Hydrogen peroxide triggered prochelator activation, subsequent metal chelation, and attenuation of the fenton reaction

Article abstract of DOI:10.1002/anie.200604859

(Chemical Equation Presented) Fenton antidote: The prochelator 2-boronobenzaldehyde isonicotinoyl hydrazone (SIH-B) reacts with H ₂O₂ to yield the active chelator salicylaldehyde isonicotinoyl hydrazone (SIH), which sequesters copper

Full text of DOI:10.1002/anie.200604859

Communications
DOI: 10.1002/anie.200604859
Bioinorganic Chemistry
Hydrogen Peroxide Triggered Prochelator Activation, Subsequent
Metal Chelation, and Attenuation of the Fenton Reaction**
Yibin Wei and Maolin Guo*
Iron and copper are redox-active metals essential for life.[1,2]
Hydrogen peroxide (H2O2) is also essential for cellular
activities[3,4] and has recently been identified as a second
messenger.[3] However, iron, copper, and H2O2 are all
“double-edged swords” because they can be extremely toxic
to cells at high concentrations. The chemical nature of this
toxicity is largely a result of the Fenton reaction [Eq. (1)],[5]
amine and clioquinol, have shown promise in AD/PD treat-
ment.[9–11] However, these chelators have troublesome draw-
backs, such as accession of intracellular iron pools or
prooxidant effects.[12,13] Their high metal affinities and
unregulated chelating properties may disrupt healthy metal
homeostasis by depleting the essential cellular labile iron
pool, thus removing metal from enzymes that rely on Fe (or
Cu) and disrupting the levels of other metal ions, such as zinc
and calcium.
FeIIðCuIÞ þ H2O2 ! FeIIIðCuIIÞ þ HOꢀ þ HOC
ð1Þ
To develop novel agents capable of attenuating the
Fenton reaction while also overcoming the drawbacks of the
uncontrolled chelators, we have synthesized prochelators that
cannot chelate metal ions by themselves but can be activated
by H2O2, with subsequent attenuation of the Fenton reaction
(Scheme 1). As the process involves consumption of H2O2,
sequestration of metal, and prevention of the production of
which generates highly deleterious hydroxyl radicals (HOC,
half-life ꢁ 1 ns). Cellular reductants, such as hydroascorbate
(AscHꢀ) and nicotinamide adenine dinucleotide (NADH),
can recycle FeIII (or CuII) back to FeII (or CuI) [Eq. (2)], thus
making the Fenton reaction catalytic when excess H2O2 is
available.
FeIIIðCuIIÞ þ AscHꢀ ! FeIIðCuIÞ þ AscCꢀ þ Hþ
ð2Þ
The occurrence of the Fenton reaction in living cells has
long been speculated on and has recently been unambigu-
ously detected by EPR spin-trap techniques.[4] It has been
revealed that iron and copper are accumulated in the brain
with age and are concentrated in certain areas of the brain in
patients with neurodegenerative diseases, such as Parkinsonꢀs
disease (PD) and Alzheimerꢀs disease (AD).[6] Moreover, the
production of H2O2 is significantly elevated and its elimina-
tion mechanisms become impaired in patients with neuro-
degenerative diseases.[7] The Fenton reaction has been
implicated as a cause of the aging process and contributes
to the pathogenesis of PD and AD,[7,8] supported by the
observation of oxygen-radical-triggered brain damage in PD/
AD patients.[9] Metal-chelating agents, such as desferriox-
Scheme 1. “Anti-Fenton reaction”.
[*] Y. Wei, Prof. Dr. M. Guo
hydroxyl radicals, we may tentatively call it an “anti-Fenton
reaction”. Our ultimate goal is to develop agents that have
minimal interference with iron (or other redox metals) under
healthy conditions, but which chelate metals and attenuate
the Fenton reaction at toxic levels of H2O2 or other reactive
oxygen species (ROS).
Herein, we report our first prochelator 2-boronobenzal-
dehyde isonicotinoyl hydrazone (SIH-B), which may be
considered as a derivative of salicylaldehyde isonicotinoyl
hydrazone (SIH). SIH has been widely investigated as one of
the orally effective tridentate iron chelators.[14] In neutral
aqueous media, SIH binds strongly to both FeIII and FeII with
the formation of Fe(SIH) and Fe(SIH)2 complexes, in which
Department of Chemistry and Biochemistry
University of Massachusetts
Dartmouth, MA 02747 (USA)
Fax: (+1)508-999-9167
E-mail: mguo@umassd.edu
Homepage: http://www.umassd.edu/cas/chemistry/guo/
guoresearch.cfm
[**] We thank the American Parkinson Disease Association, National
Parkinson Foundation, and University of Massachusetts Dartmouth
for their support of this work. This publication or project was made
possible by grant number 1 R21 AT002743-01A2 from the National
Center for Complementary and Alternative Medicine (NCCAM). Its
contents are solely the responsibility of the authors and do not
necessarily represent the official views of the NCCAM, or the
National Institutes of Health.
coordination to Fe occurs through the coplanar tridentate
ꢀ
ꢀ
=
donors: the phenolic group C6H4 O , the C O group, and the
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
[15,16]
ꢀ
NH N group of the hydrazone.
