PCET in the oxidation of ascorbate. Dramatic change of the kinetic isotope effect on the change in solvent polarity

Article abstract of DOI:10.1016/j.tetlet.2007.03.140

The first step in the oxidation of ascorbate with substituted nitrosobenzenes is a proton-coupled electron transfer (PCET) reaction and the observed kinetic isotope effects in the reaction, k_{H2 O} / k_{D2 O}, change dramatically with a change in solvent polarity, in line with known theoretical predictions.

Full text of DOI:10.1016/j.tetlet.2007.03.140

Tetrahedron Letters 48 (2007) 3633–3637
PCET in the oxidation of ascorbate. Dramatic change of the
kinetic isotope effect on the change in solvent polarity
´
´
Daniela Vuina, Viktor Pilepic, Daniel Ljubas, Kresˇimir Sankovic,
ˇ ´*
Ivana Sajenko and Stanko Ursic
Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇic´a 1., 10000 Zagreb, Croatia
Received 14 February 2007; revised 14 March 2007; accepted 23 March 2007
Available online 28 March 2007
Abstract—The first step in the oxidation of ascorbate with substituted nitrosobenzenes is a proton-coupled electron transfer (PCET)
reaction and the observed kinetic isotope effects in the reaction, kH O=kD O, change dramatically with a change in solvent polarity, in
2
2
line with known theoretical predictions.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Proton-coupled electron transfers (PCET) are processes
where proton motion can affect electron transfer.1–13
Owing to its exceptional importance in a wide range of
chemical, biochemical, and biological systems, this phe-
nomenon has been greatly studied, both experimen-
tally2,4–7 and theoretically.8–13 An important example
of how PCET works in natural systems is presented by
the well known case of oxygen production by the photo-
system II oxygen-evolving complex (PSII OEC).1,4 It is
the fundamental feature of PCET reactions that the con-
certed transfer of a proton and an electron constitute a
single chemical reaction step and the direct coupling of
the electron and proton in the transfer is the most
elementary characteristic of a PCET event.1,2 A special
class of PCET reaction is termed as hydrogen atom
transfer (HAT). Here, transfer of an electron and a pro-
ton occurs along a roughly common path; the proton is
transferred together with one of its bonding electrons.1,2
release of nitric oxide from N-nitrosated tryptophan.14f
Concerted proton–electron transfer has been inferred
for some systems14b,c,e,g on the basis of thermochemical
arguments and observed kinetic isotope effects (KIE).
Here, we present evidence in support that the first step22
in the oxidation of ascorbate with nitrosobenzenes
(Scheme 1) is a PCET reaction, and the observed kinetic
deuterium isotope effects (KIE) in the reaction depend
dramatically on the solvent polarity. The latter observa-
tion is among the first experimental confirmations of
theoretical predictions10c,d on the dependence of KIE
on solvent polarity in PCET processes. The evidence is
as follows:
(i) The observed free energy of activation DGà for the
first step of the reaction of ascorbate with nitrosobenz-
ene (Scheme 1) in water is 53.3 kJ/mol. Applying the
usual square scheme1,2 to describe the thermochemistry
of different pathways of the reaction reveals that as the
pKa of the ascorbate 2-OH group is15 11.79 and the pKa
of the nitroso group of nitrosobenzene is16 ꢀ9.5, the
expected barrier for the initial proton transfer (PT) from
ascorbate to the nitroso group in a stepwise PT/ET pro-
cess would be ca. 121.5 kJ molꢀ1, which is 68 kJ larger
than the observed DGà of the reaction. Furthermore,
since the reduction potential for the couple HAscÅ/
HAscꢀ is15a,c 774 mV, the corresponding barrier for ini-
tial electron transfer (ET) from ascorbate to the nitroso
group should be 73.8 kJ molꢀ1, ca. 21 kJ more than the
observed DGà. Therefore, concerted electron and proton
transfer seems to be the likely mechanism to avoid
higher energy intermediates during the process.
