TEMPO reacts with oxygen-centered radicals under acidic conditions

Article abstract of DOI:10.1039/c0cc00547a

In the presence of organic acids in organic media, 2,2,6,6- tetramethylpiperidine-N-oxyl (TEMPO) reacts with peroxyl radicals at nearly diffusion-controlled rates by proton-coupled electron transfer from the protonated nitroxide. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/c0cc00547a

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TEMPO reacts with oxygen-centered radicals under acidic conditionsw
Riccardo Amorati,a Gian Franco Pedulli,a Derek A. Pratt*b and Luca Valgimigli*a
Received 23rd March 2010, Accepted 7th May 2010
First published as an Advance Article on the web 11th June 2010
DOI: 10.1039/c0cc00547a
In the presence of organic acids in organic media, 2,2,6,6-
tetramethylpiperidine-N-oxyl (TEMPO) reacts with peroxyl
radicals at nearly diffusion-controlled rates by proton-coupled
electron transfer from the protonated nitroxide.
antioxidants with peroxyl radicals, formal H-atom transfer
processes, are highly medium-dependent. We wondered
whether medium effects could account for the spurious
antioxidant activity of nitroxides. Herein we report the results
of our investigations, which demonstrate that nitroxides are
among the most effective antioxidants, but only under acidic
conditions.
2,2,6,6-Tetramethylpiperidine-N-oxyl (TEMPO) is arguably
the most famous odd-electron organic species. It was first
isolated as a crystalline orange solid by Lebedev and
Kazarnovskii in 1960,1 and the characteristic three-line EPR
We first set out to establish a baseline with which to
compare medium effects, and found that when TEMPO
(5–100 mM) was used as an inhibitor in the AIBN-initiated
autoxidation of styrene (in MeCN or chlorobenzene),15,16 only
a modest retarding of the rate of autoxidation was observed,
which was consistent with previous reports.12 Interestingly,
when we added trifluoroacetic acid (TFA) to the autoxidation
mixture (in MeCN, see Fig. 1A), a very well-defined inhibition
spectrum was observed by Il’yasov2 and Rassat’s group3
a
short while later. Its persistence makes TEMPO and related
nitroxides particularly versatile, e.g. as catalysts for controlled
radical polymerization reactions,4 spin labels,5 radical
probes,6 or catalysts for the controlled oxidation of primary
alcohols.7 Being one electron away from hydroxylamines and
oxoammonium ions (Scheme 1), nitroxides have a privileged
redox position that is responsible for their role as oxidation
catalysts. Paradoxically, the same chemistry is also the basis
for the superoxide dismutase mimetic activity of TEMPO.8
Over the past two decades, considerable interest has
emerged in the biological activities of nitroxides, which have
been reported to range from neuroprotective to anticancer.9
Many of these properties have been ascribed to the anti-
oxidant activity of nitroxides, but the mechanistic rationale
behind it is far from clear.10 While it is quite clear that
TEMPO and its analogs react readily with alkyl radicals
period was observed, and the inhibition rate constant (kinh
)
derived from the slope of the inhibited rate of oxidation16
was proportional to the concentration of the acid (Fig. 1B).
In fact, the addition of TFA yielded values of kinh as high as
2.2 Â 107 MÀ1 sÀ1 (when 0.1 M TFA was included) thus
outperforming the reference antioxidant, a-tocopherol, by
more than 30-fold (kinh = 6.8 Â 105 MÀ1 sÀ1 in MeCN15).
Qualitatively similar results were obtained by adding different
acids (Table 1), including the weaker acetic acid or the
stronger p-toluenesulfonic acid (p-TSA). In fact, the addition
of p-TSA, at a concentration of 10 mM, resulted in the
essentially diffusion-controlled quenching of peroxyl radicals,
(1 Â 108–1 Â 109 MÀ1
s
À1),11 they have been found to be quite
with kinh = 1.8 Â 108 MÀ1
s
À1, equalling the performance of
unreactive toward peroxyl radicals.12 Since peroxyl radicals are
the predominant chain-carrying species under atmospheric
pressures of O2, owing to the diffusion-controlled reactions of
the most effective chain-breaking antioxidants known to
date.17 Extension of the investigation to a series of organic
acids of different strengths showed that kinh is clearly dependent
on the pKa of the acid (see Supporting Informationw). Acids
themselves (without TEMPO) did not affect the oxidizability
of styrene, and we obtained similar results when cumene was
employed as the oxidizable substrate.
alkyl radicals with O2 (2–5 Â 109 MÀ1
s
À1),13 nitroxides should
be useless as antioxidants under most conditions.
