Isocyanide or nitrosyl complexation to hemes with varying tethered axial base ligand donors: synthesis and characterization

Article abstract of DOI:10.1007/s00775-016-1369-4

Abstract: A series of ferrous-heme 2,6-dimethylphenyl isocyanide (DIMPI) and ferrous-heme mononitrosyl complexes have been synthesized and characterized. The heme portion of the complexes studied is varied with respect to the nature of the axial ligand, including complexes, where it is covalently tethered to the porphyrinate periphery. Reduced heme complexes, [(F₈)Fe^II], [(P^Py)Fe^II], [(P^Im)Fe^II], and [(P^ImH)Fe^II], where F₈?=?tetrakis(2,6-difluorophenyl)-porphyrinate and P^Py, P^Im, and P^ImH are partially fluorinated tetraaryl porphyrinates with covalently appended axial base pyridyl/imidazolyl or histamine moieties, were employed; P^ImH is a new construct. Room temperature addition of DIMPI to these iron(II) complexes affords the bis-isocyanide species [(F₈)Fe^II-(DIMPI)₂] in the case of [(F₈)Fe^II], while for the other hemes, mono-DIMPI compounds are obtained, [(P^Py)Fe^II-(DIMPI)] [(2)-DIMPI], [(P^Im)Fe^II-(DIMPI)] [(3)-DIMPI], and [(P^ImH)Fe^II-(DIMPI)] [(4)-DIMPI]. The structures of complexes (3)-DIMPI and (4)-DIMPI have been determined by single crystal X-ray crystallography, where interesting H…F(porphryinate aryl group) interactions are observed. ¹⁹F-NMR spectra determined for these complexes suggest that H…F(porphyrinate aryl groups) attractions also occur in solution, the H atom coming either from the DIMPI methyl groups or from a porphyinate axial base imidazole or porphyrinate pyrrole. Similarly, we have used nitrogen monoxide to generate ferrous-nitrosyl complexes, a five-coordinate species for F₈, [(F₈)Fe^II-(NO)], or low-spin six-coordinate compounds [(P^Py)Fe^II-(NO)], [(P^Im)Fe^II-(NO)], and [(P^ImH)Fe^II-(NO)]. The DIMPI and mononitrosyl complexes have also been characterized using UV–Vis, IR, ¹H-NMR, and EPR spectroscopies. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00775-016-1369-4

J Biol Inorg Chem
DOI 10.1007/s00775-016-1369-4
ORIGINAL PAPER
Isocyanide or nitrosyl complexation to hemes with varying
tethered axial base ligand donors: synthesis and characterization
Savita K. Sharma1 · Hyun Kim1 · Patrick J. Rogler1 · Maxime A. Siegler1 ·
Kenneth D. Karlin1
Received: 6 April 2016 / Accepted: 8 June 2016
© SBIC 2016
Abstract A series of ferrous-heme 2,6-dimethylphe-
nyl isocyanide (DIMPI) and ferrous-heme mononitrosyl
complexes have been synthesized and characterized. The
heme portion of the complexes studied is varied with
respect to the nature of the axial ligand, including com-
plexes, where it is covalently tethered to the porphyrinate
periphery. Reduced heme complexes, [(F8)FeII], [(PPy)
FeII], [(PIm)FeII], and [(PImH)FeII], where F8 = tetrakis(2,6-
difluorophenyl)-porphyrinate and PPy, PIm, and PImH are
partially fluorinated tetraaryl porphyrinates with covalently
appended axial base pyridyl/imidazolyl or histamine moie-
ties, were employed; PImH is a new construct. Room tem-
perature addition of DIMPI to these iron(II) complexes
affords the bis-isocyanide species [(F8)FeII-(DIMPI)2] in
the case of [(F8)FeII], while for the other hemes, mono-
DIMPI compounds are obtained, [(PPy)FeII-(DIMPI)] [(2)-
monoxide to generate ferrous-nitrosyl complexes, a five-
coordinate species for F8, [(F8)FeII-(NO)], or low-spin six-
coordinate compounds [(PPy)FeII-(NO)], [(PIm)FeII-(NO)],
and [(PImH)FeII-(NO)]. The DIMPI and mononitrosyl com-
plexes have also been characterized using UV–Vis, IR, 1H-
NMR, and EPR spectroscopies.
Graphical abstract
DIMPI], [(PIm)FeII-(DIMPI)] [(3)-DIMPI], and [(PImH
)
Keywords Ferrous heme · Heme isocyanide · X-ray
structures · Heme nitrosyl · H…F(porphyrinate)
interactions
FeII-(DIMPI)] [(4)-DIMPI]. The structures of complexes
(3)-DIMPI and (4)-DIMPI have been determined by
single crystal X-ray crystallography, where interesting
H…F(porphryinate aryl group) interactions are observed.
19F-NMR spectra determined for these complexes suggest
that H…F(porphyrinate aryl groups) attractions also occur
in solution, the H atom coming either from the DIMPI
methyl groups or from a porphyinate axial base imidazole
or porphyrinate pyrrole. Similarly, we have used nitrogen
Introduction
Heme containing proteins participate in critical and diverse
biological functions which include electron transfer, cataly-
sis, and signaling. For the latter two subjects, small mol-
ecule diatomic gases are often involved, such as O2, NO,
and CO [1, 2]. There exist classes of proteins which serve
to discriminate between these molecules for purposes,
including detection, signaling, and/or function [3, 4]. For
molecular oxygen, roles include storage transfer, or activa-
tion of O2 for substrate oxidation or oxygenation chemis-
tries [5]. Nitric oxide (nitrogen monoxide) is a signaling
molecule [6] such as in its interaction with the heme center
in guanylate cyclase, wherein binding leads to a signaling
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-016-1369-4) contains supplementary
material, which is available to authorized users.