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As shown in Scheme 1, the structural difference between
the prochelator SIH-B and the chelator SIH is that the phenol
oxygen in SIH, a key chelating atom, is replaced by a boronic
acid group in the prochelator SIH-B. The hypothesis is that
the steric hindrance and poor donor property of the boronic
acid group may cause SIH-B to be a poor chelator. The
protecting boronic acid group in SIH-B may be sensitive to
H2O2, and thus may be converted to a phenol group by H2O2,
that is, be “activated” to produce the active chelator SIH.
SIH is synthesized by the Schiff base condensation
reaction of isoniozid with 2-formylphenol, as described
previously.[15] SIH-B is synthesized similarly but with 2-
formylphenylboronic acid instead of 2-formylphenol (see
Supporting Information). As a result of the marked differ-
ences in the NMR and UV/Vis characteristics of SIH and
SIH-B (Figures S1 and S2 in the Supporting Information),
1H NMR and UV/Vis experiments were carried out to probe
the reaction of SIH-B and H2O2. As shown in Figure 1, upon
Next, we investigated whether SIH-B can chelate iron (or
copper) under physiologically relevant conditions and if the
chelation can be triggered by H2O2. As controls, we also
monitored the coordination of SIH with FeIII or CuII under
similar conditions. Titration of FeIII or CuII into SIH solution
immediately produces new broad absorption bands in the
visible region (360–500 nm) (Figures S4 and S5 in the
Supporting Information), which match those reported pre-
viously.[16,18] These bands were assigned to O!metal charge-
transfer (CT) absorption and internal ligand transitions, thus
suggesting the formation of specific SIH–metal complexes.
However, when similar titration experiments were carried out
with the prochelator SIH-B (Figure S6 in the Supporting
Information), no absorption band was observed in the visible
region when FeIII or CuII was titrated. The absence of ligand–
metal CT (LMCT) absorption suggests little or no interaction
between the prochelator SIH-B with FeIII or CuII under the
conditions applied.
Interestingly, upon addition of H2O2 to the SIH-B/FeIII or
SIH-B/CuII mixture, characteristic LMCT bands (Figure 2)
emerged and increased in intensity over 40 and 20 min,
respectively. The spectroscopic changes are consistent with
the formation of SIH–FeIII or SIH–CuII complexes, thus
Figure 1. 1H NMR spectra (in [D4]MeOH) of a) SIH (1 mm), the
reaction of SIH-B (1 mm) with H2O2 (10 mm) after b) 6 and c) 1 h at
293 K, and d) SIH-B (1 mm).
1
addition of H2O2 to SIH-B in methanol, the H NMR peaks
=
for SIH-B [d = 8.45 (s; -CH N-), 7.63(t), 7.37–7.45 ppm (m;
aromatic ring)] gradually decreased in intensity while the
=
peaks corresponding to SIH [d = 8.55 (s; -CH N-), 7.48 (d),
7.35 (t), 6.94 ppm (m; aromatic ring)] appeared simultane-
ously and increased in intensity with time. Other peaks [d =
8.75 (d) and 7.88 ppm (d; pyridine ring)] also underwent
similar conversions but with very small changes in chemical
shifts. In about 2 h, SIH-B was cleanly converted to SIH with
no intermediate formed, as indicated by the 1H NMR spectra.
The reaction was also followed by UV/Vis difference
spectroscopy (Figure S3in the Supporting Information) in
N,N-dimethylformamide (DMF)/potassium phosphate buffer
(KPB; 20 mm, pH 7.2; 1:1 v/v). With tenfold excess H2O2, the
conversion reaction is pseudo-first order with an apparent
rate constant (kobs) of 1.33 ” 10ꢀ3 sꢀ1, comparable to that of a
boronic ester analogue.[17] These results demonstrate that
SIH-B can be cleanly converted to SIH by H2O2 under
physiological pH conditions.
Figure 2. UV/Vis spectra of the time course of the reactions after the
addition of H2O2 (0.5 mm) to a) a mixture of SIH-B (50 mm) and FeCl3
(25 mm), and b) a mixture of SIH-B (50 mm) and CuCl2 (25 mm). The
H2O2-triggered reactions were incubated in DMF/KPB (pH 7.2) at
298 K with stirring and measured at intervals of 5 min.
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Communications
implying that H2O2 “activates” SIH-B to SIH which subse-
quently chelates the metal ions.