Recent work by Ingold and co-workers,14 as well as some
of our own efforts,15 has shown that reactions of phenolic
Scheme 1 Redox behaviour of TEMPO.
a Department of Organic Chemistry ‘‘A. Mangini’’, University of
Bologna, Via San Giacomo 11, 40126, Bologna, Italy.
E-mail: luca.valgimigli@unibo.it; Fax: +39 051 2095688;
Tel: +39 051 2095683
b Department of Chemistry, Queen’s University, 90 Bader Crescent
Lane, Kingston, Ontario, Canada K7L 3N6.
E-mail: pratt@chem.queensu.ca; Fax: +1 613 533 6669;
Tel: +1 613 533 3287
w Electronic supplementary information (ESI) available: Cartesian
coordinates, electronic energies and thermochemical corrections for
all relevant structures. See DOI: 10.1039/c0cc00547a
Fig. 1 (A) Oxygen uptake kinetics during the autoxidation of 4.3 M
styrene in MeCN initiated by 0.05 M AIBN at 303 K inhibited by
12.5 mM TEMPO with variable amounts of trifluoroacetic acid, and
(B) dependence of the measured inhibition rate constant (kinh) on the
concentration of acid.
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Table 1 Rate constants for the reaction of peroxyl radicals with
TEMPO obtained from inhibited autoxidations of styrene at 303 K in
the presence of various organic acids
PhCl
kinhMeCN(MÀ1 sÀ1) kinhMeCN(MÀ1
s
À1)/
kinh
nc kinh
/
MeCN d
Acida pKa [Ac] = 4.3 mM
[Acid (M)]
b
Scheme 2 Proposed mechanism for the reaction of TEMPO with
peroxyl radicals under acidic conditions.23
AA
BA
23.5 2.6 Â 105
3.1 Â 107
>1 1.2
>1 —
21.5 3.8 Â 105
—
—
(pKa of TEMPOH+ꢁ is À5.8 in water),20 may yield an effective
H-atom donor (Scheme 2). In fact, in a preliminary set of
calculations carried out using the CBS-QB3 approach,21 we
found that the O–H bond dissociation enthalpies (BDEs) of
protonated nitroxides are very low—even lower than those in
equivalently substituted hydroxylamines, which are commonly
thought of as having the weakest O–H bonds of any closed
shell molecule. For example, while N,N-di-tert-butylhydroxy-
DCA 13.2 5.6 Â 105
TFA 12.7 1.0 Â 106
TCA 10.8 9.6 Â 105
p-TSA 8.0 7.0 Â 106
1
1
1
1
—
2.9
—
—
7.0 Â 108
—
1.4 Â 1010
a
AA = acetic acid; BA = benzoic acid; DCA = dichloroacetic
acid; TFA = trifluoroacetic acid; TCA = trichloroacetic acid;
b
p-toluenesulfonic acid. Measured in MeCN from
p-TSA
ref. 19. Stoichiometric coefficient = moles of peroxyl radicals
d
trapped by one mole of TEMPO. Kinetic solvent effect.
=
c
lamine has a calculated O–H BDE of 69.0 kcal molÀ1 22
O–H BDE calculated for the corresponding protonated
N,N-di-tert-butyl nitroxide is 58.2 kcal molÀ1
,
the
.
