An iron(iii) tetradentate monoamido complex as a nonheme iron-based peroxidase mimetic

Article abstract of DOI:10.1039/c3dt51483h

A mononuclear iron(iii) complex of a noncyclic tetradentate monoamido ligand, Fe(iii)mpaq, catalyses the oxidation of Orange II, guaiacol, ABTS and Amplex Red with H₂O₂ in aqueous solutions at neutral pH. Under identical conditions, other structurally related nonheme iron complexes showed only negligible activities.

Full text of DOI:10.1039/c3dt51483h

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An iron(III) tetradentate monoamido complex as a
nonheme iron-based peroxidase mimetic†
Cite this: Dalton Trans., 2013, 42, 12878
Received 4th June 2013,
Accepted 20th July 2013
Yutaka Hitomi,* Kazuaki Hiramatsu, Kengo Arakawa, Toshiyuki Takeyasu,
Masashi Hata and Masahito Kodera
DOI: 10.1039/c3dt51483h
www.rsc.org/dalton
A mononuclear iron(III) complex of a noncyclic tetradentate mono- conjugates in aqueous media,17,18 but the activity of these
amido ligand, Fe(III)mpaq, catalyses the oxidation of Orange II, complexes was much lower than those of the Fe-TAMLs.
guaiacol, ABTS and Amplex Red with H2O2 in aqueous solutions
During our research to develop nonheme iron-based oxi-
at neutral pH. Under identical conditions, other structurally related dation catalysts, we discovered that a mononuclear nonheme
nonheme iron complexes showed only negligible activities.
iron(III) complex of a noncyclic tetradentate monoamido
ligand, Fe(III)mpaq, exhibits exceptional peroxidase-like activity
at neutral pH when compared with the other mononuclear
nonheme iron complexes supported by polydentate nitrogen
ligands shown in Chart 1 such as [FeIII(dpaq)(H2O)](ClO4)2
(2),20 [FeII(tpa)(CH3CN)2](OTf)2 (3),21 and [FeII(N4Py)(CH3CN)]-
(ClO4)2 (4).22 Herein, we report the synthesis, structure, and
peroxidase-like activity of Fe(III)mpaq.
The tetradentate monoamido ligand H-mpaq was syn-
thesized in 19% overall yield (5 steps) according to a procedure
similar to that described for the preparation of H-dpaq.20 Reac-
tion of H-mpaq with Fe(ClO4)3·6H2O or FeCl3 in CH3OH
afforded the corresponding iron(III) complexes, [FeIII(mpaq)-
(H2O)2](ClO4)2 (1) and FeIII(mpaq)Cl2 as green powders,
respectively. Analytically pure crystals were obtained after
recrystallization from CH3OH–diethyl ether. Single crystals
suitable for X-ray crystal analysis were obtained for FeIII(mpaq)-
Cl2, but not for 1. A thermal ellipsoid plot corresponding to
the X-ray crystallographic characterization of FeIII(mpaq)Cl2 is
shown in Fig. 1. The complex contains an iron center in a
Peroxidases are enzymes that catalyse oxidation reactions with
hydrogen peroxide (H2O2) or alkyl peroxides. Heme-containing
peroxidases, such as horseradish peroxidase (HRP), are cur-
rently used for a wide range of applications such as electro-
chemical sensors, waste water cleaning, immunohistochemistry,
and ELISA studies.‡1 Synthetic compounds that show peroxi-
dase-like activity have attracted attention from the mechanistic
and practical points of view.2 Thus, a number of iron com-
plexes have been developed that exhibit peroxidase-like activity
in aqueous media, most of which have porphyrin or macro-
cyclic ligands.3–6 For instance, a series of tetraamido macro-
cyclic ligands (TAMLs) have been developed by the Collins group
for the preparation of nonheme iron complexes that are capable
of promoting the decomposition of azo dyes, chlorinated
phenols, and even chemical weapons in aqueous media using
H2O2.7–9
Recent studies have demonstrated that mononuclear
nonheme iron complexes supported by noncyclic polydentate
nitrogen ligands are capable of catalyzing various oxidation
reactions with H2O2 in organic solvents, such as hydroxy-
lations, epoxidations, and cis-dihydroxylations,10–16 but only a
few studies have been conducted in aqueous media.17–19
Notably, Feringa, Choma and co-workers reported the peroxi-
dase-like activity of a nonheme iron complex supported by a
noncyclic pentadentate ligand, Fe(II)N4Py, and its peptide
Department of Molecular Chemistry and Biochemistry, Faculty of Science and
Engineering, Doshisha University, 1-3 Tatara Miyakodani, Kyotanabe,
Kyoto 610-0321, Japan. E-mail: yhitomi@doshisha.ac.jp
†Electronic supplementary information (ESI) available: Synthetic procedures of
Fe(III)mpaq, details of X-ray experiment and kinetic measurements, and Fig. S1
to S4. CCDC 942692. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c3dt51483h
Chart 1 Chemical structures of supporting ligands.
