An improved model for malaria pigment and β-hematin: Fe(OEP)picrate

Article abstract of DOI:10.1002/jrs.5176

Synthesis and crystallographic and spectroscopic structural characterization, along with spectral band assignments calculated by using density functional theory (M06L/cc-pVDZ and B3LYP/6-31G(d)), are reported for Fe(OEP)picrate as a model for hemozoin (malaria pigment) and its synthetic analog β-hematin, which are spectroscopically identical. The average Fe–N distance, 2.044(12)??, and the Raman modes indicate a high-spin five-coordinate iron(III) complex. Resonance enhancement is observed for the totally symmetric oxidation state marker band ν₄ along with the characteristic bands of C_β-substituted hemes in the ranges of 1621–1639?cm^?1 for ν_as(C_αC_m) and 750–756?cm^?1 for ν(pyr breathing) similar to β-hematin and hemozoin when using near-IR excitation wavelengths. The similarity of the resonance Raman spectral profiles for Fe(OEP)picrate and β-hematin, along with the structural results, indicates that Fe(OEP)picrate is an excellent model for understanding the stereochemistry and intermolecular interaction of β-hematin. Copyright

Full text of DOI:10.1002/jrs.5176

Research article
Received: 15 August 2016
Revised: 6 April 2017
Accepted: 23 April 2017
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jrs.5176
An improved model for malaria pigment and
β-hematin: Fe(OEP)picrate
Ratchadaporn Puntharod,a Kenneth J. Haller,b* Evan G. Robertson,c
Eunice S. H. Gwee,d Ekaterina I. Izgorodinad and Bayden R. Woodd*
Synthesis and crystallographic and spectroscopic structural characterization, along with spectral band assignments calculated by
using density functional theory (M06L/cc-pVDZ and B3LYP/6-31G(d)), are reported for Fe(OEP)picrate as a model for hemozoin
(malaria pigment) and its synthetic analog β-hematin, which are spectroscopically identical. The average Fe–N distance, 2.044
(12) Å, and the Raman modes indicate a high-spin five-coordinate iron(III) complex. Resonance enhancement is observed for
the totally symmetric oxidation state marker band ν4 along with the characteristic bands of Cβ-substituted hemes in the ranges
of 1621–1639 cmꢀ1 for νas(CαCm) and 750–756 cmꢀ1 for ν(pyr breathing) similar to β-hematin and hemozoin when using near-IR
excitation wavelengths. The similarity of the resonance Raman spectral profiles for Fe(OEP)picrate and β-hematin, along with
the structural results, indicates that Fe(OEP)picrate is an excellent model for understanding the stereochemistry and
intermolecular interaction of β-hematin. Copyright © 2017 John Wiley & Sons, Ltd.
Metalloporphyrins as mimics for biologically functional
Introduction
molecules have been extensively studied.[8–15] In this context, both
Strategies for antimalarial drug design aim at producing effective,
nontoxic, and inexpensive agents for use against malaria in the
developing world, where the combined mortality rate is over one
million per year with some 300–500 million people afflicted with
the disease.[1] Most deaths occur in children under 5 years of age,
and child mortality from cerebral malaria is ~20%.[2] Of the children
that survive malaria, 10% are left with severe neurological defects
affecting their education and long-term employment prospects.[2]
Moreover, ineffective treatments can result in anemia and low birth
weight[3] compromising the health of children and adults infected
with Plasmodium falciparum or Plasmodium vivax and making them
more susceptible to other infections.[4] During the intra-erythrocytic
phase of the malaria parasite’s life cycle, the parasite can consume
up to 75% of an infected cell’s hemoglobin, releasing toxic free
heme, which can kill the parasite by disrupting cell membranes.
Malaria parasites have evolved a detoxification method, which
results in the formation of an inert crystallized heme aggregate
known as hemozoin (Hmz). Hmz has a spectroscopically identical
synthetic analog known as β-hematin (β-H).[5] The structure of β-H
has been interpreted from powder synchrotron X-ray diffraction
data to be an array of dimers linked through reciprocal iron–
carboxylate bonds to one of the propionate side chains, with the
dimers, in turn, linked by hydrogen bonds.[6] At least two models
have been proposed to explain how quinine-based antimalarial
drugs such as chloroquine and amodiaquine inhibit heme
aggregation. Crystal structure data of β-H indicate that chloroquine
absorbs onto the Hmz surface, resulting in inhibition of further
heme aggregation.[6] The second model proposes inhibition
involving noncovalent binding of chloroquine to the μ-oxo dimer.[7]
The drug : dimer complex is thought to ‘cap’ the formation of Hmz
resulting in a higher concentration of free heme in the food
vacuole, which ultimately causes membrane lysis. Consequently,
knowledge on the molecular and electronic structure of Hmz and
model compounds is critically important in developing new
antimalarial drugs.
tetraphenylprophyrin (TPP) and octaethylprophyrin (OEP) iron
complexes have been the conventional models for heme systems.