On changing the solvent from the relatively polar, H-bond
accepting (HBA) solvent MeCN to the relatively non-polar,
non-H-bond accepting chlorobenzene, the effect was maintained
and, interestingly, the inhibition rate constants became even
larger. This type of kinetic solvent effect (KSE) is typical of
phenolic antioxidants, since in HBA solvents, the phenolic
H-atom is H-bonded to the solvent, making it unavailable for
abstraction by the peroxyl radical.14 This finding was particularly
intriguing since TEMPO itself does not possess an acidic
hydrogen, or for that matter, any abstractable hydrogen. A
similarly surprising result arose when we measured the isotope
effect on the kinetics of the reaction. When parallel sets of
autoxidations inhibited by 12.5 mM TEMPO were run in
MeCN to which either 4.4 mM CD3COOD/CH3COOH or
3.3 mM CF3COOD/CF3COOH was added,18 deuterium
kinetic isotope effects (DKIEs), kH/kD, of 2.4 and 2.2,
respectively, were obtained. These values happen to be quite
close to the values reported for the reaction of peroxyl radicals
with structurally related hydroxylamines (kH/kD = 2–3).12
Since TEMPO is known to disproportionate in acidic media
to yield TEMPO-H and the corresponding oxoammonium ion
(TEMPOnium),7,20 we figured that the DKIE values and
solvent effects could be explained by the intervention of
TEMPO-H as the antioxidant. In fact, the structurally related
4-oxo-TEMPO-H has been reported to react rapidly with
Although it is gratifying that the thermodynamics are quite
reasonable (the reaction enthalpy would be B30 kcal molÀ1
exothermic, since the O–H BDE in hydroperoxides are ca.
88 kcal molÀ1),24 it is not obvious that the kinetics would be.
Therefore, we next computed the transition state (TS) structure
corresponding to the transfer of the H-atom from a protonated
nitroxide to a peroxyl radical.25 The TS structure (Fig. 2A and B)
is characterized by a co-planar arrangement of all of the
heteroatoms and the H-atom being transferred. This geometry
is consistent with a proton-coupled electron transfer (PCET)26
between these two species: with proton transfer taking place
between the lone pairs of the terminal oxygen atoms of the
nitroxide and peroxyl, which are separated by only 2.46 A
(the highest doubly-occupied MO is shown in Fig. 2C), and
electron transfer across the 2p orbitals of the nitroxide and
peroxyl SOMOs which are orthogonal to the molecular
framework (one of the two singly occupied MOs is shown in
peroxyl radicals (kinh = 5.0 Â 105 MÀ1
s
À1).12 In our hands,
rate constants of kinh = 2.4 Â 106 MÀ1 sÀ1 and 3.0 Â 106 MÀ1 sÀ1
were measured at 303 K for TEMPO-H in MeCN and
chlorobenzene, respectively (kinhPhCl/kinh
MeCN
= 1.3), with a
stoichiometric coefficient (n) of 1. No acid catalysis was
observed for the reaction of TEMPO-H with peroxyl radicals.
While this result confirms that TEMPO-H is an effective
antioxidant, it cannot account for the much greater reactivity
of TEMPO under acidic conditions. When we monitored
TEMPO disproportionation by EPR under the conditions of
our inhibited autoxidations, we found that the kinetics were
much too slow to be relevant (see Supporting Information for
further detailsw). In fact, with the stronger acids, less than 1%
of TEMPO would be converted to TEMPO-H during the
inhibited period of the autoxidations (B2000 s).
Fig. 2 (A,B) Transition state structure for the formal H-atom
transfer from a protonated nitroxide to a peroxyl radical, and (C,D)
its corresponding highest occupied molecular orbitals. (E) Transition
state structure for the formal H-atom transfer from the equivalently-
substituted hydroxylamine to a peroxyl radical, and its corresponding
(F) highest doubly-occupied molecular orbital, for comparison.
We next considered the possibility that protonation of the
nitroxide under the reaction conditions, although unfavourable
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Fig. 2D).27 This structure lies 23.3 kcal molÀ1 lower in
enthalpy than the separated reactants, and is connected to
4 E. Saldivar-Guerra, J. Bonilla, G. Zacahua and M. Albores-
Velasco, J. Polym. Sci., Part A: Polym. Chem., 2006, 44, 6962.