Transfer of an electron and a proton (net hydrogen
atom transfer) from ascorbate (HAscꢀ) is important in
the chemistry and biochemistry of this ubiquitous, sim-
ple biomolecule. Examples include ascorbate peroxi-
14b
dase,14a cytochrome b561
,
a secretory-vesicle redox
chain,14c the biologically important interaction of ascor-
bate with peroxynitrite,14d the recycling of a-tocopher-
oxyl radicals by ascorbate,14e and ascorbate-induced
Keywords: Proton-coupled electron transfer; Kinetic isotope effects;
Ascorbate.
*
Corresponding author. Tel.: +385 1 4818 306; fax: +385 1 4856
201; e-mail: stu@pharma.hr
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.140

3634
D. Vuina et al. / Tetrahedron Letters 48 (2007) 3633–3637
O
O
H
OH
OH
H
OH
OH
•
N
H
N
H
H
O
O
O
O
H
H
+
+
•
-
-
O
O
O
OH
Asc• -
PhNHO•
-
HAsc
Scheme 1.
Table 1. Kinetic isotope effects kH O=kD in the reaction of ascorbate with substituted nitrosobenzenes in water and in water–dioxane at 298 Ka
2O
2
p-CH3
p-H
p-Cl
p-Br
m-Cl
Water
Water–dioxaneb
2.63 (0.17)
9.42 (0.11)
2.43 (0.07)
8.51 (0.08)
2.30 (0.10)
8.70 (0.26)
2.07 (0.14)
8.40 (0.28)
2.20 (0.11)
6.40 (0.05)
a All the data are related22 to the process defined by Scheme 1.
b In 1:1 v/v water–dioxane and 1:1 v/v D2O–dioxane.
(ii) Kinetic isotope effects kH O=kD O ranging from 2.63
state. Analogous Hammett correlations are not uncom-
mon in PCET reactions. For example, in the oxidation
of meta-substituted phenols by oxochromium(IV)
ions19b the observed slope (q) is ꢀ1.72, and the
observed kH O=kD O is 14.7, a result indicative of tunnel-
2
2
(p-CH3 nitrosobenzene) to 2.20 (m-Cl nitrosobenzene)
were observed (Table 1) in the reaction (Scheme 1). If
there is a rate-limiting electron transfer step in the reac-
tion, only significantly smaller secondary isotope effects
would be expected.17 The kinetic evidence22 clearly indi-
cates that ascorbate anions and the nitroso compound
are the only reactants in the process, and the above ther-
mochemical analysis strongly suggests a concerted pro-
cess. Hence, the observed isotope effects should be
consistent with a PCET process. The observed KIE for
the reaction in water are not large. PCET processes
are considered to occur by tunneling1,8–10,12,13a (see also
below) but KIE (referred to as the H/D kinetic isotope
effects) in PCET reactions are not necessarily large, as
demonstrated both experimentally5a,6 and theoreti-
cally.9,10c,d,13a Thus, for example, the observed KIE in
the range 1.2–2.1 are not uncommon5a,6 for a PCET
reaction.
2
2
ing in the PCET reaction.
(v) A dramatic change of the KIE in the reaction on
going from water to water–dioxane solvent was
observed (Table 1). To our knowledge, this observation
presents the first experimental confirmation of the theo-
retical predictions on the influence of solvent polarity on
the magnitude of KIE in PCET reactions.10c,d Accord-
ing to this theoretical explanation, a complex interplay
between several factors, including the localization of,
and the distance of the reactant and product proton
vibrational wave function and the electron donor–accep-
tor distance (which influences electron–proton electro-
static interactions) and the solvent polarity control the
magnitude of the corresponding KIE. In general, the
KIE in a PCET process increases as the probability of
the PCET mechanism increases. In particular, the KIE
increases as the proton donor–acceptor distance
increases and the electron donor–acceptor distance
decreases and as solvent polarity decreases in a PCET
process. Here, since the observed Hammett logk/r
parameters correlation suggests some charge reorganiza-
tion/separation in the activation process, the observed
change of KIE could be interpreted as a consequence
of a decrease in the electron donor–acceptor distance
in the PCET processà due to lowering of the solvent
dielectric constant. This should not contradict the fact
that the proton donor–acceptor distance also possibly
(iii) The observed difference in the enthalpies of activa-
tion in H2O and D2O, DHz ¼ 8:9 ð0:3Þ kJ molꢀ1
H2O
and DHzD O ¼ 31:6 ð1:7Þ kJ molꢀ1
,
respectively, are
2
substantially larger than the expected semiclassical val-
ues calculated from the observed KIE. This observation
indicates that tunneling18 is likely in the proton transfer.
