Stable Iron Porphyrin Intramolecularly Coordinated by Alcoholate Anion: Synthesis and Evaluation of Axial Ligand Effect of Alcoholate on Spectroscopy and Catalytic Activity

Article abstract of DOI:10.1021/acs.inorgchem.8b03384

We synthesized intramolecularly aliphatic alcoholate-coordinated iron porphyrins (1a, 1b) that retain their axial coordination in the presence of another ligand or oxidant. The electron-donative character of alcoholate was less than that of thiolate, and

Full text of DOI:10.1021/acs.inorgchem.8b03384

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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Stable Iron Porphyrin Intramolecularly Coordinated by Alcoholate
Anion: Synthesis and Evaluation of Axial Ligand Effect of Alcoholate
on Spectroscopy and Catalytic Activity
Yoshinori Shirakawa, Yuuki Yano, Yuki Niwa, Kanako Inabe, Naoki Umezawa, Nobuki Kato,
Yosuke Hisamatsu, and Tsunehiko Higuchi*
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
S
* Supporting Information
ABSTRACT: We synthesized intramolecularly aliphatic
alcoholate-coordinated iron porphyrins (1a, 1b) that retain
their axial coordination in the presence of another ligand or
oxidant. The electron-donative character of alcoholate was less
than that of thiolate, and the coordination ability of a sixth
ligand to 1a and 1b was very much lower than in the case of
the thiolate-coordinated compounds. Density functional
theory calculations indicated that the marked difference in
coordination ability could be explained in terms of
thermodynamic and steric factors. The catalytic oxidizing
ability of the thiolate-coordinated compound, SR complex,
was much higher than that of 1a.
and alcoholate. This issue has not previously been studied, and
so the basis for the emergence of thiolate as an axial ligand of
heme of cytochrome P450 during the course of evolution,
rather than alcoholate, remains unclear. Another reason for the
present work was to establish how axial coordination of
alcoholate affects the structure and function of iron porphyrin.
In addition, we wished to test the validity of a reported
prediction (see below) that heme alcoholate complex would be
a more active oxidation catalyst than heme thiolate.
A number of iron porphyrins coordinated by external
alkoxide anion have been prepared, and their physico-chemical
properties such as UV−vis,5a EPR,5b electrochemistry,5c and
crystal structure5d,e have been investigated. Morishima et al.
predicted that heme alcoholate complex would be a more
active catalyst for oxidation with peroxide than heme thiolate,
based on the results of their density functional theory (DFT)
calculations.6 However, the relative effects of the axial
alcoholate ligand on coordination chemistry and catalytic
oxidizing activity have never been evaluated by using an
isolable heme alcoholate complex. This is probably because the
highly basic alcoholate ligand is usually exchangeable by
another ligand or acidic hydroperoxide as a result of
protonation to form a weak alcohol ligand with an acidic
component. Furthermore, previously reported heme alcoholate
complexes needed an excess of highly basic alcoholate anion,
which seriously affects evaluation of the chemical properties.
Interestingly, Dawson and coauthors have reported preparation
1. INTRODUCTION
Cytochrome P450s catalyze various types of oxidative reactions
involved in steroid biosynthesis, fatty acid metabolism, and
detoxification of xenobiotic compounds, most of which are
monooxygenase-type NADPH/NADH- and O2-dependent
reactions.1 Because of the extremely strong oxidizing ability
of cytochrome P450 enzymes, much interest has been focused
on their chemistry, and identification of the reactive
intermediate responsible for oxygen atom transfer to the
substrate in the catalytic oxidation reaction has been a major
goal in bioinorganic chemistry.1 Cytochrome P450 and NO
synthase (NOS) have strong oxidizing ability and unusual
structure among heme enzymes, in that their heme iron has
thiolate coordination. Consequently, attention has mainly been
focused on their structure−function relationship. We pre-
viously synthesized the first thiolate-coordinated iron porphyr-
in (named SR complex (Figure 1)) that retains its thiolate
coordination during catalytic oxidizing reactions, and com-
pared the effects of axial thiolate, imidazole, and chloride
ligands on the catalytic oxidation reaction mediated by the SR
complex.2 On the other hand, heme of catalase is coordinated
by phenolate, which has relatively high ligand field strength,
and several model complexes having iron porphyrin coordi-
nated by phenolate have been reported.3 Electrons of the
oxyanion of phenolate conjugate with the π-electron system of
the attached benzene ring. On the other hand, alcoholate, in
which an oxy anion is attached to a saturated carbon atom,4
does not conjugate with any π-electron system, as the case of
the sulfur anion of thiolate ligand of P450. Therefore, it is of
interest to compare the relative axial ligand effects of thiolate
Received: December 4, 2018
© XXXX American Chemical Society
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Figure 1. Structures of SR complex, SR-HB complex, and heme alcoholates 1a and 1b.