Finally, we tested whether SIH-B can inhibit the Fenton
reaction under physiologically relevant conditions. SIH,
which is known to inhibit the Fenton reaction,[19] was used
for comparison. Fenton reactions were generated according
to Equations (1) and (2) by incubating FeCl3, FeCl2, or CuCl2
with H2O2 in the presence of hydroascorbate in KPB (20 mm,
pH 7.2). 2-Deoxyribose (10 mm) was also added as a substrate
for hydroxyl radicals in the assay. The production of HOC
radicals was monitored by quantification of the 2-deoxyribose
degradation product, malonaldehyde (MDA), by its conden-
sation with thiobarbituric acid (TBA) to form a chromophore
with characteristic absorption at 532 nm.[19] We used the 2-
deoxyribose degradation assay to measure the ability of SIH-
B to prohibit the formation of HOC radicals. As shown in
Figure 3and Figure S7 in the Supporting Information, it is
apparent that at a ligand-to-metal ratio of 2:1, both SIH-B
and SIH significantly prevent 2-deoxyribose degradation
caused by hydroxyl radicals that are generated by either Fe-
or Cu-promoted Fenton chemistry. However, if a ligand-to-
metal ratio of 1:1 was used under similar conditions, a marked
decrease in protection was observed (Figure S8 in the
Supporting Information). SIH is a tridentate ligand, and
thus metal–SIH (1:1) complexes may offer open coordination
sites for H2O2 to access the metal, while the metal–(SIH)2
complexes are coordination saturated, which may completely
block the access of H2O2. This finding suggests that a “caged”
metal configuration without any open coordination sites is
important for attenuating the Fenton reaction.
As shown in Figure 3c, the effectiveness of SIH-B in the
attenuation of Fe-promoted Fenton chemistry is correlated
with the SIH-B/Fe ratio and also its preincubation time with
H2O2. At a low SIH-B/Fe ratio (< 10), a preincubation period
( ꢁ 10 min) between SIH-B and H2O2 is important for SIH-B
to be effective in attenuating the Fenton reaction. Better
attenuation is observed with increasing preincubation time
from 0 to 40 min. This observation may result from the fact
that the conversion of SIH-B to SIH by H2O2 is the rate-
limiting step. However, at high SIH-B/Fe ratio (> 10), the
attenuation is more effective and preincubation appears less
important. Taken together, the results support H2O2-triggered
prochelator SIH-B activation followed by metal sequestering
as a likely mechanism for attenuating the Fe (or Cu)-
promoted Fenton reaction. Studies performed with iron
concentrations from 0.5 to 20 mm (Figure S9 in the Supporting
Information) and over the pH range of 5.84 to 9.10
(Figure S10 in the Supporting Information) demonstrate
that SIH-B is effective in inhibiting the Fenton reaction at
physiologically relevant iron concentrations and pH range.
In summary, we have developed a prochelator SIH-B,
which can be converted to the active chelator SIH by H2O2 for
subsequent sequestration of iron and copper. This process can
effectively attenuate both Fe- and Cu-promoted Fenton
reactions under physiologically relevant conditions. The
H2O2-sensed chelating reactivity has interesting character-
istics that allow its consideration as a strategy to develop
novel compounds for attenuating the Fenton reaction under
oxidative stress conditions without disturbing healthy metal
Figure 3. Effect of SIH and SIH-B on the oxidative degradation of 2-
deoxyribose promoted by a) FeIII or b) CuII in the presence of H2O2 and
hydroascorbate. SIH-B (50 mm) was preincubated with H2O2 for
45 min at 298 K, then the solution (0.5 mL) was added to the assay
system containing FeIII (or CuII; 25 mm), 2-deoxyribose (10 mm), and
hydroascorbate (200 mm) in KPB (20 mm, pH 7.2). c) Effect of preincu-
bation time (0, 5, 10, 20, 40 min) and [SIH-B]/[Fe] ratio (r) on
attenuation of FeII-promoted Fenton chemistry in KPB buffer (20 mm,
pH 7.2). [Fe]=10 mm, [H2O2]=200 mm; A and A0 are the absorbance at
532 nm in the presence and absence of SIH-B, respectively.
homeostasis. Given the high levels of Fe (or Cu) and ROS in
brain tissues with certain neurodegenerative diseases, as well
as their critical roles in cardiovascular disease and certain
cancers, reagents capable of producing an “anti-Fenton
reaction” may be promising candidates for potential ther-
apeutics. Notably, the activation step of the current system is
relatively slow and thus improvement is warranted.
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Experimental Section
Freshly prepared solutions of FeCl3 (or CuCl2) in methanol and FeCl2
in dilute HCl were used. For titration experiments, a solution of the
metal ion was added to SIH-B or SIH (stock solutions in MeOH) and
the mixture was equilibrated at 298 K for 10 min. All titrations were
performed in KPB/DMF solvent unless otherwise noted.
UV/Vis spectra were recorded on a PerkinElmer Lambda 25
spectrometer at 298 K. UV difference spectra were recorded imme-
diately after addition of H2O2 to SIH-B and at different time intervals.
1H and 13C NMR spectra were recorded on a Bruker AC 300
spectrometer, and FTIR spectra were recorded on a Nicolet 4700
FTIR spectrometer. The 2-deoxyribose degradation assays were
performed similarly to the method reported by Lopes et al.[19]
Received: November 30, 2006
Revised: March 20, 2007
Published online: May 15, 2007
Keywords: bioinorganic chemistry · copper · Fenton reaction ·
.
hydrogen peroxide · iron
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