Crystallography and several spectroscopic techniques have been
applied to investigate the molecular and electronic structure of
heme compounds. Among the spectroscopic techniques,
resonance Raman (RR) spectroscopy is uniquely sensitive to the
stereochemical and bonding arrangements of atoms in
metalloporphyrin molecules,[16] and the technique has been used
to establish the correlation between heme structure and the
structurally sensitive high-wavenumber Raman marker bands of
D
4h and D2d symmetry forms of Ni(OEP).[8,10–12] RR spectroscopy is
also a powerful technique to investigate evidence for electronic
interaction between π systems[17,18] including π–π excitonic
*
*
Correspondence to: Kenneth J. Haller, School of Chemistry, Institute of Science,
Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
E-mail: ken.haller@gmail.com
Bayden R. Wood, Centre for Biospectroscopy and School of Chemistry, Monash
University, Victoria 3800, Australia.
E-mail: bayden.wood@monash.edu
a Department of Chemistry, Faculty of Science, Maejo University, Chiang Mai 50290,
Thailand
b School of Chemistry, Institute of Science, Suranaree University of Technology,
Nakhon Ratchasima 30000, Thailand
c
Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La
Trobe University, Melbourne, VIC, 3086, Australia
d Centre for Biospectroscopy and School of Chemistry, Monash University, VIC, 3800,
Australia
Abbreviations: Fe(OEP)picrate, 2,3,7,8,12,13,17,18-Octaethylporphyrinato(picrato)
iron(III); H2OEP, 2,3,7,8,12,13,17,18-Octaethylporphyrin; H2PPIX, 3,8,13,17-
Tetramethyl-7,12-divinylporphyrin-2,18 dipropionic acid; for
a generalized
porphyrin; Cα, Cβ, and Cm refer to the porphyrin core α, β, and meso carbon
positions, respectively.; H2TPP, 5,10,15,20-Tetraphenylporphyrin
J. Raman Spectrosc. (2017)
Copyright © 2017 John Wiley & Sons, Ltd.

R. Puntharod et al.
interactions, and for analyzing the effects of π–π stacking.[13,19]
Excitation in near-resonance with the B (Soret) band in metal
porphyrins results in polarized (Alg) totally symmetric modes
becoming enhanced, while excitation into the Q0 band (~575 nm)
results in the enhancement of depolarized (Blg and B2g) modes.
Enhancement in between the Q0 and Q1 transitions results in
anomalously polarized bands (A2g). Li et al.[20] undertook a study
to examine the contribution of ethyl substituent involvement in
the Ni(OEP) vibrational spectra by replacing the methylene
hydrogen atoms with deuterium. The spectra showed that the
strongest bands were observed in the 1000-cmꢀ1 region, which
were assigned to modes that were predominantly ethyl in
character, as revealed by their methylene-d shifts and normal
coordinate analysis.[20]
Specific RR bands can be used to identify Cα and Cβ substituents.
The ν2(A1g) and ν11(B1g) modes are sensitive to the nature of Cβ
substituents, while the ν3(A1g), ν19(A1g), and ν10(B1g) modes are
characteristic of Cα stretching vibrations.[21] Consequently, RR
spectroscopy is a powerful technique to gain structural information
on the various metalloporphyrins.
applied to help understand the dramatic enhancement of the
totally symmetric modes of β-H observed in the Raman profile
when applying near-IR excitation wavelengths.
Experimental
Synthesis of Fe(OEP)picrate
Fe(OEP)Cl [0.0153 g (0.025 mmol)] (Sigma-Aldrich) was dissolved in
5 mL of dichloromethane followed by addition of 2 mL of 0.0185 M
(0.037 mmol) picric acid solution. Subsequently, ~4 mL of ethanol
and ~6–8 mL of dimethyl sulfoxide were added. The mixture was
stirred vigorously until it was homogenous; then, 1 mL of methanol
was added, and the solution allowed to stand at room temperature
for 3 days. The dark blue solid product was filtered off and washed
with ethanol.