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Fig. 1 ORTEP diagram of FeIII(mpaq)Cl2. The H-atoms are not shown for clarity.
The thermal ellipsoids indicate a 50% probability level. Selected bond distances:
Fe–N1: 2.111(2), Fe–N2: 2.174(2), Fe–N3: 2.051(2), Fe–N4: 2.196(2), Fe–Cl1:
2.3138(10), Fe–Cl2: 2.3073(9) Å.
Fig. 3 Rate dependence of the H2O2 oxidation of ABTS catalysed by 1 (2 μM)
as a function of the H2O2 and ABTS concentrations at pH 7.0 and 25 °C. The
y-axis represents the initial rates (v) divided by the catalyst concentration.
distorted octahedral coordination environment. The tetra-
dentate ligand leaves two coordination sites for the chloride
ligands.
An azo dye, Orange II, was first chosen as the substrate for
the peroxidase reaction catalysed by nonheme iron com-
plexes.23 The spectrum of Orange II did not change upon
addition of H2O2 in the presence of catalysts 2–4 or the simple
iron salt (NH4)2Fe(SO4)2. On the other hand, catalyst 1 efficien-
tly promoted the decomposition of Orange II with H2O2
(Fig. 2A). Catalyst 1 also mediated the oxidation of guaiacol
and ABTS using H2O2, while with complexes 2–4, virtually no
reaction was observed (Fig. 2B and 2C). The total turnover
numbers (TONs) of the catalyst were 10, 34, and 2.7 for oxi-
dation of Orange II, guaiacol and ABTS, respectively. The
initial rate of ABTS oxidation catalysed by 1 increased linearly
as a function of the concentration of 1 up to 10 μM, suggesting
that this catalyst generates a monomeric oxidizing species, at
least under these catalytic reaction conditions (Fig. S1†).
Next, we investigated the peroxidase activity of 1 at varied
concentrations of ABTS and H2O2. The initial rate of formation
of the ABTS cation radical was saturated as the concentrations
of ABTS and H2O2 increased (Fig. 3). Assuming that the follow-
ing peroxidase-like reactions occur,
k1
FeðiiiÞL þ H2O2 ! FeðvÞL;
k2
FeðvÞL þ ABTS ! FeðivÞL þ ABTS•þ
;
and
k3
FeðivÞL þ ABTS ! FeðiiiÞL þ ABTS•þ
;
eqn (1) can be derived under steady state conditions:
d½ABTS•þꢀ
dt
2k1k2k3½H2O2ꢀ½ABTSꢀ½1ꢀ0
¼
;
ð1Þ
k2k3½ABTSꢀ þ k1ðk2 þ k3Þ½H2O2ꢀ
where [1]0 represents the total concentration of complex 1.
Each complex in the rate equations may have aqua, hydroxo,
and/or oxo ligands, but those ligands are omitted in the above
equations. Especially, Fe(III)mpaq may exist as a mixture of
species having two aqua and/or hydroxo ligands depending on
operating pH (vide infra). Thus, the rate constants are appar-
ent. By fitting the 3D plot in Fig. 3 using eqn (1), the apparent
second-order reaction rate constant with H2O2 and the harmo-
nic mean of the rate constant towards ABTS were determined
to be k1 = 0.31
0.02 mM−1 s−1 and k2k3/(k2 + k3) = 18
Fig. 2 Initial rates for the oxidation of Orange II (A, 20 μM), guaiacol (B,
2 mM), and ABTS (C, 0.1 mM) using 1 mM H2O2 catalysed by 2 μM complexes
1–4 at pH 7.0 and 25 °C.