* Kenneth D. Karlin
karlin@jhu.edu
1
Department of Chemistry, The Johns Hopkins University,
Baltimore, MD 21218, USA
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J Biol Inorg Chem
cascade resulting in smooth muscle relaxation [7–14]. Car-
bon monoxide is also a diatomic gas which is biosynthe-
sized through heme O2-activation chemistry (i.e., in heme
oxygenases [15–17]); CO can also act in biological signal-
ing via heme protein binding [18].
In the history of the study of O2 interactions with hemo-
proteins, the investigation of the binding of diatomic surro-
gate ligands, mainly CO and NO, has received considerable
attention. These have been utilized as structural models, but
also are useful in the study of ligand binding dynamics and
electronic structure of the ligated reduced hemes [19–23].
For example, CO bound hemes are amenable to vibra-
tional spectrosopic analyses, along with CO photoejection
and CO rebinding study [21, 24]. As well, reduced hemes
with NO bound are active for EPR spectroscopic interro-
gation. The replacement of CO with isocyanide (RNC:)
ligands has also been found to be a useful probe to investi-
gate vibrational spectroscopy and binding kinetics or heme-
ligand photodissociation and time-resolved rebinding. The
strong isocyanide N–C triple-bond stretching vibration can
be monitored, whereas variation in the size or nature of the
isocyanide R-group, e.g., R = aryl vs –Me or –tBu, pro-
vides insights concerning steric effects or issues of small
ligand binding to iron relative to the size or shape of a pro-
tein active-site pocket [25–30].
One of our research group’s major foci has been and
continues to be the study of dioxygen binding and reduc-
tion at heme-copper heterobinuclear metal ion centers [31].
We seek to determine how neighboring copper-ligand moi-
eties influence the binding of O2 to hemes, and in a com-
plementary manner, see how hemes affect O2 binding to
copper ion in varying ligand environments. Then, as such
synthetic heme-O2-Cu assemblies can be compared and
related to the active-site chemistry of heme-copper oxi-
dases which bind and reduce O2 to two water molecules
(while also translocating protons through a mitochondrial
membrane which downstream facilitates ATP biosynthe-
sis), we are interested in elucidating detailed insights into
the O–O reductive cleavage process, as a function of the
exact nature (structure and electronics/bonding behavior)
of the heme, the copper ligand and the source of electrons
(E° value) and protons (pKa). Additional factors include
heme or copper-ligand electron-donating ability (and thus
the FeIII/FeII and/or CuII/CuI E° value), nature of porphyri-
nate peripheral groups, and/or copper-ligand denticity and
their possible steric influences or affects upon the entire
heme-O2-Cu(ligand) structure, for example the Fe…Cu
distance in the heme-O2-Cu assembly [31–35].
Fig. 1 Synthetic steps involved in generation of a low-spin heme-
peroxo-copper complex
depend on the detailed nature of the heme, the copper
ligand, the axial base, etc., as mentioned above [31, 36].
As such, it is critical that complementary investiga-
tions be carried out on surrogate ligand binding to the vari-
ous components of our assemblies. One such aspect is the
investigation of O2, CO, NO, and/or RNC binding to varying
designed porphyrinoids, wherein the axial base ligand is var-
ied between a weak O-donor (as solvent), such as tetrahydro-
furan (THF), or N-donor ligands, such as DCHIm (Fig. 2),
or a covalently linked imidazolyl or pyridyl ligand. To bet-
ter our understanding of the chemistry of full heme-O2-Cu
assemblies, it is very useful to understand the structural
aspects, physical properties, and reactivity patterns of just
these heme-containing moieties with varying axial ligand
base, with O2, CO, NO, and RNCs. In fact, some of this
information has been obtained and previously published, in
particular, for heme-O2 (FeIII-superoxide) complexes and for
four of the five species, as shown in Fig. 2 [37, 38]. In addi-
tion, some of the related (heme)FeII-CO and (heme)FeII-NO
compounds have also been described [37]. Here, we report
on advances made from the study of new (heme)FeII derived
adducts with DIMPI along with nitric oxide, using the F8,
PIm, PPy, and a brand new heme PImH possessing a covalently
linked histamine moiety, see Fig. 2 and Eqs. 3 and 4. New
insights have been obtained based on the X-ray structures
and physical properties which are described and also com-
pared with the corresponding adducts using F8, which does
not incorporate a tethered axial ligand:
(P)FeII + DIMPI → (P)FeII − DIMPI
(3)
A specific example of such a synthetic construct is
shown in Fig. 1, where also an added heme axial ‘base’,
dicyclohexylimidazole (DCHIm), is present [35, 36]. As
anticipated, the structural, spectroscopic properties and
reactivity of this and other such assemblies significantly
(P)FeII + ·NO(g) → (P)FeII − NO
(4)
(P) = F8, PPy, PIm, PImH
.
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J Biol Inorg Chem
Fig. 2 Ferric heme-superoxo complexes previously characterized and a new five-coordinate ferrous heme
peak. Electron paramagnetic resonance (EPR) spectra were
Materials and methods
recorded with a Bruker EMX spectrometer equipped with
a Bruker ER 041 × G microwave bridge and a continuous-
flow liquid helium cryostat (ESR900) coupled to an Oxford
Instruments TC503 temperature controller. Spectra were
obtained at 8 K under nonsaturating microwave power
conditions (ν = 9.428 GHz, microwave power = 0.201
mW, modulation amplitude = 10 G, microwave fre-
quency = 100 kHz, and receiver gain = 5.02 × 103). EPR
spectra were simulated using the Easy Spin (see ESI).
The compounds (F8)FeII (1) [35, 40, 41], (PPy)FeII (2)
[37, 38], and (PIm)FeII (3) [37, 42] were synthesized as pre-
viously described.