It should be noted that picric acid is a known skin and eye irritant
and is hazardous to ingest and inhale. It also should be noted that
picric acid should never be allowed to dry out and avoid crystals
forming around the cap. Picric acid becomes unstable at high
temperatures and reacts strongly with oxidizing materials and can
ignite in the presence of certain metals and water. Contact between
picric acid and concrete floors can result in the formation of
explosive salts.
In terms of coordination sphere stereochemistry, μ-oxo iron(III)
TPP and OEP porphyrins, both containing five-coordinate high-spin
iron(III) coordinated to an oxygen atom from the axial ligand, are
good model systems to study the molecular and electronic
structure of β-H.[22]
We have previously reported the RR spectra of Fe(OEP)Cl, Fe
(TPP)Cl, and hemin.[22] While these compounds have similar
structural properties, the most similar compound in terms of
the RR spectral profile is the Fe(OEP)picrate complex. As will
be discussed, this can be attributed to a similar type of crystal
packing of the picrate complex to β-H and Hmz and the number
of hydrogen bonding interactions. The RR spectra of β-H, and of
Cβ-substituted hemes, show characteristic bands of Cβ-
substituted hemes in the ranges of 1621–1639 and 750–
756 cmꢀ1 assigned to the νas(CαCm) and ν(pyr breathing) modes,
respectively.[14] These bands do not appear in the spectra of Cm-
substituted hemes. This work provides electronic and structural
Attenuated total reflection Fourier transform infrared
spectroscopy
Attenuated total reflection Fourier transform infrared spectroscopy
spectra were recorded on a Bruker Equinox spectrometer. Solid Fe
(OEP)picrate and Fe(OEP)Cl were placed on the window of an
attenuated total reflection cell, while picric acid solution was dried
onto the window. Spectra were recorded in absorption mode at
5-cmꢀ1 resolution with 60 interferograms co-added.
UV–Vis absorbance spectroscopy
information by using Fe(OEP)picrate (Fig. 1) as
a model
UV–Vis absorbance spectra were recorded by using a Cary 100
Bio UV–Vis spectrometer. Dichloromethane was used as the
solvent to dissolve both Fe(OEP)picrate and Fe(OEP)Cl, while the
picric acid solution was prepared by using distilled water as the
solvent.
compound for β-H and Hmz. Fe(OEP)picrate was chosen as a
model compound for β-H because the solid phase, unlike that
of [Fe(OEP)]2O discussed in our recent work,[22] shows the
enhancement of the oxidation state marker band, ν4, when
applying near-IR excitation laser wavelengths. The results
indicate that the solid-state electronic structure of β-H is
different to that of [Fe(TPP)]2O and [Fe(OEP)]2O, but similar to
Resonance Raman spectroscopy
that
of Fe(OEP)picrate complex. Single-crystal
X-ray
Resonance Raman spectra of the products were recorded on a
Renishaw system 2000 spectrometer by using 413 and 514-nm
excitation lines generated by a Spectra Physics Kr+–Ar+ Stabilite
2017 laser system, 633-nm excitation line from a helium–neon laser,
782-nm excitation line generated by a diode laser, and 830-nm
excitation line from a diode laser. The approximate laser power at
the sample is 0.039, 1.3, 1.6, 0.5, and 4.0 mW for the excitation
wavelengths 413, 514, 633, 782, and 830 nm, respectively. The
system was equipped with an Olympus optical microscope and a
Zeiss ×60 water immersion objective to enable spectral acquisition
in water to minimize thermal degradation of the solid porphyrin
samples. Picric acid was dried in air before recording the RR
spectrum by using the ×40 objective in the dry state. Spectra were
recorded between 1800 and 230 cmꢀ1. Each spectrum was
accumulated as one scan with a laser exposure of 10 s. The spectral
resolution is between 1 and 2 cmꢀ1. The percentage of power at
the sample was 10% power with 4 pixels of the charge-coupled
crystallography of the molecular and supramolecular structure
of Fe(OEP)picrate and UV–Vis absorbance spectroscopy was
Figure 1. Schematic diagram of the structure of Fe(OEP)picrate.
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J. Raman Spectrosc. (2017)

An improved model for malaria pigment and β-hematin
device binned together to improve the signal-to-noise ratio. The
instrument was calibrated to the 520.5-cmꢀ1 Si band. No change
was observed in the surfaces of the samples based on microscopic
examination before and at the end of data collection. The
enhancement factors were calculated relative to the largest band
in the spectrum located at 1558 cmꢀ1, which remained invariant
for all laser wavelengths investigated except 413 nm. This band
was consequently chosen as the normalization band, and the
enhancement factors calculated relative to its intensity. For the
413-nm spectrum, the normalization band was 1580 cmꢀ1, which
was the most intense band in the spectrum at this excitation laser
line.