The same is true with regard to the correspond-
ing observed entropies of activation, DSHz O
¼
2
ꢀ148:8 ð3:5Þ J Kꢀ1 molꢀ1
,
and DSzD O ¼ ꢀ80:0 ð3:8Þ
2
J Kꢀ1 molꢀ1, respectively, which suggest a ratio of isoto-
pic Arrhenius pre-exponential factors substantially dif-
ferent from the semiclassical expectation of unity. The
observation of tunneling is as expected1,8–10,12,13a for a
PCET process. Eyring and co-workers19a have observed
closely similar isotopic differences in the enthalpies and
entropies of activation in the ferrate(VI) oxidation of
aqueous phenol and the evidence clearly suggests a
PCET process. The kH O=kD O value of 2.4 observed is
The rate of reaction in 50:50 water–dioxane is reduced from
2900 molꢀ1 sꢀ1 in water to 1800 molꢀ1 sꢀ1 in water–dioxane.
à A referee has suggested that it is not clear that the electron donor–
acceptor distance in the PCET process would decrease, and that it is
not necessary for this distance to be decreased for the observed
change of KIE. He has argued that the analysis in Ref. 10c indicates
that, even with a constant electron donor–acceptor distance, the KIE
is expected to increase as the solvent polarity decreases because of
changes in the curvatures and relative energies of the free energy
curves along the collective solvent coordinate.
2
2
very similar to the KIE observed in our reaction in
water.
(iv) The Hammett plot of logk versus r parameters
for the reaction gave a slope (q) of 1.64, consistent
with a charge redistribution and the presence of a
partial negative charge on the acceptor in the transition

D. Vuina et al. / Tetrahedron Letters 48 (2007) 3633–3637
3635
There is a question– whether the reaction is a HAT pro-
cess, or a PCET where the electron and proton are trans-
ferred between different sets of orbitals.1,2,20a,b Several
lines of evidence can be interpreted to be more in sup-
port of PCET than a HAT process. The rate of the reac-
tion depends markedly on the solvent polarity (see
Fig. 1) while only small variations of reaction rates on
the solvent polarity are expected for HAT reactions.21a
The observed entropy of activation in the reaction,
DSà = ꢀ 148.8 J Kꢀ1 molꢀ1 can be compared, for exam-
ple, with the value of DSà = ꢀ 144 J Kꢀ1 molꢀ1 in the
PCET reaction of phenols with oxochromium(IV)
ions19b and the values for numerous formal HAT reac-
tions for which DiLabio and Ingold have proposed20b
to occur by PCET. In the HAT reaction21b,c of PhCH2Å
with PhCH3, DSà ꢁ ꢀ 50 J Kꢀ1 molꢀ1. Furthermore, in
the PCET and ET self-exchange reactions of iron-bi-
3.8
3.3
2.8
2.3
1.8
imidazoline
complexes,21c
DSà(PCET) = ꢀ 109
J Kꢀ1 molꢀ1 and DSà(ET) = ꢀ 105 J Kꢀ1 molꢀ1, while
-0.2
0
0.2
0.4
2þ
in the self-exchange reaction between FeðH2OÞ6 and
σ
3þ
FeðH2OÞ , DSà(ET) = ꢀ 105 J Kꢀ1 molꢀ1 and similar
6
values were observed for other self-exchange ET reac-
tions21c of iron(II)/iron(III) complexes. The change in
charge distribution is larger for ET than for the corre-
sponding PCET11a (except when the electron and proton
in a PCET are transferred in opposite directions)11b and
the similarities in the magnitude of the entropies of acti-
vation in the ET and the PCET reactions may support
the expectation that the entropy change in the latter is
largely caused by charge redistribution originating from
the motion of the electron in transfer. It should be added
that the proton in a PCET is transferred over a much
smaller distance than the electron1,8–12 thus requiring
correspondingly less solvent reorganization. In a HAT
process, accompanied transfer of a proton and an elec-
tron is expected to cause less charge redistribution9
and consequently, less solvent reorganization21c which
would be reflected in a smaller entropy of activation
and a smaller influence of solvent polarity on the rate
of reaction.