All chemicals and solvents were purchased as commer-
cially available analytical grade unless otherwise speci-
fied. Tetrahydrofuran (THF, inhibitor free) was dried over
sodium/benzophenone ketyl, and purified by distillation
under argon. Pentane was dried by distillation over calcium
hydride. Toluene was used after passing through a 60 cm
long column of activated alumina (Innovative Technolo-
gies), under argon. 2,6-Dimethylphenyl isocyanide (DIMPI)
was purchased from Sigma Aldrich. ·NO gas was obtained
from Matheson Gases and purified following methods pre-
viously described in the literature [39]. A three-way syringe
was used for the addition of ·NO gas to all metal complex
solutions. Preparation and handling of air-sensitive com-
pounds was performed under an argon atmosphere using the
standard Schlenk techniques or in an MBraun Labmaster
130 inert atmosphere (less than 1 ppm O2, less than 1 ppm
H2O) drybox filled with nitrogen. Deoxygenation of all sol-
vents was accomplished by either repeated freeze/pump/
thaw cycles or bubbling with argon for 45–60 min.
Synthesis of (PImH)FeII (4)
The synthesis of complex (PImH)FeII (4) involves multiple
steps,, as shown in Scheme 1, where F6(NHCOCH2CH2Br)
TPPH2 [43] and LN3Tr [44] were synthesized as previously
described.
PImTr: To a 50 mL round bottom flask containing 1.8 g
(2.0 mmol) of F6(NHC(O)CH2CH2Br)TPPH2 and 0.7 mL
of diisopropylethyl amine was added to 1.7 g (4 mmol) of
LN3Tr in 15 mL of THF. The resulting mixture was heated
to reflux overnight. After cooling to room temperature, the
solvent was removed under vacuum. The solid residue was
re-dissolved in dichloromethane and washed several times
with distilled water, from which the organic layer was dried
over anhydrous sodium sulfate. Column chromatography of
the crude material on alumina (ethyl acetate:hexane = 97:3,
Rf = 0.4) yielded 2.3 g (1.9 mmol, 65 %) of the desired prod-
uct as a purple solid. ESI–MS (m/z): 1236.11 (M+H)+ (Fig.
Instrumentation: Benchtop UV–Vis measurements were
carried out using a Hewlett Packard 8453 diode array spec-
trophotometer equipped with HP Chemstation software
and a Unisoku thermostated cell holder for low-tempera-
ture experiments. A 10 mm path length quartz cell cuvette
modified with an extended glass neck with a female 14/19
joint, and stopcock was used to perform all UV–Vis experi-
ments. ESI–MS were acquired using a Finnigan LCQ Duo
ion-trap mass spectrometer equipped with an electrospray
ionization source (Thermo Finnigan, San Jose, CA). The
heated capillary temperature was 250 °C, and the spray
voltage was 5 keV. Spectra were recorded continuously
after injection. Infrared (IR) spectra were obtained on solid
samples using a Thermo Scientific Nicolet Nexus 670 Fou-
rier transform IR (FT-IR) spectrophotometer with ATR
1
S1); H-NMR (CD3CN, 300 MHz; δ, ppm, RT): 8.88–8.78
(m, 8H, pyrrole-H), 8.73 (s, 1H, pyridine-H), 8.29 (d, 1H,
pyridine-H), 8.02 (d, 1H, pyridine-H), 7.91–7.73 (m, 4H,
Ar–H, pyridine-H), 7.47–7.19 (m, 20H, Ar–H, Tr-H), 6.94-
6.92 (m, 6H, Ar–H), 6.87 (s, 1H, Imidazole-H), 6.04 (s, 1H,
Imidazole-H), 1.68 (t, 2H, (–CH2–)), 1.49 (t, 2H, (–CH2–)),
1.32 (t, 2H, (–CH2–)), 1.20 (t, 2H, (–CH2–)), 1.04 (s, 2H, (–
CH2–)py), and –2.87 (s, 2H, NHpyrrole) (Fig. S2).
1
attachment. H-NMR and 19F-NMR spectra were acquired
using a Bruker 300-MHz NMR spectrometer. Chemical
shifts were reported as δ (ppm) values relative to an internal
standard (tetramethylsilane) and the residual solvent proton
1 3

J Biol Inorg Chem
Scheme 1 Steps involved in the
synthesis of the (PImH)FeII (4)
complex
PImTr-d8: The pyrrole deuterated porphyrin ligand
PImTr-d8 was prepared using a procedure identical to that
described above for PImTr, but employing the pyrrole deu-
terated porphyrin F6(NH2)-d8 [36] instead of F6(NH2).
[(PImH)FeIII-Cl]: The ligand PImTr (1.3 g, 1.05 mmol)
was dissolved in 20 mL THF under an argon atmosphere.
Iron(II) chloride tetrahydrate (7 g, 55.2 mmol) was added,
and the solution was heated to reflux at 60 °C under argon
for 3 h. After cooling to room temperature, the solution
was exposed to air and stirred for 3 h. The solvent was
removed by rotary evaporation, and the residue obtained
was re-dissolved in 100 mL CH2Cl2 followed by filtering
the insoluble solid present. The solution was stirred with
HCl (1 M, 100 mL) for 3 h and then neutralized using solid
NaHCO3. The organic layer was washed with 100 mL satu-
rated NaHCO3, and then, brine solution dried over anhy-
drous MgSO4. The desired product was purified by column
chromatography (silica, CH2Cl2:MeOH = 95:5, Rf = 0.4).
Yield: 0.8 g, 72 %. UV–Vis spectrum [λmax, nm] in THF:
419, 527, 553 (Fig. S3). ESI–MS (m/z): 1047.01 (M−
pyrrole-d) and (δ, ppm, −90 °C): 126.0 (s, br, pyrrole-D),
shown in Fig. S5.