Theoretical procedures
Vibrational spectra of picric acid and the iron picrate complex were
optimized by using the B3LYP/6-31G(d) and M06L/cc-pVDZ levels
of density functional theory to facilitate interpretation of the
experimental spectra. The B3LYP functional is widely used for its
generally reliable vibrational predictions. M06 belongs to a
Minnesota family functional and is designed to describe transition
metal complexes. It has been used to study different biological
systems.[23,24] M06L optimizations were conducted by using the
conductor-like polarizable continuum model with water as the
solvent. All calculations were performed with the Gaussian09
suite of programs. Scaling for both B3LYP and M06L calculations
is necessary to negate shortcomings in the prediction of stretching
vibrations in particular such as basis set limitations.[25] These factors
were determined individually for both levels of theory by
calculating the average ratio between the experimental and
predicted values.[26] Geometry optimizations preceded vibrational
wavenumber value calculations, with Fe(OEP)picrate complex; the
crystal structure was used as the starting structure. B3LYP geometry
optimization of the free Fe(OEP)picrate monomer results in the
picrate ring tilting at a greater angle to the porphyrin ring than in
the more constrained crystal environment, with the Cpara(picrate)–
Cα(heme) distance increased from 4.42 to 5.71 Å. Simulated Raman
spectra were computed with Raman intensities calculated for 830-
nm excitation (v0 = 12048 cmꢀ1) by using the relation:
Figure 2. UV-Vis spectra of Fe(OEP)picrate, Fe(OEP)Cl, and picric acid.
[Colour figure can be viewed at wileyonlinelibrary.com]
700 and 900 nm. In an earlier study,[22] we presented UV–Vis spectra
of Fe(TPP)Cl, Fe(OEP)Cl, [Fe(TPP)]2O, and [Fe(OEP)]2O. All spectra
showed a band between 600 and 690 nm assigned to a charge-
transfer transition indicative of high-spin ferric hemes. However,
no obvious electronic transition was observed in the near-IR region
(750–900 nm) where the totally symmetric modes are dramatically
enhanced.[22] The UV–Vis spectra of hemin converted to β-H have
also been reported by our group.[14] These spectra also show a
charge-transfer transition indicative of high-spin ferric hemes, in
this case shifting from 612 nm in hemin to 649 nm in β-H. Upon
conversion to β-H, we noted a small broad low-lying transition
centered at 867 nm that we assigned to a z-polarized charge-
transfer transition dxy → eg(π*). The extraordinary band
enhancement observed when exciting with near-infrared excitation
wavelengths in β-H when compared with hemin was explained in
terms of an aggregated enhanced Raman scattering hypothesis
based on the intermolecular excitonic interactions between
porphyrinic units that occurs upon excitation into this broad low-
lying electronic transition centered at 867 nm.[28] Ali and
Openeer[29] recently performed high-level time-dependent density
4
Ii ¼ fðv0 ꢀ viÞ Si=vi½1 ꢀ expðꢀhcvi=kTÞꢁ
where Si is the calculated Raman activity for the ith mode at vi
cmꢀ1; h, c, and k are the fundamental constants; temperature T
was assumed to be 298 K, and f is a normalization factor. A
Lorentzian lineshape of full-width half-maximum (FWHM) =
10 cmꢀ1 was applied.
Results
UV–Vis spectra
Figure 2 shows the UV–Vis–near-infrared spectra of picric acid, Fe
(OEP)Cl, and Fe(OEP)picrate. The Soret band for Fe(OEP)picrate is
slightly red-shifted from the Fe(OEP)Cl complex from 378 to
384 nm. The Fe(OEP)picrate complex contains the O atom from
the picrate, which is a π-bonding ligand resulting in the Soret
absorption band appearing at a longer wavelength. Picric acid, on
the other hand, absorbs very strongly at 354 nm. Both Fe(OEP)Cl
and Fe(OEP)picrate show weak charge-transfer bands at
approximately 630 nm indicative of high-spin ferric hemes.[27] Fe
(OEP)Cl and Fe(OEP)picrate absorb very weakly in the near-IR and
show evidence of a low-lying charge-transfer transition between
Figure 3. Attenuated total reflection Fourier transform infrared spectra of
Fe(OEP)picrate, picric acid, and Fe(OEP)Cl. [Colour figure can be viewed at
wileyonlinelibrary.com]
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R. Puntharod et al.