Figure 2. Synthesis of complex 1a. Reagents and conditions: (a) NBS, BPO, hν; (b) NaOAc in DMF, 59% (2 steps); (c) TFA-AcOH, 75%; (d) (1)
2-chloro-1-methylpyridinium iodide, Et3N in THF/CH2Cl2; (2) α,α,α,α-meso-tetrakis(2-aminophenyl)porphyrin in THF/CH2Cl2, 60%; (e)
pivaloyl chloride, pyridine in CH2Cl2, 65%; (f) Fe(CO)5, I2, 2,4,6-collidine intoluene, 82%; (g) sodium methoxide/MeOH intoluene, 54%.
Figure 3. Electronic absorption spectra of 1a(Fe(III)) and its reduced states and 1b(Fe(III)) at 298 K. [1] = 10 μM in toluene. (A) a: the
spectrum of 1a(Fe(III)); b: the spectrum observed immediately after the addition of 1a to toluene containing an excess of NaBH4 and 15-Crown-5
under Ar; c: the spectrum 16 min after the start of reduction. (B) Electronic absorption spectrum of 1b(Fe(III)) at 298 K.
B
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of a P450 mutant (ΔC436S CYP2B4) that is indicated to have
a heme-alcoholate coordination structure judging from
magnetic circular dichroism (MCD) data and electronic
absorption spectra.7 However, the protein has not been fully
characterized, e.g., in terms of vibration spectroscopy, electro-
chemical properties, and catalytic activity.
Here we report the synthesis of the first isolable alcoholate-
coordinated iron porphyrins (1a and 1b in Figure 1) that
retain their axial ligands during catalytic oxidation. We also
describe their chemical properties including catalytic oxidiza-
tion activity.
any change of λmax, although the reason for this is unclear. We
show only the spectrum of 1b (Fe(III)) in Figure 3.
EPR Spectra. Both of these complexes were found to
contain Fe(III) in a high-spin state from the results of EPR and
electronic absorption spectroscopy. EPR spectra of 1a and 1b
showed three signals assignable to Fe(III)porphyrin in a high-
spin state (Figure 4 and Table 2).
Complex 1a was chromatographically and mass spectro-
metrically homogeneous. Therefore, 1a appears to be a mixture
of two conformers at 4 K, although NMR data could not be
used to support this idea due to the paramagnetic property of
1a.
Cyclic Voltammetry. The cyclic voltammograms of 1a and
1b gave reversible redox couples (Figure S3). The E1/2 (FeII/
FeIII) of 1a (−0.63 V) was ca. 0.2 V higher than that of SR, and
the E1/2 (FeII/FeIII) of 1b was similar (Figure S3 and Table 2).
These results indicate that the electronic Push effect of
alcoholate is lower than that of thiolate.
2. RESULTS AND DISCUSSION
Synthesis. We expected that the synthetic method of SR
complexes that we developed2a would be directly applicable to
the heme alcoholate complex (Figure 2). Indeed, the coupling
reaction of (o-acetoxymethylphenoxy)acetic acid (4a) and an
amino group of α,α,α,α-meso-tetrakis(2-aminophenyl)-
porphyrin, and the subsequent synthetic steps proceeded as
in the case of the SR complex.2a Complex 1a was successfully
purified by silica gel column chromatography under air, even
though alkoxide anion is highly basic and should be protonated
by acidic silanols on silica gel. The reason for the axial
coordination stability is probably that the alkoxide oxygen is
deeply buried in bulky pivalamido groups of the porphyrin, and
therefore, alkoxide and silanol cannot come into direct contact.
We also prepared 1b, an alcoholate-coordinated iron porphyrin
with a NH−O hydrogen bond on the axial ligand analogously
to our previous synthesis of SR-HB, an iron porphyrin
coordinated by thiolate with a NH−S hydrogen bond (Figure
S1).2d High-resolution mass (ESI) spectra of 1a (m/z
1166.4135 [M + Na]+) and 1b (m/z 1223.4353 [M + Na]+)
together with the results of elemental analysis supported these
structures.
