Invention of the First Azo Compound To Serve as a Superoxide Thermal Source under Physiological Conditions: Concept, Synthesis, and Chemical Properties

Full text of DOI:10.1021/ja972886l

12364
J. Am. Chem. Soc. 1997, 119, 12364-12365
periods of time.16 Unfortunately, such methods do not mimic
the in ViVo situation where superoxide is produced very slowly
but continuously over the entire lifetime of a cell, and it should
be remembered that some cells (e.g., neurons) are as old as the
organism itself. Thus, current methods generate superoxide in
relatively high concentrations which will favor reaction 2, the
uncatalyzed bimolecular dismutation. The biological damage
due to superoxide itself may therefore be partly or wholly
disguised by damage due to H2O2 and the highly reactive HO•
radical.16
To overcome the potential problems inherent in earlier
methods, we have invented a procedure for generating super-
oxide at a known, slow and well-defined rate by the thermal
(or, if desired, photochemical) decomposition of a suitable
precursor. These superoxide thermal sources (SOTSs) are azo
compounds which decompose to yield, either directly or
indirectly, electron-rich, carbon-centered radicals, many of which
are known to react with dioxygen to yield carbocations and
superoxide.19
Invention of the First Azo Compound To Serve as a
Superoxide Thermal Source under Physiological
Conditions: Concept, Synthesis, and Chemical
Properties1
K. U. Ingold,* Thomas Paul,2a Mary Jane Young,2b and
Leanne Doiron2c
Steacie Institute for Molecular Sciences
National Research Council of Canada
Ottawa, Ontario, Canada K1A 0R6
ReceiVed August 18, 1997
In quantitative terms, the superoxide radical anion (O•2-) is
the most important radical formed in aerobic organisms.3
Indeed, a significant fraction (1-4%) of the oxygen metabolized
in the mitochondrial respiratory chain escapes complete 4-elec-
tron reduction to water after accepting the initial electron.
Superoxide is also formed in ViVo as a direct (or side)
consequence of various enzymatic processes4,5 and by the
autoxidation of a number of biologically significant compounds.6
Although superoxide can inactivate certain enzymes,7 it is very
unreactive in typical free radical reactions such as hydrogen
atom abstraction. However, its conjugate acid, the hydroperoxyl
radical (pKa ≈ 4.7), has a hydrogen-atom-abstracting ability
comparable to that of the alkylperoxyl radicals which are
responsible for lipid peroxidation.11 Superoxide and the reactive
or
The first of a novel family of compounds which meets these
stringent requirements is di(4-carboxybenzyl)hyponitrite, SOTS-
1, which was synthesized from R-bromotoluic acid by esteri-
fication, reaction with silver hyponitrite20 and de-esterification.21
The decomposition of SOTS-1 in water at physiological
temperature (37 °C) and pH 7 (where SOTS-1 is ionized) occurs
by the mechanism shown in Scheme 1.
The critical step in the overall reaction involves an unusual
1,2-H-atom shift22 (reaction 9) which converts the primary
alkoxyl radical formed from the hyponitrite into the desired
electron-rich carbon-centered radical, -O2CC6H4C4 HOH. This
oxygen species derived therefrom (HOO•, H2O2, and HO•) are
believed to be responsible for a host of pathological processes
ranging from simple inflammation, through atherosclerosis and
DNA damage, all the way to the very aging process itself.12,13
Indeed, aerobic life would not be possible without Nature’s
defenses against superoxide.14 These defenses are provided by
enzymes known as superoxide dismutases (SODs) which
catalyze the dismutation of superoxide to oxygen and hydrogen
peroxide (which is then dismutated by catalase, CAT, to water
and oxygen).
(13) Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and
Medicine, 2nd ed.; Clarendon Press: Oxford, U.K. 1989. OxidatiVe Stress;
Sies, H., Ed.; Academic: London, U.K. 1985. OxidatiVe Stress, Oxidants
and Antioxidants; Sies, H., Ed.; Academic: London, U.K. 1991.
(14) See, e.g., ref 5 and Longo, V. D.; Gralla, E. B.; Valentine, J. S. J.