Figure 4. Experimental Raman spectra of deep yellow solid-state picric acid along with a normal Raman simulation from the B3LYP/6-31G(d) calculation.
Table 1. Observed peak positions (cmꢀ1) compared with predicted band positions, strengths, and local coordinate assignments for picric acid
Experimental
Calculated B3LYP/6-31G*
Raman activity Mode description
Å4 amuꢀ1
Raman
413 nm 514 nm 633 nm 782 nm 830 nm
IR
Scaled ×0.970
cmꢀ1
IR int
km molꢀ1
1632
—
1632
1614
—
1633
—
1633
1610
—
1636
1613
—
—
1612
—
1648
1622
1606
1581
1565
1478
1441
1393
1358
278
298
110
16
7.5
0.7
15
Ring CC str
NO2 antisym (para)
—
—
NO2 antisym (ortho) + OH bend
CC ring str + OH bend
NO2 antisym + CC ring str
CO str (and CH in plane bend)
OH bend
1562
1531
—
1563
1532
—
1563
1532
—
—
—
—
73
1530
—
1534
—
1540
—
41
42
87
9.3
3.1
8.7
201
—
—
—
—
—
1426
—
220
24
—
—
—
—
—
Ring str (+ OH bend)
1344
1344
1346
1345
1348
—
149
NO2 symstr (sym comb of para +
non-H-bonded)
—
—
—
—
—
1339
1347
349
98
NO2 symstr (antisym comb of para +
non-H-bonded)
—
—
—
—
—
—
—
—
—
—
—
1324
1296
1284
196
102
158
14
23
Ring CC str
—
Ring CC, OH, and CH in plane bends
NO2 symstr (H-bonded ortho) + OH
and CH bends
1280
1280
1281
1278
1280
1270
140
1179
—
1178
—
1178
—
1177
—
1179
—
—
—
1163
1151
1069
949
941
928
906
815
812
769
17
44
88
0.8
18
32
65
0.3
2.3
44
33
9.7
3.0
2.1
1.1
16
Sym CN str (all three)
CH and OH in plane bend
CH2 in plane bend
1092
—
1092
—
1092
—
1088
—
1090
—
1087
—
CH2 out of plane (antisym)
CH2 out of plane (sym)
para-CN str (+ other 2)
ortho-CN antisymstr
—
—
—
—
—
—
942
—
942
—
944
—
940
—
941
—
—
—
5.0
23
832
—
832
—
832
—
830
—
831
—
—
In plane ring def + δ (ONO) sym
δ (ONO) para (+ in plane ring def)
HO out of plane def
—
3.8
2.1
—
—
—
—
—
—
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An improved model for malaria pigment and β-hematin
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R. Puntharod et al.
functional theory (DFT) calculations to simulate the UV–Vis
absorption spectrum of Hmz. The theoretical spectra matched very
well with the available experimental spectra. The lowest energy
electronic transition at 880 nm was found to be completely due
to spin-polarized electronic transitions of one spin type only. The
authors also used calculations to predict a high-spin S = 5/2 on each
of the Fe centers, in addition to
a weak intramolecular
antiferromagnetic exchange coupling between the two Fe centers,
which results in a singlet state for the minimum of the potential
energy surface. It appears that compounds including β-H and Fe
(OEP)picrate, which exhibit enhanced totally symmetric modes
when exciting with near-IR, have broad low-energy electronic
transitions in the 700 to 900-nm region of the electromagnetic
spectrum.