(PImH)FeII: The degased solution of [(PImH)FeIII-Cl]
(500 mg, 0.5 mmol) in 120 mL CH2Cl2 was added to a
degassed 50 mL saturated Na2S2O4 (aq) solution under an
argon atmosphere. The two solutions were mixed using
argon bubbling for 30 min in an additional funnel. The
reaction mixture was allowed to sit for 20 min until the
two layers separated. The organic layer was separated and
passed through anhydrous Na2SO4 powder loaded in a fil-
ter tube (one end connecting to the additional funnel and
the other end connecting to a Schlenk flask) under an argon
atmosphere. Then, the solvent was removed and dried in
vacuo for 3 h. The resulting solid was kept in glove box.
Yield: 437.1 mg, 93 %. UV–Vis spectrum [λmax, nm] in
THF: 419, 525, 552. 19F-NMR (THF-d8, 282 MHz; δ,
ppm): −109.7, −110.3, −111.0, −111.6, −112.4.
(PImH)FeII-d8: The pyrrole deuterated heme–FeII (PImH
)
FeII-d8 was prepared using an identical procedure to that
described above for (PImH)FeII, but employing pyrrole
Cl−)+ (Fig. S4) H-NMR (300 MHz, THF, δ, ppm, RT):
deuterated porphyrin [(PImH)FeIII-Cl]-d8 instead of [(PImH
)
1
82 (s, br, pyrrole-H). EPR spectra (X-band spectrometer,
ν = 9.428 GHz): g = 6.0, 1.98 in THF at 7 K.
FeIII-Cl]. 2H NMR (300 MHz, THF): δ 57.0 (s, 1H, pyrrole-
d), 49.0 (s, 1H, pyrrole-d), 19.0 (s, 1H, pyrrole-d), 15.7
[(PImH)FeIII-Cl]-d8: The pyrrole deuterated heme–FeIII
[(PImH)FeIII-Cl]-d8 was prepared using an identical pro-
cedure to that described above for [(PImH)FeIII-Cl], but
employing pyrrole deuterated porphyrin PImTr-d8 instead
(s, 1H, pyrrole-d), 8.3 (s, 4H, (pyrrole-d). H-NMR (THF,
2
300 MHz; δ, ppm, −90 °C): 9.80 (Fig. S6).
[(F8)FeII-(DIMPI)2], (1)-DIMPI: In the drybox, to
a solution of (F8)FeII (1) (10.0 mg, 0.024 mmol) in THF
(5 mL) was added 2,6-dimethylphenyl isocyanide (6.3 mg,
2
of PImTr. H NMR (THF, 300 MHz, δ, ppm, RT): 82 (s, br,
1 3

J Biol Inorg Chem
0.048 mmol). After stirring the reaction mixture for 30 min,
the solvent was removed under vaccum to yield a red solid.
The crude solid obtained was further dissolved in THF
and layered with pentane to obtain a very fine crystalline
material. UV–Vis spectrum [λmax, nm] in THF: 430, 527.
1H-NMR (THF-d8, 300 MHz; δ, ppm): 10.45 (pyrrole-H),
2.43 (s, –CH3 (DIMPI)), 7.2 (m, ArH (DIMPI)); 19F-NMR
(THF-d8, 282 MHz; δ, ppm): −109 (d). FT-IR spectrum
complex (2)-NO. Excess of NO gas was bubbled through
the 2 mM THF solution of (PPy)FeII (2). UV–Vis spec-
trum [λmax, nm] in THF: 417, 543. 1H-NMR (THF-d8,
300 MHz; δ, ppm): 8.0 (pyrrole-H). FT-IR spectrum
(solid): νNO = 1648 cm−1. EPR spectra (X-band spectrom-
eter, ν = 9.428 GHz): g = 2.07, 2.01 (br-hyperfine), 1.98 in
THF at 7 K.
[(PIm)FeII-NO], (3)-NO [46]: This complex was prepared
in the same manner as complexes (1)-NO and (2)-NO. UV–
Vis spectrum [λmax, nm] in THF: 423, 542. 1H-NMR (THF-
d8, 300 MHz; δ, ppm): 8.8 (pyrrole-H); 19F-NMR (THF-d8,
282 MHz; δ, ppm): −106.2 (br), −107.9 (br), −110.9 (br).
FT-IR spectrum (solid): νNO = 1650 cm−1. EPR spectra
(X-band spectrometer, ν = 9.428 GHz): g = 2.07, 2.00
(hyperfine), 1.97 in THF at 7 K.
(solid): νCN = 2124 cm−1
.
[(PPy)FeII-(DIMPI)], (2)-DIMPI: In the dry box, to the
THF solution of (PPy)FeII, (2) (10.0 mg, 0.011 mmol) in a
10 mL Schlenk flask, we added one equivalent of DIMPI
(1.5 mg, 0.012 mmol) and the reaction mixture was stirred
for half an hour. The solvent was removed under vacuum
to yield a deep red colored solid, which was further recrys-
tallized by dissolving in a minimal amount of THF and
layering it with pentane to obtain the fine crystalline mate-
rial. UV–Vis spectrum [λmax, nm] in THF: 430, 534. FT-IR
[(PImH)FeII-NO], (4)-NO: Synthesis of this complex
was accomplished as mentioned above for the prepara-
tion of (1)-NO. UV–Vis spectrum [λmax, nm] in THF: 425,
spectrum (solid): νCN = 2104 cm−1
.
541. H-NMR (THF-d8, 300 MHz; δ, ppm): 9.65 (pyrrole-
1
[(PIm)FeII-(DIMPI)], (3)-DIMPI: This complex was
synthesized in a similar manner to complex (2)-DIMPI.
X-ray quality crystals were obtained from the solution of
MeTHF/pentane. UV–Vis spectrum [λmax, nm] in THF:
H); 19F-NMR (THF-d8, 282 MHz; δ, ppm): −104.9 (br),
−108.1 (br). FT-IR spectrum (solid): νNO = 1650 cm−1
.
EPR spectra (X-band spectrometer, ν = 9.428 GHz):
g = 2.06, 1.99 (hyperfine), 1.96 in THF at 7 K.