2 mM−1 s−1, respectively. These values are close to that
reported for Fe-TAML activators at pH 7.24
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Fig. 4 CSI-MS spectra of a reaction mixture of 1 and 100 equiv. of H2O2 (B) or
H218O2 (D) in CH3CN–H2O (9 : 1) at 0 °C. The isotope pattern shown in panel B is
assignable to [FeIII(mpaq)(OOH)]+ or [FeV(mpaq)(O)(OH)]+, whose simulated
spectrum is shown in panel A. The isotope pattern shown in panel D is assign-
able to a mixture of mono-18O-labeled and bis-18O-labeled species (77 : 23).
Scheme 1 Proposed mechanism for the reaction of 1 and 2 with H2O2.
It has been reported that mononuclear nonheme iron com-
plexes containing two cis-oriented labile coordination sites can was reversed when the pH was once again adjusted to 3. Singu-
generate monomeric FeV(O)(OH) species that efficiently acti- lar value decomposition of the observed spectral changes
vate H2O2 with the assistance of
a
coordinated water using the program SPECFIT32 indicated the existence of at
molecule.25–28 Recently, Costas, Cronin and co-workers suc- least three different species that interconverted with pKa values
cessfully provided clear evidence for an FeV(O)(OH) species by of 3.4 and 7.0 (Fig. S3†). Nearly identical pKa values were
using cold-spray mass spectroscopy (CSI-MS).29 Inspired by obtained independently on the basis of the concentrations of
this pioneering study, we herein report the results of the 1, which indicates that the observed spectral changes are
CSI-MS analysis of a reaction mixture containing 1 and 100 caused only by protonation equilibria and do not involve con-
equivalents of H2O2 in CH3CN–H2O (9 : 1) at 0 °C. The posi- centration-dependent processes such as μ-oxo dimer for-
tive-ion CSI-MS spectrum showed an ion signal at a mass-to- mation. On the other hand, the structurally similar iron(III)
charge (m/z) ratio of 394.2, the mass and isotope distribution pentadentate monoamido complex 2 showed a single pH-
patterns of which were consistent with the chemical formula dependent spectral change with a pKa value of 5.2 (Fig. S4†).
[FeIII(mpaq)(OOH)]+ or [FeV(mpaq)(O)(OH)]+ (both calculated This spectral change is assignable to the protonation equili-
m/z = 394.2) (Fig. 4). When H218O2 was used instead of H2O2, brium between [FeIII(dpaq)(H2O)]2+ and [FeIII(dpaq)(OH)]+. On
the position of the ion signal shifted to 396.2 (a change of two the basis of the result obtained for 2, we propose that the spec-
mass units and the major species) and to 398.2 (a change of tral changes with pKa values of 3.4 and 7.0 for 1 correspond to
four mass units and a minor species). The reaction of 1 with the protonation equilibria between [FeIII(mpaq)(H2O)2]2+ and
H2O2 in CH3CN–H218O (9 : 1) afforded ion signals assignable [FeIII(mpaq)(H2O)(OH)]+ and [FeIII(mpaq)(H2O)(OH)]+ and
to mono-18O-labeled species at m/z = 396.2 together with FeIII(mpaq)(OH)2, respectively. Thus, it is most likely that 1
unlabeled species at m/z = 394.2 (Fig. S2†). Taken together, exists as a mixture of [FeIII(mpaq)(H2O)(OH)]+ and FeIII(mpaq)-
these results indicate that an FeV(O)(OH) derivative of 1 is gen- (OH)2 at pH 7.0.
erated from an FeIII(H2O)(OOH) species with the assistance of
Compound 1 is soluble in aqueous solutions over a wide
a coordinated water molecule, as reported for mononuclear pH range. Therefore, the initial rate of oxidation of Orange II
nonheme iron complexes containing two cis-oriented labile at pH values ranging from 4 to 9 was measured. The pH profile
coordination sites (Scheme 1).25–29
showed a bell shape with a maximum near pH 7 (Fig. 5). As
To provide further insight into the solution structures of mentioned above, 1 should exist as FeIII(mpaq)(OH)2 at pH
catalyst 1 in aqueous media, the UV-vis absorption spectrum values greater than 6 (Fig. S3D†), which should readily react
of 1 was measured at various pH values. Complex 1 showed a with H2O2 to generate the corresponding FeIIIOOH species.