Infrared spectra
Attenuated total reflection Fourier transform infrared spectra
are shown in Fig. 3. In this section the results discussed are
the B3LYP calculations. The spectrum of the complex contains
features derived from both the picric acid and the OEP
components, but is not a simple linear addition. According to the
DFT calculations, the normal coordinate composition of the NO2
and CC ring stretch modes changes in the complex, in part due to
changes in the bonding and possibly due to the sterically induced
rotation of the ortho-NO2 functional groups. This leads to shifts in
the band positions and intensities between picric acid and the
complex. For example, the band at 1612 cmꢀ1 in picric acid,
tentatively assigned to two close lying νas(NO2) modes, is mixed
with the CC ring stretching from the porphyrin. Bands at 1339
and 1270 cmꢀ1 are assigned to the nitro group symmetric
stretches, νs(NO2), in picric acid.[18] The νas(NO2) modes in the Fe
(OEP)picrate spectrum are split into two bands at 1604 and
1582 cmꢀ1 due to the different molecular environments of the
ortho and para-substituted NO2 groups, and a further pair of bands
appears at 1533 and 1520 cmꢀ1. The corresponding ortho-νs(NO2)
split modes appear at 1331 cmꢀ1 along with a shoulder feature at
~1320 cmꢀ1. A number of bands can be directly assigned to the
OEP that appear both in Fe(OEP)picrate and Fe(OEP)Cl. These
include bands at 1470, 1446, 1370, 1315, 1110, 1054, 1014, 980,
957, 879, and 842 cmꢀ1, which are absent in the RR spectra. Fonda
et al.[30] reported that the IR spectrum of Cu(octaethylchlorin)
exhibited seven modes 1464, 1452, 1375, 1064, 1058, 1015, and
956 cmꢀ1, which are in very similar positions to the bands observed
in Fe(OEP)picrate. These bands were insensitive to metal
substitution, methine deuteration, and changes in peripheral
substituents and hence were assigned to ethyl group vibrations.[30]
They also noted that these modes did not appear in the RR spectra,
except for the 1464-cmꢀ1 band that was present in nearly all of the
RR spectra of the chlorin complexes that they examined.[30] Kincaid
et al.[31] reported high-resolution infrared spectra of divalent metal
(Mn, Fe, Co, Ni, Cu, Zn) complexes of octaethylporphine obtained at
ꢀ15 K in argon matrices or as thin films. They observed bands at
1473 and 1456 cmꢀ1 in Ni(OEP), similar in position to bands
observed at 1470 and 1446 cmꢀ1 in the Fe(OEP)picrate complex
in this work, that they assigned to ethyl group vibrations. They
found that the δsym(CH3) moiety of the ethyl group was observed
as a medium-intensity band within the 1375 to 1385-cmꢀ1 region,
whereas we observe a similar band at 1370 cmꢀ1, which is also
assigned to this mode in the Fe(OEP)picrate complex. Kincaid
et al.[21] assigned bands at 1119, 1069, 1061, and 1021 cmꢀ1 to ethyl
group vibrations in Ni(OEP), which correlate to bands at 1110, 1054,
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An improved model for malaria pigment and β-hematin
and 1014 cmꢀ1 in the Fe(OEP)picrate complex. Kincaid et al.[31] also
assigned a band at 959 cmꢀ1 to an ethyl group vibration, and we
observe a similar intensity band at 954 cmꢀ1 corresponding to this
assignment.
bonding is weaker. The agreement of observed and predicted
spectral shifts between bands in picric acid and the picrate complex
confirms the assignments.
The B3YLP predictions of the Raman bands seen in the picrate
complex are assigned to porphyrin modes. The spin-state marker
band, ν10 (Cα–Cm stretch), at ~1630 cmꢀ1 is in agreement with the
empirical correlation between the position of ν10 for five-coordinate
high-spin Fe(OEP) derivatives[35] and is also a normal value for an Fe
(III) heme with a strong π acceptor axial ligand such as picrate.[35] ν4,
which is a pyrrole half-ring stretching mode, is observed at 1375–
1378 cmꢀ1, indicating iron(III).[35] The ν4 band is shifted to
1372 cmꢀ1 in Fe(OEP)Cl due to the effect of retention of planar
pyrrole units on the geometry of the 16-membered ring and the
resulting induced vibrational wavenumber value shifts.[10] Binding
of the picrate O atom as a π acceptor axial ligand to Fe competes
with Fe dπ → porphyrin π* backbonding, thereby slightly increasing
the wavenumber value of the ν4 band,[36–38] and changes the
symmetry of the heme complex. It should be noted that not only
is the ν4 band sensitive to the back donation into porphyrin π*
and π–acid ligand orbitals of iron π electrons,[37] but it is also
sensitive to the oxidation and spin state.[36] The predicted B3LYP
spectrum shows ν4 at 1366 cmꢀ1, which is in fairly good agreement
with the experimental spectrum. The band at 1480 cmꢀ1
dominates the calculated spectrum but appears very weak and
shifted to ~1495 cmꢀ1 in the experimental spectrum. This mode
is a complex mode involving a number of ethyl CH2 deformation
modes. The reason for its diminished intensity in the experimental
spectrum is that the ethyl deformation modes are not subject to
resonance effects that enhance the heme modes. Alternatively,
the intensity calculation fails as there are so many degenerate
Raman spectra
Experimental Raman spectra of deep yellow solid-state picric acid
are compared with a normal Raman calculation from B3LYP/6-
31G(d) results in Fig. 4. Observed and predicted band positions
are listed in Tables 1 and 2, respectively. DFT calculations have
previously been performed on β-H by using BP86/SDD level of
theory.[32,33] We have applied a higher level of theory to examine
the Fe(OEP)picrate complex. The bands at approximately 1610,
1345, and 832 cmꢀ1 are assigned to the νas(NO2), νs(NO2), and the
in-plane (scissoring) deformation δ(NO2), respectively, of the
aromatic NO2 groups of picric acid. These bands appear at 1604,
1348, and 814 cmꢀ1 in the calculated spectrum. Figure 5 compares
experimental RR spectra of Fe(OEP)picrate with the B3LYP-
predicted spectrum computed. Perhaps rather surprisingly, this
level of theory appeared to reproduce the observed band profile
better than M06L/cc-pVDZ. Table 2 compares the band positions
observed in the spectrum of the complex with corresponding
values computed at both B3LYP/6-31G(d) and M06L/cc-pVDZ levels
of theory. The Fe(OEP)picrate complex shows Raman bands at 1607
and 1333–1339 cmꢀ1 in the region of νas(NO2) and νs(NO2),[34]
observed when using near-IR excitation wavelengths that are
shifted compared with the respective positions for picric acid
(1610–1613 and 1345–1348 cmꢀ1), indicating that the N–O
Figure 5. Resonance Raman spectra of Fe(OEP)picrate along with a normal Raman simulation from the B3LYP/6-31G(d) calculation.