1
430, 532. H-NMR (THF-d8, 300 MHz; δ, ppm): 9.1 (pyr-
role-H), 7.2 (m, ArH (DIMPI)), 2.43 (s, –CH3 (DIMPI));
19F-NMR (THF-d8, 282 MHz; δ, ppm): −110.6 (d), −110.8
(d), −111.0 (d), −111.7 (d). FT-IR spectrum (solid):
X‑ray crystal structure determination
X-ray structure determination of (3)-DIMPI and (4)-DIMPI
was performed at the X-ray diffraction facility at Johns
Hopkins University. CIF files have been deposited with the
Cambridge Crystallographic Data Centre (CCDC). CCDC
1455862 and 1455863 contain the supplementary crystallo-
graphic data for this article. These data can be obtained free
of charge from the CCDC via http://www.ccdc.cam.ac.uk/
data_request/cif.
All reflection intensities were measured at 110(2)
K using a SuperNova diffractometer (equipped with
Atlas detector) with Cu Kα radiation (λ = 1.54178 Å)
under the program CrysAlisPro (version 1.171.36.32
Agilent Technologies, 2013). The program CrysAlis-
Pro (version 1.171.36.32 Agilent Technologies, 2013)
was used to refine the cell dimensions and for data
reduction. The structures were solved with the program
SHELXS-2013 (Sheldrick 2013) and were refined on
F2 with SHELXL-2013 (Sheldrick 2013). Analytical
numeric absorption correction based on a multifaceted
crystal model was applied using CrysAlisPro (version
1.171.36.32 Agilent Technologies, 2013). The tempera-
ture of the data collection was controlled using the sys-
tem Cryojet (manufactured by Oxford Instruments). The
H atoms were placed at calculated positions using the
instructions AFIX 23, AFIX 43, or AFIX 137 with iso-
tropic displacement parameters having values 1.2 or 1.5
times Ueq of the attached C or N atoms.
νCN = 2098 cm−1
.
[(PImH)FeII-(DIMPI)], (4)-DIMPI: This complex was
also synthesized in a similar manner as the aforementioned
complexes, (2)-DIMPI and (3)-DIMPI. X-ray quality crys-
tals were obtained from a solution of Toluene/pentane.
1
UV–Vis spectrum [λmax, nm] in THF: 430, 533. H-NMR
(THF-d8, 300 MHz; δ, ppm): 9.78 (pyrrole-H), 7.2 (m, ArH
(DIMPI)), 2.27 (m, –CH3 (DIMPI)); 19F-NMR (THF-d8,
282 MHz; δ, ppm): -110.55 (d), −110.8 (d), −110.9 (m),
−111.6 (d). FT-IR spectrum (solid): νCN = 2112 cm−1
[(F8)FeII-NO], (1)-NO [45]: The ferrous mononitrosyl
complex (1)-NO was generated by bubbling excess NO
gas through the THF solution of (F8)FeII (1) (2 mM) under
argon atmosphere at room temperature. After the reaction
mixture stirred for 2 h, the solvent was removed under vac-
uum to obtain a dark red solid. A highly pure material was
obtained by dissolving red solid in the minimum amount
of THF and layering it with pentane inside the dry box.
1
UV–Vis spectrum [λmax, nm] in THF: 408, 547. H-NMR
(THF-d8, 300 MHz; δ, ppm): 6.9 (br, pyrrole-H); 19F-NMR
(THF-d8, 282 MHz; δ, ppm): −106 (br). FT-IR spectrum
(solid): νNO = 1688 cm−1. EPR spectra (X-band spectrom-
eter, ν = 9.428 GHz): g = 2.09, 2.02, 1.99 (hyperfine) in
THF at 7 K.
[(PPy)FeII-NO], (2)-NO [46]: A method similar to that
used to synthesize complex (1)-NO was used to make
1 3

J Biol Inorg Chem
refine to 0.561(16) and 0.722(4), respectively (see the Elec-
tronic Supplementary Material, i.e., the appropriate CIF
file).
Results and discussion
Stable heme–isocyanide complex formation
DIMPI reacts immediately with the reduced synthetic fer-
rous-heme complexes, [(F8)FeII], [(PPy)FeII], [(PIm)FeII],
and [(PImH)FeII], to yield six-coordinate low-spin ferrous-
heme isonitrile species, as shown in Schemes 2 and 3.
Scheme 2 Generation of bis-isocyanide ferrous-heme complex; (1)-
DIMPI at room temperature
Generation of bis‑isocyanide‑porphyrin complex [(F8)
FeII‑(DIMPI)2]
Crystals of (3)-DIMPI were obtained from a MeTHF
solution of complex and layered with pentane, while crys-
tals of (4)-DIMPI were obtained from saturated solution of
toluene. The structure of (3)-DIMPI is partly disordered.
Some unresolved electron density—i.e., a very disordered
lattice methyl THF solvent molecule—has been taken out
in the final refinement (SQUEEZE details are provided in
the CIF file, Spek, 2009) [47]. In addition, the imidazole/
amide arm may be slightly disordered, but the disorder is
not significant enough to model it in the final refinement.