FT-IR Spectroscopy of NO-1a(FeIII). N-O Stretching
mode of the NO-1a(FeIII) complex was measured by FT-IR
spectroscopy (Figure S4 and Table 2). The assignment was
confirmed by the observation of an isotope shift when 15NO
was used (1863 cm−1). The N-O stretching mode of NO-1a
(1897 cm−1) was clearly lower than the mode of NO-FeIII
meso-tetraphenylporphyrin axially coordinated by iso-amyl
alcohol (1935 cm−1) or H2O (1937 cm−1).8a,b The difference
in the two modes supports the idea that complex 1a is not a
heme-alcohol form, but a heme-alcoholate form. The N-O
stretching mode of NO-1a (1897 cm−1, this work) was much
higher than that of the NO-SR complex (1828 cm−1).9
Electrochemistry and FT-IR both indicate that the axial
alcoholate ligand is a poorer π-electron donor than thiolate
toward iron porphyrin.
Coordination Chemistry of 1a with 1-Methylimida-
zole Evaluated by Electronic Absorption Spectral
Titration, DFT Calculation, and Measurement of
Effective Magnetic Moment. The binding constant of 1-
methylimidazole (1-MeIm) as a sixth ligand to complex 1a was
evaluated by electronic absorption spectral titration. Only 10
equiv of 1-MeIm was necessary for almost complete formation
of the 1-MeIm-SR complex (Figure 5a). In contrast, to our
surprise, addition of even 2000 equiv of 1-MeIm did not alter
the absorption spectra of the 1a complex, indicating that it is in
a high-spin state (at 298 K, Figure 5b), even though the ligand
field strength of 1-MeIm is high. The reported 1-MeIm-
coordinated iron porphyrin methoxide is known to be in the
low-spin state from its EPR spectra at 77 K.10 However, it was
also reported that no visible spectral change was observed at
room temperature when 30 equiv of 1-MeIm was added to iron
meso-tetraphenylporphyrin methoxide solution.10 Our data
clearly indicate that there is a very marked difference between
the effect of thiolate and that of alcoholate as a fifth ligand of
heme upon sixth ligand coordination.
Electronic Absorption Spectra. The electronic absorp-
tion spectra of 1a(Fe(III)) and 1a(Fe(II)) were measured
(Figure 3 and Table 1). The spectral pattern of 1a(Fe(III))
Table 1. Electronic Absorption Spectra of Complexes 1a
a
and 1b
complex
band maxima, nm (ε, M−1 cm−1 × 10−3
)
1a(Fe(III)) 349 (32.7), 418 (80.3), 481 (sh, 20.3), 581 (11.9), 637
(sh, 6.66)
1a(Fe(II))
438 (94.5), 536 (10.8), 570 (12.2), 636 (sh, 6.73)
1b(Fe(III)) 350 (33.3), 420 (88.6), 500 (sh, 12.3), 594 (5.92),
649 (3.46)
a
Toluene, 298 K. sh: shoulder.
(spectrum a in Figure 3, λmax = 418 nm) was similar to that of
Fe(III) meso-tetramesitylporphyrin methoxide.5a Next, the
complex 1a in toluene was reduced with NaBH4-15-Crown-5
couple. The spectrum of the reduced product showed a
bathochromically shifted Soret band (spectrum b, λmax = 438
nm), but gradually changed to spectrum c (λmax = 432 nm).
Spectrum c is similar to the spectrum obtained when Fe(III)
meso-tetraphenylporphyrin chloride (Fe(III)(TPP)Cl) was
reduced in the same manner (λmax = 425 nm, Figure S2).
Therefore, spectrum b should be that of 1a(Fe(II)) and
spectrum c would be that of Fe(II) porphyrin coordinated by
alcohol or crown ether as the fifth ligand. In the case of 1b,
reaction with NaBH4-15-Crown-5 caused a considerable
decrease of intensity throughout the whole spectrum without
Coordination of a sixth ligand to the heme iron is a
thermodynamically controlled process. We mixed 1a and 1-
MeIm in CH2Cl2 at 298 K and measured the heat of formation
by using isothermal titration calorimetry (ITC). As expected,
almost no measurable heat other than heat of dilution was
observed (Figure S5). The coordination equilibrium constant
depends on the energy difference between the noncoordina-
tion state and coordination state, because the chemical
equilibrium obeys the Arrhenius equation. Therefore, we
carried out DFT calculation for each complex in order to
C
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Figure 4. EPR Spectra of 1a (a) and 1b (b). [1a], [1b]: 100 μM in toluene at 4 K.