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R. Puntharod et al.
modes. The M062X spectrum is in good agreement with the
assignment of experimental vibrational modes based on the
B3LYP spectrum (for more detail, refer to Table 2).
patterns of relative enhancement factors when comparing the
same applied Raman excitation wavelength especially when
applying near-IR excitation (Fig. S1). Figure 6 displays the relative
enhancement factors of Fe(OEP)picrate and β-H for the totally
symmetric A1g mode, ν4, assigned to 1370–1375 cmꢀ1 [ν(pyr-half-
ring)sym] and the nontotally symmetric B1g mode, ν15, assigned to
750 cmꢀ1 [ν(pyr-breathing)]. For both modes, the enhancement
factors are comparable between the two complexes. The ν4 band
is enhanced when exciting with 413-nm wavelength laser source
but is weaker when excited with 514 nm and is much weaker when
applying 633-nm excitation, while it is stronger when using 782-nm
excitation wavelength and completely dominates the Raman
spectrum when the 830-nm excitation wavelength is applied.
Moreover, Fe(OEP)picrate and β-H are also comparable in the
appearance of ring and pyrrole breathing modes such as the bands
at 970–976 cmꢀ1, δas(pyr deformation), 670–678 cmꢀ1, δs(pyr
deformation), 821–822 cmꢀ1, γ(CmH), and 801–802 and 751–
752 cmꢀ1, ν(pyr breathing). Thus, the enhancement of Fe(OEP)
picrate modes throughout the spectrum is very similar to that of
β-H for all modes related to the conformation of the porphyrin
plane at all excitation wavelengths investigated.
Early studies suggested that saturated alkyl substituents are not
generally thought to couple electronically to aromatic
chromophores, although the influence of inductive and
hyperconjugative effects is readily seen in shifts of the electronic
transition energies.[39] Li et al.,[20] on the other hand, reported that
the orientation of the ethyl substituents places the σ orbital in the
proper orientation to interact with the π* orbital. Consequently,
the ethyl coordinates, especially C1C2 stretching, can induce
significant excited-state origin shifts and Q/B vibronic coupling.[20]
The mean spectra of Fe(OEP)picrate and β-H synthesized by the
acidification of hematin with glacial acetic acid[22] show similar
Discussion
Fe(OEP)picrate serves as a good model compound for β-H and Hmz
because it has similar bands in terms of intensity and position to
these compounds, especially when exciting with 830 nm. Fe(OEP)
picrate shows bands at 1627 and 751 cmꢀ1 that are characteristic
of beta-substituted hemes, which are observed in OEP derivatives.
Neither of these bands appear in the spectra of Fe(TPP)Cl, [Fe
(TPP)]2O, and dimethyl ester of protoheme. Another reason for
choosing Fe(OEP)picrate as a model compound for β-H and Hmz
Figure 6. Enhancement factors of ν4 and ν2 following vector normalization
over the 1800–2000 cmꢀ1 for Fe(OEP)picrate and β-hematin.
Figure 7. The crystal packing of Fe(OEP)picrate.