The structure (4)-DIMPI is partly disordered. One difluo-
rophenyl group and the lattice toluene solvent molecule
are found to be disordered over two orientations, and the
occupancy factors of the major components of the disorder
When one equivalent DIMPI is added to a THF solution
of [(F8)FeII] at room temperature, a new UV–Vis peak at
430 nm is observed, but the absorption at 422 nm char-
acteristic of the starting complex still remains. How-
ever, the addition of another equivalent of DIMPI leads
to the full formation of the 430 nm peak in the Soret
region (Fig. 3; Scheme 2). Additional DIMPI added to
the solution does not change the UV–Vis spectral fea-
tures. Based on these observations, we postulate that two
DIMPI molecules are bound to the iron(II) center, as also
1
confirmed by integration of peaks in the H-NMR spec-
trum of [(F8)FeII-(DIMPI)2]. Similar UV–Vis spectral
Scheme 3 Generation of six-
coordinate ferrous-heme isocya-
nide complexes; (2)-, (3)-, and
(4)-DIMPI
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J Biol Inorg Chem
Fig. 3 UV–Vis spectroscopic data for the ferrous DIMPI and NO complexes of (1), (2), (3), and (4) in THF at room temperature. black—
reduced Fe(II) species; red—Fe(II)-DIMPI and blue—Fe(II)-NO complexes
features were observed for a structurally characterized
bis-isocyanide iron(II) complex with tetraphenylporphy-
rin (TPP), [(TPP)FeII-(tBuNC)2] [48]. The reactivity of
DIMPI with reduced synthetic hemes is similar to that
in general and previously observed for carbon monoxide
(CO) as the heme ligand [37, 49]. Complex (1)-DIMPI
was further characterized by FT-IR spectroscopy, where a
single ν(C≡N) stretch is observed at 2124 cm−1 (vide infra,
Fig. 6; Table 3), as would be expected for this highly
symmetric compound, even with the presence of two
DIMPI ligands per molecule. In addition, the 19F-NMR
spectrum of (1)-DIMPI shows one sharp absorbance at
−109.0 ppm for the o-difluoro substituted phenyl rings of
the F8 porphyrin [50].
Generation of six‑coordinate (PPy/PIm/PImH
iron(II)‑DIMPI complexes
)
[(PPy)FeII] and [(PIm)FeII] in THF solution at RT exhibit
different structures in this solvent, as previously deduced
by the observation of the positions of NMR spectroscopic
pyrrole resonances. [(PPy)FeII] and [(PIm)FeII] are both
five-coordinate high spin, the former at all temperatures
between RT and −90 °C. However, at low temperature,
[(PIm)FeII] is six-coordinate low spin (S = 0), with the
pyrrole resonances appearing in the diamagnetic region,
indicating that both the imidazolyl group and a THF sol-
vent molecule act as axial ligands [37]. The new complex
[(PImH)FeII] behaves similarly; at room temperature, it is
1 3

J Biol Inorg Chem
Fig. 4 Displacement ellip-
soid plots (50 % probability
level) of [(PIm)FeII-(DIMPI)]
(left; 3-DIMPI) and [(PImH
)
FeII-(DIMPI)] (right; 4-DIMPI),
in both the cases showing the
imidazolyl and 2,6-dimeth-
ylphenyl isocyanide ligands
bound to Fe(II) center. Lattice
solvent molecules and H atoms
have been omitted for the sake
of clarity. Selected bond lengths
and bond angles are reported in
Table 2
five-coordinate high spin, where the tethered imidazolyl is
axially bound to the Fe(II) center. This results in an asym-
metry, and four different pyrrole resonances appear in a
ratio of 4:2:1:1 (see Fig. S6). By contrast, at lower temper-
atures, [(PImH)FeII] forms a six-coordinate low spin (S = 0)
species, again postulated to have both a THF and imidazole
bound to the Fe(II) center, as δpyrrole = 9.80 ppm, Fig. S6.
The addition of one equivalent of DIMPI solution to
each of the reduced Fe(II) porphyrinates ([(PPy)FeII], [(PIm)
FeII] and [(PImH)FeII]) in THF at room temperature leads to
a substantial change in the UV–Vis Soret region, with the
formation of a band at 430 nm in all three cases. Additional
equivalents of DIMPI do not yield any change in the UV–
Vis spectra (Fig. 3; Scheme 3). This may indicate that only
one DIMPI molecule is bound to the iron(II) center axially,
while the covalently linked axial base imidazole/pyridine is
coordinated to the iron(II) center giving an overall six-coor-
dinate low-spin ferrous-DIMPI complex. These conclu-
sions are borne out by the X-ray structures determined for
complexes (3)-DIMPI and (4)-DIMPI (see below, Fig. 4).
We have also carried out experiments to determine
binding constants of DIMPI with the ferrous hemes with
covalently attached axial ligand bases. Titrations with
DIMPI were performed using (PPy)FeII (2), (PIm)FeII (3),
and (PImH)FeII (4), and isosbestic behavior was seen for all
the titrations (Figs. S9–S11). A plot of the absorbance at
430 nm versus [DIMPI] (Fig. S9–S11, in the ESI) reaches
a maximum at ∼1 equiv of DIMPI, and no further spec-
tral changes are observed with the addition of more DIMPI.
Assuming that DIMPI reversibly binds to the ferrous-heme
complexes (PPy)FeII (2), (PIm)FeII (3), and (PImH)FeII (4)
under equilibrium conditions, a good fit of the data can be
obtained with a model for a one-to-one binding isotherm.
This fit gives association constants (Ka) of 2.29 × 107,
1.19 × 107, and 1.29 × 107 M−1, for (2), (3), and (4),
respectively. These values are comparable with those meas-
ured for DIMPI binding to hemoglobin (Hb) and myoglo-
bin (Mb) [Ka = 1.0 × 108 M−1] [51]. The binding constant
for complex (2)–DIMPI is twofold greater than (3)–DIMPI
and (4)–DIMPI, which is consistent with a less strong bind-
ing of the pyridine axial base in (2)–DIMPI when compared
to the imidazole and histamine containing complexes.
Crystal structures of isocyanide complexes
To understand the similarities and differences in the coordi-
nation geometry of these iron(II)-DIMPI complexes, crystal
structures of (3)-DIMPI and (4)-DIMPI were determined.