Biol. Chem. 1996, 271, 12275-12280.
(15) E.g., by addition of organic solutions of KO2 and a crown ether to
the aqueous medium or by pulse radiolysis of formate solutions.
(16) The method most favored by biochemists is the traditional aerobic
X/XO system3,4 (or aerobic acetaldehyde (AA)/XO).17 However, at high
XO concentrations, the reaction is soon over because of a relatively rapid
depletion of the substrate. In some systems there may also be a deactivation
of the XO. In addition, commercial XO may be contaminated with iron
which can initiate hydroxyl radical chemistry by reaction with the H2O2
Various methods have been developed for generating super-
oxide for in Vitro and ex ViVo studies. These methods either
produce superoxide “instantaneously”15 or over relatively short
formed in reaction 2. As a consequence, any biological effects of O•2- can
be masked by effects due to the much more aggressive HO• radical. Heroic
efforts to remove XO-associated iron and to chelate any remaining iron
have been reported,17,18 but its complete absence in a particular XO
preparation is, of course, impossible to prove.
(1) Issued as NRCC No. 40846.
(2) (a) NRCC Research Associate. (b) NRCC Postdoctoral Fellow. (c)
AICR/NFCR Summer Student.
(3) The first report on superoxide in biology involved the demonstration
that the aerobic xanthine/xanthine oxidase (X/XO) system produced
4
(17) Dix, T. A.; Hess, K. M.; Medina, M. A.; Sullivan, R. W.; Tilly, S.
L.; Webb, T. L. L. Biochemistry 1996, 35, 4578-4583.
(18) Lloyd, R. V.; Mason, R. P. J. Biol. Chem. 1990, 265, 16733-16736.
Britigan, B. E.; Pou, S.; Rosen, G. M.; Lilleg, D. M.; Buettner, G. R. J.
Biol. Chem. 1990, 265, 17533-17538.
O•2-
. A 1995 review by Fridovich cites 54 (!) earlier reviews on the
biology of O•2- and the superoxide dismutase enzymes which remove it.5
(4) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1968, 243, 5753-5760.
(5) Fridovich, I. Ann. ReV. Biochem. 1995, 64, 97-112.
(6) E.g., ascorbate, thiols, sugars, ubiquinol, etc.
(19) von Sonntag, C.; Schuchmann, H.-P. Angew. Chem., Int. Ed. Engl.
1991, 30, 1229-1253.
(20) Ogle, C. A.; Martin, S. W.; Dziobak, M. P.; Urban, M. W.;
Mendenhall, G. D. J. Org. Chem. 1983, 48, 3728-3733.
(7) E.g., 6-phosphogluconate dehydratase,8 aconitase,9 and ribonucleotide
reductase10 (the rate-limiting enzyme for DNA synthesis).
(8) Gardner, P. R.; Fridovich, I. J. Biol. Chem. 1991, 266, 1478-1483.
(9) Gardner, P. R.; Fridovich, I. J. Biol. Chem. 1991, 266, 19328-19333.
Flint, D. H.; Tuminello, J. F.; Emptage, M. H. J. Biol. Chem. 1993, 268,
22369-22376. Hausladen, A.; Fridovich, I. J. Biol. Chem. 1994, 269,
29405-29408.
(21) Following standard procedures, R-bromotoluic acid was converted
to its acid chloride which was reacted with benzyl alcohol to produce the
benzyl ester which was then converted to the hyponitrite. Ester cleavage
was performed under aqueous basic conditions at 0 °C to yield SOTS-1:
1H NMR (600 MHz, Na phosphate buffer in D2O, 278 K, acetone as internal
standard) δ 7.90-7.86 (2H, m (AB system), arom H), 7.47-7.43 (2H, m
(AB system), arom H), 5.35 (2H, s, CH2); 13C NMR (150 MHz, Na
phosphate buffer in D2O, 278 K, acetone as internal standard) δ 176.3
(COOH), 139.5 and 137.1 (arom C-C), 130.0 and 129.3 (arom C-H),
75.7 (CH2). Full details of the synthesis will be published later.
(22) Gilbert, B. C.; Holmes, R. G. G.; Laue, H. A. H.; Norman, R. O.