broad band at 660 nm at pH 3, assignable to a charge-transfer The resulting [FeIII(mpaq)(H2O)(OOH)]+ species should then
band from the amido group to the iron(III) ion. Similar absorp- be smoothly converted to the corresponding FeV(O)(OH)
tion bands have been reported for other iron(III)–monoamido species, as proposed for other iron complexes having cis-
complexes, such as 2 and Fe(III)PaPy3.20,30,31 As the pH value oriented labile coordination sites (Scheme 1a). The bell-
increased up to 10, the charge-transfer band at 660 nm shaped pH–initial rate profile can be fitted by an equation
decreased in intensity (Fig. S3†). Notably, this spectral change consisting of two pKa values (Fig. S5†). However, currently, we
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Fig. 5 pH dependence of the initial reaction rate for the decomposition of
Orange II (20 μM) using H2O2 (0.5 mM) in the presence of 2.0 μM complex 1
(red circle) or 2 (blue triangle).
cannot explain why the activity of 1 decreases as the pH
increases above 7.2. In contrast to 1, compound 2 showed vir-
tually no reactivity in the pH range of 4–9. Compound 2
should react with H2O2 to give the corresponding FeIIIOOH
species; however, we speculate that the resulting [FeIII(dpaq)-
(OOH)]+ species cannot be efficiently transformed to an FeV(O)
species probably because H2O cannot act as an efficient proton
donor, and thus reverts to [FeIII(dpaq)(H2O)]2+ and/or [FeIII-
(dpaq)(OH)]+, depending on operating pH (Scheme 1b). This
may be the reason why 2 is able to catalyse oxidation reactions
using H2O2 in CH3CN, but not in H2O.20
Fig. 6 Oxidation of Amplex Red using H2O2 in the presence of varied concen-
trations of catalysts 1–4. (A) Photograph of a 96-well plate after 30 min. (B) Fluo-
rescence intensity of the solution at 550 nm with excitation at 595 nm after
100 s. 1: red; 2: blue; 3: yellow; and 4: green.
Finally, we evaluated the minimum concentration of
1 required to catalyse a fluorescence reaction using H2O2. To
this end, we chose the substrate 10-acetyl-3,7-dihydroxy-
phenoxazine, known as Amplex Red, which is frequently used as
a substrate for H2O2 oxidation using HRP because it is converted
to red fluorescent resorufin. This transformation has been
widely used for various biological assays, including ELISA
studies. Therefore, as in ELISA experiments, this study was
conducted using a 96-well microplate in which each well con-
tained various concentrations of 1 and 50 μM Amplex Red.
Upon the addition of 2.0 mM H2O2, the fluorescence was
This work was supported by Grant-in-Aid for Scientific
Research on Priority Areas (no. 19028033 and 20037039,
“Chemistry of Concerto Catalysis”) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan.
Notes and references
‡ELISA, enzyme-linked immunosorbent assay; mpaq, 2-[N-methyl-N-pyridin-2-
ylmethyl]-amino-N′-quinolin-8-yl-acetamido; N4Py, N,N-bis(2-pyridylmethyl)-N-
bis(2-pyridyl)methylamine; dpaq, 2-[N,N-bis(pyridin-2-ylmethyl)]-amino-N′-qui-
nolin-8-yl-acetamido; tpa = tris(2-pyridylmethyl)amine; PaPy3, N,N-bis(2-pyridyl-
methyl)amine-N-ethyl-2-pyridine-2-carboxamido; ABTS, 2,2′-azino-bis(3-ethyl-
benzothiazoline-6-sulphonic acid).
detected with
a normal fluorescence multiplate reader
(Fig. 6A). Although the total TON of the catalyst decreases as
the catalyst concentration decreases: 26 at 500 nM of 1 to 1.6
at 100 nM of 1, it was found that even 100 nM of 1 was enough
to lead to significant fluorescence intensity after 100 s
(Fig. 6B).
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