The structures are shown in Fig. 4, and important structural
parameters are listed in Tables 1 and 2. In both structures,
the geometry around the iron is octahedral, where the fifth
ligand is the covalently linked imidazole (or pyridine),
while the sixth ligand is the isocyanide (DIMPI). The metal
center is in the porphyrinate plane. The observed bond dis-
tances and angles for both indicate very little strain within
the linker arm. A slight perturbation from the expected lin-
ear Fe–C–N bond angle is seen for complex (3)‑DIMPI,
with a ∠Fe–C–N value of 173.8(4)°. This is notably less
linear when compared with (4)-DIMPI, where ∠Fe–C–N,
179.3(3)° is nearly linear. This difference could arise due
to crystal packing effects [52]. The Fe–C(DIMPI) bond dis-
tances are ca. 1.82 Å for both (3)-DIMPI and (4)-DIMPI.
The Fe–N(imidazole) bond distances are ca. 2.02 Å, while
the average Fe–N(porphyrin) bond distances are 1.99 Å,
similar to our previously reported [(F8)FeII]·2THF complex
[53]. Both structures are well ordered except the difluo-
rophenyl group and toluene solvent molecule in complex
(4)-DIMPI (see “Materials and methods”). These appear to
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J Biol Inorg Chem
Table 1 Crystallographic data for complex [(PIm)FeII-(DIMPI)] and [(PImH)FeII-(DIMPI)]
Compounds
(3)-DIMPI
(4)-DIMPI·Toluene
Formula weight (g/mol)
1106.89
1271.13
T (K)
110(2)
110(2)
Crystal shape
Space group
a (Å)
small dark red plate (0.11 × 0.05 × 0.02 mm3)
dark red plate (0.15 × 0.08 × 0.03 mm3)
Triclinic, P-1 (no. 2)
12.3852(6)
12.5816(6)
19.3657(12)
104.424(5)
95.293(4)
Monoclinic, P21/c (no. 14)
12.4643(2)
12.3010(2)
39.9319(8)
90
b (Å)
c (Å)
α (°)
β (°)
93.9552(18)
90
γ (°)
V (Å3)
111.823(5)
2655.1(3)
6107.91(19)
4
Z
2
Dx (g cm−3
μ (mm−1
Absorption correction range
(sin θ/λ)max (Å−1
)
1.385
1.382
)
2.897
2.601
0.797–0.947
0.60
0.759–0.940
0.60
)
Total, unique, and observed reflections
24,276, 9448, 6441
0.0467
40,278, 11,998, 8909
0.0597
Rint
GOF
1.041
1.021
R1/wR2 [I > 2σ(I)]
R1/wR2
0.0703/0.1902
0.1038/0.2151
1.126, −0.478, 0.083
0.0501/0.1089
0.0760/0.1220
0.644, −0.483, 0.054
Δρmax, Δρmin, rms
be disordered over two orientations and can rotate slightly
from perpendicularity with respect to the porphyrin plane.
Lehnert and co-workers have previously published on a
crystal structure of the zinc(II) analogue, a five-coordinate
complex with the PIm ligand [46].
observed (2.3 to 2.7 Å), while very acute CH…O angles
are present (~100°–109°) [57, 58].
With these precedents in mind, we postulate that in the
solid-state structure of (3)-DIMPI, a CH…F interaction
occurs between the methyl group on the DIMPI ligand
(H64C) and F1 from the proximate o-F aryl porphyrinate
substituent (see the green line in the middle top of the
(3)-DIMPI structure in Fig. 5, left, and Fig. S7); here, the
CH…F distance and angle are 2.73 Å and 134°, respec-
tively (Table 2). A far closer to linear interaction occurs
between F6 and H41 from the ligated imidazolyl group
with a bond distance and angle of 2.99 Å and 167°, respec-
tively (Table 2; Fig. 5, bottom right). Notice F6 and F5, on
the same porphyrinate aryl group both H-bond, the latter to
the pyrrole hydrogen atom, (H2), this F5...H2C interaction
is likely synergistic with the F6...H41 interaction. Envision-
ing the iron atom as a kind-of pseudo center of symmetry,
the F3 and F4 atoms on the left-hand o-F2-phenyl ring form
two H-bonds, between F3…H8pyrrole (C8) and F4…H39imid
(C39) (Table 2). The overall result is that the H-bonding
interactions formed by the four F atoms just described
fixes the orientation of the imidazolyl axial ligand, which
is already close to perpendicular to the porphyrinate plane,
to be nearly coplanar with both the aryl rings containing
these F3, F4, F5, and F6 atoms. Notice how in viewing
Other potentially important (from a structural perspec-
tive) observations obtained from the crystal structures
of both complexes are that weak but noticeable intramo-
lecular CH…F interactions occur, as shown in Fig. 5 and
listed in Table 2. The observed range of C–H…F interac-
tions for our new structures lie between 2.73 and 3.08 Å,
which is greater than the sum of reported Van der Waals
radii for hydrogen and fluorine (approximately 2.3–2.5 Å)
[54]. Based on reported structural observations and DFT
calculations [55, 56], the very strong H-bonding ability
of fluorine gives rise to such C–H…F interactions which
vary between ~2.7 and 3.1 Å, similar to what is observed
for (3)-DIMPI, and (4)-DIMPI. Such literature examples
of organic compounds with longer distance H…F interac-
tions occur even, where the CH…F angle lies in the range
between 130° and 145°, and even in some cases, it is close
to 100° [55, 56]. Further comparisons may be made to