5 P. P. Borbat, A. J. Costa-Ficho, K. A. Earle, J. K. Moscisnki and
J. H. Freed, Science, 2001, 291, 266.
them via
a H-bonded pre-reaction complex which lies
27.9 kcal molÀ1 beneath them. In contrast, the lowest energy
TS structure for H-atom transfer between the equivalently-
substituted hydroxylamine and peroxyl radical (Fig. 2E)
shows a cyclic PCET process,28 due to the exchange of the
electron between the nominally doubly-occupied lone pair of
the hydroxylamine N-atom and the peroxyl SOMO as the
proton is passed between the terminal oxygen atoms. This TS
structure lies 4.2 kcal molÀ1 above the separated reactants,
and is connected to them by a H-bonded pre-reaction complex
that lies 3.5 kcal molÀ1 beneath them. Therefore, while theory
predicts a modest barrier for the reaction of the hydroxylamine
with the peroxyl, it predicts a diffusion-controlled reaction
between the protonated nitroxide and the peroxyl, consistent
with the foregoing experimental findings.
6 (a) L. Valgimigli, G. F. Pedulli and M. Paolini, Free Radical Biol.
Med., 2001, 31, 708; (b) C. Gentilini, P. Franchi, E. Mileo,
S. Polizzi, M. Lucarini and L. Pasquato, Angew. Chem., Int. Ed.,
2009, 48, 3060.
7 (a) G.-J. ten Brink, I. W. C. E. Arends and R. A. Sheldon, Science,
2000, 287, 1636; (b) R. A. Sheldon, I. W. C. E. Arends, G.-J. ten
Brink and A. Dijksman, Acc. Chem. Res., 2002, 35, 774;
(c) F. Minisci, F. Recupero, A. Cecchetto, C. Gambarotti,
C. Punta, R. Faletti, R. Paganelli and G. F. Pedulli, Eur. J. Org.
Chem., 2004, 109.
8 S. Goldstein, G. Merenyi, A. Russo and A. Samuni, J. Am. Chem.
Soc., 2003, 125, 789.
9 See, e.g., (a) S. M. Vaz and O. Augusto, Proc. Natl. Acad. Sci. U.
S. A., 2008, 105, 8191; (b) E. Venditti, A. Scire, F. Tanfani,
L. Greci and E. Damiani, Biochim. Biophys. Acta, Gen. Subj.,
2008, 1780, 58.
10 B. P. Soule, F. Hyodo, K. Matsumoto, N. L. Simone, J. A. Cook,
M. C. Krishna and J. B. Mitchell, Free Radical Biol. Med., 2007,
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11 See, e.g., J. Sobek, R. Martschke and H. Fischer, J. Am. Chem.
Soc., 2001, 123, 2849.
12 (a) I. T. Brownlie and K. U. Ingold, Can. J. Chem., 1967, 45, 2427;
(b) J. Lalevee, X. Allonas, J.-P. Fouassier and K. U. Ingold, J. Org.
Chem., 2008, 73, 6489.
13 B. Maillard, K. U. Ingold and J. C. Scaiano, J. Am. Chem. Soc.,
1983, 105, 5095.
An important point that remains to be clarified is the
unusually large stoichiometric factor observed with the weaker
acids we have examined. Indeed, when either acetic or benzoic
acids were used as a proton source in the TEMPO-inhibited
autoxidations, an apparently infinite inhibited period was
observed. Furthermore, when the concentration of TEMPO
was monitored in these reactions, there was nearly no decay
of the EPR spectrum of the nitroxide (see Supporting
Informationw). This result, which stands in stark contrast with
the consumption of TEMPO by the end of the ca. 2000 s
inhibited period of autoxidations in the presence of the
stronger acids, implies that TEMPO is regenerated from
TEMPOnium when the weaker acids are used as proton
source. We are currently investigating this exciting and
unexpected feature further.
14 G. Litwinienko and K. U. Ingold, Acc. Chem. Res., 2007, 40, 222.
See also: M. Jha and D. A. Pratt, Chem. Commun., 2008, 1252.
15 L. Valgimigli, R. Amorati, S. Petrucci, G. F. Pedulli, D. Hu,
J. J. Hanthorn and D. A. Pratt, Angew. Chem., Int. Ed., 2009,
48, 8348.
16 (a) G. W. Burton, T. Doba, E. J. Gabe, L. Hughes, F. Lee,
L. Prasad and K. U. Ingold, J. Am. Chem. Soc., 1985, 107, 7053;
(b) R. Amorati, G. F. Pedulli, L. Valgimigli, O. A. Attanasi,
P. Filippone, C. Fiorucci and R. Saladino, J. Chem. Soc., Perkin
Trans. 2, 2001, 2142.