a
Table 2. EPR, Cyclic Voltammogram of Iron(III)porphyrin
Thiolate or Alcoholate Axial Ligand and N-O Stretching
Mode of the NO-Coordinated Complex
Table 3. Calculated Energy and Coordination Heat
axial
ligand
ν (N-O) of
ON-Fe(Por)
a
Fe(Por)
EPR at 4 K E1/2 (FeII/FeIII)
SR
R-S−
gx = 1.96
gy = 2.21
gz = 2.32
−0.81 V
1828 cm−1
1a
R-O−
R-S−
g = 1.9, 5.5,
−0.63 V
−0.70 V
1897 cm−1
1837 cm−1
energy of 5-
coordinate Fe
porphyrin (A)
[spin state
6.0
energy of 1-MeIm-Fe heat formed
porphyrin
by 1-MeIm
coordination
(C)
SR-HB
gx = 1.96
gy = 2.20
gz = 2.32
g = 2.1, 5.4,
6.1
(6-coordinate) (B)
[spin state S = 1/2]
Fe porphyrin
1a
SR complex
Fe(Por)OMe*
Fe(Por)SMe*
S = 5/2]
−4780.9022
−5103.7762
−2367.1415
−2690.0928
−5046.3011
−5369.1662
−2632.7216
−2955.7058
0.0112
−0.0201
−0.0708
−0.1037
1b
R-O−
−0.50 V
n.d.
a
ν (N-O) data of ON-SR and ON-SR-HB: ref 9.
a
Unit: hartree. Energy of 1-MeIm: −265.4101 hartree (D). Heat (C)
obtain the energy difference. Coordination of 1-MeIm to the
SR complex was exothermic (Table 3). In contrast,
coordination of 1-MeIm to 1a was endothermic. This
endothermicity in the case of 1a may explain why 1-MeIm
hardly coordinated to 1a. Next, we calculated simple iron
was calculated by an equation: C = B − (A + D). DFT Calculation:
Geometry optimization with ωB97X-D/6-31G*, then single point
energy with M06-2X/6-31G* in the cases of 1a and SR complex. *
Geometry optimization with ωB97X-V/6-311+G(2df,2p) in the cases
of Fe(Por)OMe and Fe(Por)SMe (1-MeIm: −265.5093 hartree).
Figure 5. (a) Electronic absorption spectral changes of SR (10 μM in CH2Cl2 at 298 K) with increasing 1-MeIm concentration. [1-MeIm] (μM): 0
(black), 20 (red), 100 (blue). (b) Electronic absorption spectral changes of 1a (10 μM in CH2Cl2 at 298 K) with increasing 1-MeIm concentration.
[1-MeIm] (μM): 0 (solid line), 20 000 (dashed line).
D
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porphyrin methoxide (Fe(Por)OMe), 1-MeIm-Fe(Por)OMe,
Fe(Por)SMe, and 1-MeIm-Fe(Por)SMe with a higher-level
function set (ωB97X-V/6-311+G(2df,2p)) in order to
examine whether or not the difference in the energy profile
between 1a and SR is general (Table 3). The results indicated
that Fe(Por)SMe was more exothermic than Fe(Por)OMe,
although both gave exothermic profiles. These results suggest
that imidazole coordination to heme thiolate is generally more
advantageous than that to heme alcoholate. The difference in
the heat profile between 1a and Fe(Por)OMe may be due to a
difference in conformational restriction of the axial ligand
linker part and/or a difference in the environment around the
axial ligand. The very low coordination ability for 1-MeIm
suggests that the axial orbital of iron (dz2) repels the lone pair
of 1-MeIm due to its high electron density. This may be
interpreted as indicating that alcoholate as an axial ligand is a
high σ-electron donor to the iron dz2 orbital, in contrast to its
poor π-donative character.
(Fe(III)) in CDCl3 (2.47 mM) in the presence of 1.0 M 1-
MeIm was 4.23, which is close to that of 1a in the absence of 1-
MeIm. Therefore, this result also indicates that a large excess
amount of 1-MeIm does not alter the spin state of 1a.