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J. Raman Spectrosc. (2017)

An improved model for malaria pigment and β-hematin
Figure 8. Closest porphyrin plane to porphyrin plane contact distance for β-hematin molecules in the crystal structure. Projection diagram drawn by
DIAMOND program using XETXUP from the CSD. [Colour figure can be viewed at wileyonlinelibrary.com]
is that it shows the enhancement of the oxidation state marker
band, ν4, when applying near-IR excitation laser wavelengths.
Neither [Fe(OEP)]2O, Fe(OEP)Cl, nor hemin show this dramatic
enhancement. Furthermore, Fe(OEP)Cl has chloride as the axial
ligand, which is different from Fe(OEP)picrate that has oxygen as
axial ligand same as β-H.
The enhancement of the vibrational mode ν4 in β-H when using
near-IR laser excitation indicates that β-H contains more
supramolecular interactions compared with other heme dimers
and monomers based on examination of the number of
intermolecular bonds as deduced by the analysis of structures
generated by using ORTEP-III.[22] This is consistent with β-H having
strong hydrogen bonds to the propionate linkage. In this case, the
relative intensity of ν4 may be an indicator of both the strength of
π–π and supramolecular interactions.
Implications for intermolecular interaction of Fe(OEP)picrate in
β-hematin
Fe(OEP)picrate shows a similar Raman enhancement profile to that
of β-H in both stretching and bending modes of pyrrole breathing,
indicating that they are similar in chromophoric and electronic
structure. Moreover, Fe(OEP)picrate could resemble β-H in the
conformation of the porphyrin core.
To predict the intermolecular interaction and crystal packing of
β-H, we used the correlation of the enhancement of the ν4 band
of Fe(OEP)picrate and β-H when applying near-IR excitation
wavelengths including 782 and 830 nm. It was hypothesized that
the enhancement of the totally symmetric mode at near-IR
excitation wavelengths could in part be attributed to
supramolecular interaction. This implies that the study of crystal
structures of malaria pigment models may be a very fruitful source
of information on intermolecular interaction of β-H. Thus, it is
hypothesized that the porphyrin core planes of β-H and Fe(OEP)
picrate are very similar in terms of symmetry and intramolecular
and intermolecular hydrogen bond contacts.
Analysis of the crystal packing of Fe(OEP)picrate in Fig. 7 shows
extensive π–π stacking between adjacent porphyrin cores as well
as through the components of the axial ligand. However, there is
considerably less π–π stacking in β-H due to the propionate linkage,
but additional intermolecular interactions do occur via the strong
propionic acid hydrogen bond linkage.[22] The closest inter-
molecular distance from carbon to carbon atoms in the heme ring
between dimeric β-H is 3.435(2) Å (Fig. 8), which is almost the same
as the closest distance observed in the Fe(OEP)picrate, i.e. 3.443
(3) Å (Fig. 7), further emphasing the similarity of these structures
in terms of intermolecular interactions. The electronic spectra are
also similar with both showing a weak broad near-IR transition
between 800 and 900 nm and a charge-transfer band centered
around 630 nm.
Conclusion
Fe(OEP)picrate was synthesized by the reaction of Fe(OEP)Cl and
picric acid. The molecular structure information provided by RR
spectroscopy correlates well with that from single-crystal X-ray
crystallographic analysis[40]; both techniques verify Fe(OEP)picrate
to be a five-coordinate high-spin iron(III) complex. Fe(OEP)picrate
is a good model to understand the stereochemistry of β-H based
on the similar enhancement of the totally symmetric mode ν4 and
nontotally symmetric mode ν15 which is assigned to ν(pyrrole
breathing) at every excitation wavelength. Moreover, the
enhancement of pyrrole-related modes in the 670 to 850-cmꢀ1
range of Fe(OEP)picrate when using near-IR excitation further
demonstrates the structural similarity to β-H. The enhancement of
ν4 is greater for β-H than Fe(OEP)picrate because β-H contains a
closer intermolecular porphyrin ring and extensive hydrogen
bonding in comparison with Fe(OEP)picrate. This work indicates
that β-H contains more supramolecular interactions compared with
Fe(OEP)picrate and supports the notion that β-H is linked by the
hydrogen-bonded propionate.
Acknowledgements
R. Puntharod acknowledges financial support from the Office of the
Commission of Higher Education, Thailand. This work is supported
by an Australian Research Council (ARC) Discovery Grant
DP140102504. BRW is supported by an ARC Future Fellowship grant
FT120100926. The authors gratefully acknowledge a generous
allocation of computer resources through the Monash eResearch
Centre and the National Computational Infrastructure in Canberra.
J. Raman Spectrosc. (2017)
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R. Puntharod et al.
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