) e
5 × 104 M-1 s-1 (this value relates to both VA and VAtBu).
(47) Gilbert, B. C.; Laue, H. A. H.; Norman, R. O. C.; Sealy, R. C. J.
Chem. Soc., Perkin Trans. 2 1976, 1040. Dobbs, A. J.; Gilbert, B. C.; Laue,
H. A. H.; Norman, R. O. C. J. Chem. Soc., Perkin Trans. 2 1976, 1044.
Gilbert, B. C.; Holmes, R. G. G.; Laue, H. A. H.; Norman, R. O. C. J.
Chem. Soc., Perkin Trans. 2 1976, 1047. Gilbert, B. C.; Warren, C. J. Res.
Chem. Intermed. 1989, 11, 1. The 1,2-H shift also takes place in alcoholic
solvents. See: Elford, P. E.; Roberts, B. P. J. Chem. Soc., Perkin Trans. 2
1996, 2247.
(37) On the basis of the redox potentials (see: Wayner, D. D. M.; Sim,
B. A.; Dannenberg, J. J. J. Org. Chem. 1991, 56, 4835. Wayner, D. D. M.;
McPhee, D. J.; Griller, D. J. Am. Chem. Soc. 1988.) of the pertinent
fragments, the conceivable inVerse C-C fragmentation, that is, that leading
to the tert-butyl cation, is thermodynamically much less favorable. Also,
with the 4-methoxy analogue (see: Baciocchi, E.; Bietti, M.; Putignani,
L.; Steenken, S. J. Am. Chem. Soc. 1996, 118, 5952), the tert-butyl radical
was actually detected by product analysis.
(38) It was found that in the presence of phosphate buffer at pH ) 6.9
(10 mM KH2PO4 and 10 mM Na2HPO4), the veratryl aldehyde/ketone ratio
increased to 50 (aldehyde 98%, ketone 2%), that is, the C-C bond cleavage
pathway is strongly assisted by the base phosphate, whereas C-H
deprotonation apparently profits much less from the base (the details of
this mechanism are presently under investigation: Baciocchi, E.; Bietti,
M.; Steenken, S. To be published). At the same time, the total yield increased
by a factor of 4 as compared to that obtained at pH 4-5. In addition to
SO4•-/S2O82-, the oxidant Co(III)W12O405- (see: Baciocchi, E.; Bietti, M.;
Steenken, S. J. Chem. Soc., Perkin Trans. 2 1996, 1261 and references
(48) If the rate of the (hypothetical) electron transfer, step c, is (much)
larger than that of the protonation of the radical zwitterion, the reverse of
step a, the formation of the benzyloxyl radical, would appear to be concerted
with the reaction of OH- with the radical cation.
(49) A similar mechanism involving the formation of a benzyloxyl
radical has also been proposed for the oxidation of 3,4-(MeO)2C6H3CH-
(OH)Me with ClO2 (see ref 4).
(50) We have recently obtained evidence that this mechanism is not
restricted to the alcohol function at the R-position.