C. J. Chem. Soc., Perkin Trans. 2 1976, 1047-1052.
(10) Gaudu, P.; Nivie`re, V.; Pe´tillot, Y.; Kaupi, B.; Fontecave, M. FEBS
Lett. 1996, 387, 137-140.
(11) Zaikov, G. E.; Howard, J. A.; Ingold, K. U. Can. J. Chem. 1969,
47, 3017-3029. Korcek, S.; Chenier, J. H. B.; Howard, J. A.; Ingold, K.
U. Can. J. Chem. 1972, 50, 2285-2297.
(12) See references cited in ref. 5.
S0002-7863(97)02886-2 CCC: $14.00 Published 1997 by the American Chemical Society

Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 50, 1997 12365
Scheme 1
Further confirmation of the chemistry shown in Scheme 1
was obtained by 266 nm laser flash photolysis (LFP). Esters
(ethyl, tert-butyl) of SOTS-1 in acetonitrile subjected to LFP
showed the expected29 absorption in the visible at 460 nm which
is not quenched by O2 and is due to the benzyloxyl radicals,
ROC(O)C6H4CH2O•. By way of contrast, in the absence of O2
LFP of SOTS-1 in phosphate buffer, pH 7.6 (and in acetonitrile/
water) showed no sign of the benzyloxyl radical but instead
gave an instantaneous absorption at 320 nm with a shoulder at
360 nm and broad, weaker absorption centered at 515 nm. The
320 nm absorption disappears upon LFP at pH 12 in the ab-
sence of O2 leaving behind the 360 and 515 nm absorptions
(ratio of O.D.’s, 4:1). The 320 nm absorption is obviously due
to the ketyl radical, -O2CC6H4C4 HOH, and the 360 and 515
nm absorptions are due to the corresponding anion radical,
-O2CC6H4C4 HO-. We estimate that these two species are
present in approximately equal concentrations at pH 7.6. All
three of these absorptions are quenched by O2, implying that
they do, indeed, arise from carbon-centered radicals.
Seeking final confirmation that SOTS-1 did indeed generate
superoxide, we examined the effect of (Cu/Zn) SOD and catalase
on reaction 12. As would be expected, the initial rate of growth
of the 350 nm absorption due to -C(NO2)3 at 37 °C was
unaffected by catalase (cf., reaction 3). However, and to our
dismay in view of pioneering studies of McCord and Fridovich
on O•2-/C(NO2)4/SOD,26 the initial rate of growth of this
absorption was completely unaffected by the addition of SOD
(cf., reaction 2). Our immediate presumption was that SOTS-1
did not yield superoxide. This presumption was eventually
demonstrated to be unfounded because the AA/XO induced
growth of the -C(NO2)3 absorption was also unaffected by
SOD26b (or catalase). Furthermore, the SOTS-1 induced growth
of the reduced cytochrome c absorption (reaction 13) was,
indeed, inhibited by SOD in a dose-dependent manner26b (and
was unaffected by catalase).
In summary, the invention of SOTS-1 provides a unique and
novel tool for investigating superoxide-dependent oxidative
stress under well-defined, metal ion free, biomimetic conditions.
We are currently comparing the effect of equal fluxes of
superoxide from SOTS-1 and the aerobic XO/AA couple on
strand scission of Escherichia coli plasmid DNA (both super-
coiled and a short linear restriction fragment). We are also
investigating the effect of superoxide on certain cell lines in
culture by adding SOTS-1 to the culture medium, by injecting
it directly into the cells and (hopefully) by having it irreversibly
transported into the cells by adding it as its methyl ester in
DMSO to the medium. The ability of SOTS-1 to initiate the
peroxidation of lipids, including lipoproteins, dispersed in water
is also under study. We believe that SOTS-1 (and other SOTS)
will provide new insights into the role of superoxide in
biological systems.
class of radical rearrangements only occurs in the presence of
water (or alcohols).