Figure 1. Hammett plots for the reaction of ascorbate with substituted
nitrosobenzenes at 298 K in water and in water–dioxane. Solid circles
and open circles, in water and D2O (qH ¼ 1:51, r2 = 0.993;
2O
qD ¼ 1:67, r2 = 0.991); solid triangles and open triangles, in 1:1 v/v
2O
water dioxane and D2O–dioxane (qH
¼ 1:95, r2 = 0.990;
2O–Dioxane
qD
¼ 2:18, r2 = 0.997), respectively. Rate constants were
2O–Dioxane
obtained as described in the text.22
decreases; according to these theoretical predictions,10c,d
KIE are largest for intermediate proton donor–acceptor
distances due to the opposed effects of the ratio of the
hydrogen-to-deuterium couplings (these increase as the
proton donor–acceptor distance becomes larger, thus
increasing the KIE) and the probability of PCET (which
increases as the proton donor–acceptor distance
decreases, which can lead to a larger KIE10c,d).
(vi) The observed variation of the KIE with different
substituents on the nitrosobenzene (Fig. 1, Table 1)
may serve as further evidence in support of the above
noted theoretical predictions.10c,d Accordingly, it would
be expected10c,d that the KIE become larger as the reac-
tion becomes more endothermic (or less exothermic, in
the normal Marcus regime). Our experimental results
show that the observed KIE could increase as the reac-
tion becomes more endothermic, assuming however,
that the entropy of activation remained essentially
unchanged§ on changing a substituent. This is also illus-
trated by the difference between the slopes (q values) of
the Hammett logk versus r correlations for the exam-
In conclusion, the results obtained should be relevant
for improved understanding and interpretation of ki-
netic isotope effects in PCET processes in biochemical/
biological systems. Finally, after Mayer,20a and DiLabio
and Ingold20b demonstrated theoretically that the for-
mal HAT reaction can in fact be a PCET process, and
that some PCET reactions could involve a cyclic transi-
tion state,20b the question arises whether a multi-center,
cyclic PCET might occur in the reaction studied here.
This is currently being investigated in our laboratory.
ined reaction in H2O and D2O (q
¼ 1:51, qD O
¼
H2O
2
1:67, Dq = 0.16) and H2O–dioxane and D2O–dioxane
(qH O–Dioxane ¼ 1:95, qD O–Dioxane ¼ 2:18, Dq = 0.23). The
2
2
Acknowledgement
differences are relatively small but statistically significant
for both systems.
We thank the Croatian ministry of Science and Educa-
tion for financial support (project 006-0354).
§ The expectation that the changes of entropy of activation due to the
changes of the substituent on the electron/proton acceptor could be
relatively unimportant relies on an assumption that the solvation in
the corresponding transition state would differ only slightly within the
series of substrates differing by these uncharged substituents.
– According to the referee’s opinion, the distinction between HAT and
PCET is not rigorous because the electron and the proton behave
quantum mechanically. A point of interest in this study is, however, if
the electron and the proton are transferred between the different sites
(possibly via a cyclic transition state).