17 M. Wijtmans, D. A. Pratt, L. Valgimigli, G. A. DiLabio, G. F. Pedulli
and N. A. Porter, Angew. Chem., Int. Ed., 2003, 42, 4370.
18 1% D2O/H2O were included in these reactions.
We believe the foregoing observations to have very important
implications in both health and industry. In biological
systems, various environments within the cell have acidic
functionalities that can provide a proton to unleash the
antioxidant activity of TEMPO. For example, lipid-derived
peroxyl radicals can be expected to be trapped by TEMPO
when in proximity to acidic sidechains of transmembrane
proteins of the lipid bilayer or apoproteins of circulating
lipoproteins. These reactions may very well underlie the
biological activities of nitroxides, and may help in the design
of experiments aimed at deconvoluting the purported neuro-
protective and anticancer roles of nitroxides.
19 A. Kutt, I. Leito, I. Kaljurand, L. Soovali, V. M. Vlasov,
¨
¨
L. M. Yagupolskii and I. A. Koppel, J. Org. Chem., 2006, 71, 2829.
20 V. D. Sen and V. A. Golubev, J. Phys. Org. Chem., 2009, 22, 138.
21 J. A. Montgomery, Jr., J. W. Ochterski and G. A. Petersson,
J. Chem. Phys., 1994, 101, 5900.
22 Compare with the experimental value of 68.2 kcal molÀ1
(F. G. Bordwell and W.-Z. Liu, J. Am. Chem. Soc., 1996, 118,
10819). The O–H BDE of TEMPO-H has been determined to be
69.7 kcal molÀ1 (L. R. Mahoney, G. D. Mendenhall and
K. U. Ingold, J. Am. Chem. Soc., 1973, 95, 8610).
23 While we could not observe the very minor amount of protonated
nitroxide expected at equilibrium, EPR spectra of TEMPOH+
ꢁ
have been previously reported in concentrated acids. See:
V. Malatesta and K. U. Ingold, J. Am. Chem. Soc., 1973, 95, 6404.
24 J. Berkowitz, G. B. Ellison and D. Gutman, J. Phys. Chem., 1994,
98, 2744–2765.
25 In order to make the computations tractable at a reliable level of
theory, the model nitroxide bears only methyl groups. The O–H
BDE calculated for the protonated form of this nitroxide is
61.3 kcal molÀ1, again, substantially lower than that calculated for
the corresponding hydroxylamine (74.0 kcal molÀ1). Likewise,
iso-propylperoxyl is used as a model peroxyl radical for the secondary
chain-carrying peroxyl radicals in the styrene autoxidations.
26 J. M. Mayer, D. A. Hrovat, J. L. Thomas and W. T. Borden,
J. Am. Chem. Soc., 2002, 124, 11142.
Hindered amine light stabilizers (HALS) are derivatives of
2,2,6,6-tetramethylpiperidine and are extremely efficient stabilizers
against light-induced degradation of most polymers. Nitroxides
are observed in these reactions, and have a key role in trapping
alkyl radicals.29 Since organic acids are often generated during
the photo-induced polymer degradation, our results suggest
they may catalyze additional reactions of the nitroxides with
peroxyl radicals, and also work to regenerate them.
27 While this system is nominally a singlet, it is necessary to perform
these calculations within a spin-unrestricted formalism since
Notes and references
restricted solutions are unstable with respect to orbital rotations.
28 G. A. DiLabio and K. U. Ingold, J. Am. Chem. Soc., 2005, 127, 6693.
29 (a) E. N. Step, N. J. Turro, M. E Gande and P. P. Kelmchuk,
Macromolecules, 1994, 27, 2529; (b) M. Lucarini and G. F. Pedulli,
Angew. Makromol. Chem., 1997, 252, 179.
1 O. L. Lebedev and S. N. Kazarnovskii, Zh. Obshch. Khim., 1960,
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2 A. V. Il’yasov, Zh. Strukt. Khim., 1962, 3, 95.
3 R. Briere, H. Lemaire and A. Rassat, Tetrahedron Lett., 1964, 5,
1775.
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This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 5139–5141 | 5141