The solubility of SOTS-1 in water at pH 7 is ca. 1.5 mM at
room temperature. It decays with first-order kinetics, and its
half-life in water at physiological pH (6.5-8) and temperature
(37 °C) was found to ca. 4900 s by three independent
procedures:23 (i) H NMR (in D2O in the presence of O2, the
1
only products detected were the expected (Scheme 1) alcohol
and aldehyde which were formed in yields of roughly 40 and
60%, respectively); (ii) spectrophotometry at 350 nm using
tetranitromethane;19,25,26a (iii) spectrophotometry at 550 nm using
FeIII cytochrome c.26a Methods ii and iii depend on the
formation of O•2- and have a simple 1:1 stoichiometry, reac-
tions 12 and 13.
These two reactions therefore provide a method for determin-
ing the molar efficiency, 2e,27 of superoxide formation from
SOTS-1 (i.e., 2e ) moles of O•2-/mole of SOTS-1) since the
extinction coefficients of the absorbing species are known, viz.,
25b
ꢀ350 ) 15 000 M-1 cm-1 for -C(NO2)3 and ꢀ550 (reduced-
oxidized) ) 21 000 M-1 cm-1 for cytochrome c.28 Comparison
of the initial rates (first 5-10 min) of growth of these
absorptions with the measured initial rate of SOTS-1 decom-
position gave 2e ) 0.46 ( 0.10 and 0.32 ( 0.06 for -C(NO2)3
and cytochrome c, respectively, while the maximum absorbance
reached after ca. 6 h gave 2e ) 0.42 ( 0.006 and 0.44 ( 0.02,
respectively. We conclude that 2e ) 0.41 which is ca. 20% of
its theoretical maximum value (in the absence of cage effects)
of 2.0.
(23) The O•2--induced absorption at 530 nm using nitro blue tetrazo-
lium24 was useful only in a diagnostic sense. With both SOTS-1 and XO/
AA superoxide sources, this absorption grows in for only a few minutes,
perhaps because the absorbing species is insoluble.
(24) Beauchamp, C.; Fridovich, I. Anal. Biochem. 1971, 44, 276-287.
Bielski, B. H. J.; Shiue, G. G.; Bajuk, S. J. Phys. Chem. 1980, 84, 830-
833.
Acknowledgment. This paper is dedicated to the father of super-
oxide in biology, Professor Irwin Fridovich, on the occasion of his
attainment of emeritus status. This work would not have been
undertaken nor the goal pursued for several years without the continued
and generous support of the Association for International Cancer
Research and the National Foundation for Cancer Research. T. Paul
thanks the Alexander von Humboldt Foundation for a Feodor Lynen
Fellowship. We sincerely thank the following friends and colleagues
for the contributions indicated: S. J. Garden (exploratory syntheses);
S. Lin (synthesis); J. P. MacManus (DNA and cell culture); I. Rasquinha
(DNA); I. Hill and H. de Groot (cell culture).
(25) (a) Rabani, J.; Mulac, W. A.; Matheson, M. S. J. Phys. Chem. 1965,
69, 53-70. (b) Bielski, B. H. J.; Allen, A. O. J. Phys. Chem. 1967, 71,
4544-4549.
(26) (a) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244, 6049-
6055. (b) In ref 26a, a solution of O•2- in dimethylformamide was infused
into aqueous C(NO2)4 at 25 °C, and the growth of the 350 nm absorption
was shown to be retarded by SOD. The critical difference between the
McCord and Fridovich experiment with C(NO2)4/SOD and our own using
both SOTS-1 and the XO/AA couple has not yet been explored. However,
Professor Fridovich has kindly suggested that it relates to the fact that we
were unable in our experiments to add sufficient SOD to compete with the
very fast O•2-/C(NO2)4 reaction. This problem does not occur in the
cytochrome c experiments because its reaction with O•2- is very much
slower.
JA972886L
(27) The efficiency of escape of the initial radical pair from the solvent
cage is e ()k8/(k7 + k8)).
(28) Massey, V. Biochim. Biophys. Acta 1959, 34, 255-256.
(29) Avila, D. V.; Ingold, K. U.; Di Nardo, A. A.; Zerbetto, F.; Zgierski,
M. Z.; Lusztyk, J. J. Am. Chem. Soc. 1995, 117, 2711-2718.