3636
D. Vuina et al. / Tetrahedron Letters 48 (2007) 3633–3637
15. (a) Davies, M. B.; Austin, J.; Partridge, D. A. Vitamin C,
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(Scheme 1). The reverse reaction (the recombination of
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AscÅꢀ and PhNHOÅ, k
)
ꢀ1
k1
HAscꢀ þ PhNO ¢ AscÅꢀ þ PhNHOÅ
ð1Þ
k
ꢀ1
should be insignificant under the conditions applied. This
is probably due to a 103-fold increase in the rate of dispro-
portionation of an ascorbyl radical to ascorbate and
dehydroascorbic acid on going from the neutral to the
acidic range (see for example Ref. 15b, pp 78–79, and
references cited therein), since the disproportionation com-
petes with the reverse reaction. The net chemical reaction
is14g
HAscꢀ þ PhNO þ Hþ ! DHA þ PhNHOH
ð2Þ
(DHA = dehydroascorbic acid, PhNHOH = phenylhydr-
oxylamine), as confirmed spectroscopically and by product
analysis, according to previous findings.14g The reaction
goes to completion, under all the conditions employed.
The findings relevant to the above are (i) the kinetics are
second order overall and first order with respect to both
ascorbate and the nitroso compound. This is true in acid-
ic14g as well in the neutral range; (ii) in the acidic range,
the kinetics follow the rate law14g
12. Hammes-Schiffer, S. Acc. Chem. Res. 2006, 39, 93, and
references cited therein.
´
13. (a) Presse, S.; Silbey, R. J. Chem. Phys. 2006, 124, 164504;
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Chem. Phys. 2005, 7, 614.
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Schlegel, H. B. Biochemistry 2001, 40, 11905; (c) Njus, D.;
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Kytzia, A.; Korth, H. G.; Sustmann, R.; de Groot, H.;
ꢂ
ꢀ
ꢁ
ꢃ
Ka
Ka þ ½Hþꢂ
ꢀ
kobs
¼
ðkHA ꢀ kH A
Þ
þ KH A ½H2Aꢂo
2
2
ꢀ
where kHA and kH A are the second-order rate constants
2
for the reactions of the nitroso compound with ascorbate
and ascorbic acid, respectively, Ka is the first dissociation
3
constant of ascorbic acid, and since kHA is14g 10 greater
´
Kirsch, M. Chem. Eur. J. 2006, 12, 8786; (g) Ursˇic, S.;
ꢀ
´
Vrcˇek, V.; Ljubas, D.; Vinkovic, I. New J. Chem. 1998,
than kH A, the latter can, to a good approximation, be
2
neglected at H+ concentrations greater than 10ꢀ3 mol dmꢀ3
(P98% ascorbate reaction); (iii) the second-order rate
constants (k1, 2) calculated from the observed pseudo-
first-order rate constants using the above rate law are
221; (h) Mottley, C.; Kalyanaraman, B.; Mason, R. P.
´
´
FEBS Lett. 1981, 130, 12; (i) Brezova, V.; Tarabek, P.;
´
ˇ
ˇ
Dvoranova, D.; Stasko, A.; Biskupic, S. J. Photochem.
Photobiol. A 2003, 155, 179.

D. Vuina et al. / Tetrahedron Letters 48 (2007) 3633–3637
3637
invariably the same in the acidic range from 0.1 to
0.0002 mol dmꢀ3 of H+; (iv) the ratio kobs/[HAscꢀ] de-
creases steadily from 2.9 · 103 at 0.0002 mol dmꢀ3 of
H+ to 5.5 · 102 at pH 6; (v) the obtained ESR spectra
confirmed the formation of ascorbyl and phenylnitroxyl
radicals in the reaction, in line with early14h and the recent
ESR studies (for PhNHOÅ see also Ref. 14i; for AscÅꢀ see
Refs. 15a, pp 123–126 and 15b, Chapter 4).