Catalytic Oxidation Activity of 1a and 1b. We
previously reported that the SR complex showed high catalytic
activity in the O−O bond cleavage of alkyl hydroperoxides,2a
peroxyacids,2b−f and endoperoxide.2g At first, cumene hydro-
peroxide was examined as an oxidant for 2,4,6-tri-tert-
butylphenol (TBPH) in order to evaluate the catalytic activity
of heme alcoholate 1a. However, no activity was observed in
this case. Next, the catalytic oxidative activity of 1a for TBPH
oxidation with peroxyphenylacetic acid (PPAA), a more
powerful oxidant than cumene hydroperoxide, was compared
with that of SR in order to evaluate the relative axial ligand
effect of alcoholate (Table 4). The initial reaction rate
catalyzed by 1a was 130-fold lower than the rate in the case
of SR. This large difference is probably due to the small
equilibrium constant of PPAA coordination to the iron of 1a
and also poorer electron donation of alcoholate to iron than
that of thiolate.
A comparison of the structures of the two iron porphyrins
obtained by the above-mentioned calculation (Figure 6)
In summary, we have succeeded in the synthesis of
intramolecularly aliphatic alcoholate-coordinated iron porphyr-
ins, which were confirmed to retain alcoholate coordination
during catalytic oxidization reaction. We also evaluated their
sixth ligand coordination ability. Complex 1a is simply an O-
substituted analogue of the SR complex, but notably, its
chemical properties are very different from those of SR.
Complex 1a in a low-spin state was hardly formed, in spite of
addition of an extremely large excess of 1-MeIm, whereas SR in
low-spin state was readily formed by the addition of only 10
equiv of 1-MeIm to SR. DFT calculations indicated that this
marked difference in coordination ability can be explained in
terms of thermodynamic and steric factors. The cyclic
voltammogram of 1 and IR spectrum of 1a-NO complex
indicate that the electron donation of alcoholate to heme is
much weaker than that of thiolate. These results may explain
why nature has selected thiolate as an axial ligand of heme in
cytochrome P450, not alcoholate.
Figure 6. Calculated structures of Fe(Por)OMe and Fe(Por)SMe
obtained by geometry optimization (gas phase) using ωB97X-V/6-
311+G(2df,2p) function set.
indicates that the form of the porphyrin plane of Fe(Por)OMe
is dome-like. This is consistent with the crystal structure of
iron octaethylporphyrin methoxide.5e In contrast, the porphyr-
in of Fe(Por)SMe is almost completely planar. The cone angle
(∠N21−Fe−N23) of the former (144.3°) is considerably
narrower than that of the latter (163.9°). The narrow cone
angle should sterically hinder the approach of 1-MeIm to the
iron. Thus, the shape of the porphyrin plane can explain the
resistance of 1a to accepting a sixth ligand.
Finally, the values of effective magnetic moment (μeff) of
complex 1a in the absence and presence of 1-MeIm were
determined using the Evans method in order to confirm the
spin state at 298 K.11 The μeff of 1a (Fe(III)) in CDCl3 was
4.68 at 298 K, showing mainly a high-spin state. The μeff of 1a
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b03384.
Table 4. Observed Initial Rates of TBP• Formation in the Oxidation of TBPH with Peroxyphenylacetic Acid (PPAA)
a
Catalyzed by 1 or SR Complex
b
Fe(Por)
turnover number freq (ν/s)
νSR/νFe(Por)
SR
1a
1b
39
0.29
1.0
1
130
39
a
Conditions: solvent = CH2Cl2: [TBPH] = 0.2 M; [PPAA] = 5 × 10−3 M: [SR] = [1a or 1b] = 10−4 M. These reactions were carried out at 298 K
b
under an argon atmosphere. Observed initial rates of the reactions were based on catalysts (turnover number of catalyst/s).
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AUTHOR INFORMATION
Corresponding Author
*E-mail: higuchi@phar.nagoya-cu.ac.jp.
■
ORCID
Naoki Umezawa: 0000-0003-3966-2303
Tsunehiko Higuchi: 0000-0002-3586-4680
Present Address
N. Kato: Graduate School of Science, Tohoku University, 6-3,
Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan.
Author Contributions
All authors contributed to writing the manuscript. All authors
have given approval to the final version of the manuscript.
Funding
This work was supported in part by Grants-in-Aid for Scientific
Research (A) (No. 20249006) from the Japan Society for the
Promotion of Science (JSPS). We are also grateful for support
by the Platform Project for Supporting Drug Discovery and
Life Science Research (No. 12760016, No. 16am0101055j-
0005) from MEXT, and by the Japan Agency for Medical
Research and Development (AMED).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to Dr. Takuya Kurahashi (Institute for
Molecular Science) for cooperation in EPR measurement.
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