examples of non-bonded CH…O contacts made between an
O atom in an Fe(IV)-oxo complex with surrounding ligand
methyl group H atoms; there, short CH…O distances are
1 3

J Biol Inorg Chem
Table 2 Selected bond lengths (Å) and bond angles (°) for (3)-DIMPI and (4)-DIMPI. The proposed H-bonds are also listed
Compound:
(3)-DIMPI
Compound:
(4)-DIMPI.Toluene
Bond length (Å)
Bond length (Å)
Fe–N1
Fe–N2
Fe–N3
Fe–N4
Fe–N5
Fe–C56
N8–C56
1.998 (4)
2.000 (3)
1.990 (4)
2.001 (3)
2.034 (4)
1.824 (4)
1.163 (6)
Fe–N1
Fe–N2
Fe–N3
Fe–N4
Fe–N8
Fe–C59
N11–C59
1.994 (2)
1.991 (2)
1.995 (2)
1.985 (2)
2.024 (2)
1.835 (3)
1.166 (3)
Compound:
(3)-DIMPI
Compound:
(4)-DIMPI·Toluene
Bond angle (°)
Bond angle (°)
N1–Fe–N5
N2–Fe–N5
N3–Fe–N5
N4–Fe–N5
N5–Fe–C56
Fe–C56–N8
91.38 (15)
89.77 (15)
88.85 (15)
88.58 (14)
176.03 (16)
173.8 (4)
N1–Fe–N8
N2–Fe–N8
N3–Fe–N8
N4–Fe–N8
N8–Fe–C59
Fe–C59–N11
85.99 (8)
89.90 (9)
91.10 (8)
89.03 (9)
177.61 (9)
179.3 (3)
Weak C–H…F interaction
Bond length (Å)/bond Angles (°)
Weak C–H…F interaction
Bond length (Å)/bond angles (°)
F1…H64methyl (C64)
F2…H7pyrrole (C7)
2.731/(133.93)
3.490/(95.04)
F1…H35py (C35)
F1…H40imid (C40)
3.089/(129.88)
2.914/(135.45)
F3…H8pyrrole (C8)
F4…H39imid (C39)
F5…H2pyrrole (C2)
2.966/(95.57)
3.117/(153.44)
2.926/(100.23)
F2…H67methyl (C67)
F3…H12pyrrole (C12)
F4…H8pyrrole (C8)
3.148/(146.75)
3.163/(91.25)
2.883/(101.78)
F6…H41imid (C41)
2.991/(166.53)
F5…H17pyrrole (C17)
2.903/(100.89)
F6…H66methyl(C66)
N9H…N7pyridine
2.539/(176.34)
1.986/(173.74)
Fig. 5 Crystal structures showing weak intramolecular CH…F interaction identified from the green lines shown. (Left) (3)-DIMPI and (Right)
(4)-DIMPI. See text for further discussion
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J Biol Inorg Chem
group to the lower right side of (4)-DIMPI, as shown in
Fig. 5, right. To the left side (Fig. 5, right), the F5 atom that
on the same ring as F6 is involved in H-bonding to a pyr-
role H atom (H17) (Table 2). For F1, on the right-hand aryl
group, we postulate that multiple H…F interactions occur,
to H40 from the ligated imidazolyl group as well as H35
from the dangling free pyridyl group (see the green lines to
the upper right of the (4)-DIMPI structure in Fig. 5, Right);
here, the CH…F distances and angles are 2.91 Å and 135°
(F1…H40), and 3.08 Å and 130° (F1…H35), respectively.
Tilting of the o-F2-aryl group containing F3 and F4 allows
for two H-bonds to form to pyrrole H atoms (Table 2).
Thus, all six fluorine atoms in (4)-DIMPI may participate
in H-bonding. In part, we postulate that these solid-state
interactions persist in solution, as indicated by 19F-NMR
spectroscopy, where almost all of the fluorine resonances
exist as doublets due to H-atom coupling, as described
below.
Fig. 6 Solid-state FT-IR spectra for Fe(II)-DIMPI complexes of (1),
(2), (3), and (4)
Another H-bond observed in this X-ray structure of
(4)-DIMPI is a very strong one, between the H atom on
the uncoordinated N atom of the imidazolyl group, to the
N atom of the dangling pyridine (Fig. 5-Right). Here, the
N9H…N7pyridine angle is near linear (173.7°) with a N9H…
N7 distance of 1.986 Å (and where N9…N7 = 2.863 Å)
(Table 2).
Table 3 Properties of ferrous-heme-DIMPI and ferrous-heme-NO
model complexes
Compound
UV–Vis (nm) IR (cm−1
)
EPR (g values)
ν(C≡N)/ν(N–O)
[(F8)FeII-
(DIMPI)2]
[(PPy)FeII-
(DIMPI)]
[(PIm)FeII-
(DIMPI)]
[(PImH)FeII-
430
430
430
430
2124
Silent
Silent
Silent
Silent
Complexes (2)-DIMPI, (3)-DIMPI, and (4)-DIMPI were
also characterized by FT-IR spectroscopy (Fig. 6; Table 3).
The IR spectrum for (1)-DIMPI shows a single sharp ν(C≡N)
band (2124 cm−1), corresponding to the absorption for both
isonitrile ligands in [(F8)FeII-(DIMPI)2], which is shifted
4 cm−1 higher in energy relative to the stretching frequency
of the uncomplexed ligand. The shift to higher energy is
relatively small and could be attributed to a ligand (σ2NC) to
metal (dσ) interaction, consistent with the expected behav-
ior for an isonitrile ligand acting as a σ-donor to a metal
center. [(TPP)FeII-(tBuNC)2] [48], as mentioned above,
was structurally characterized, but no IR data were given.
On the other hand, for all three porphyrin Fe(II)-DIMPI
complexes with covalently attached axial bases (pyridine/
imidazole/histamine moieties), we observe a shift in the
C≡N bond stretch of the isonitrile ligand to lower energy
with respect to the uncomplexed ligand. The ν(C≡N) band
for (2)-DIMPI is 2104 and 16 cm−1 lower in energy com-
pared to that of free DIMPI, and also 20 cm−1 lower in
energy with respect to (1)-DIMPI. For (3)-DIMPI, the value
of ν(C≡N) is 2098 cm−1, which is 23 cm−1 lower in energy
with free ligand. In (4)-DIMPI [ν(C≡N) band (2112 cm−1)],
we again observe a lower energy C≡N bond stretch
compared to free DIMPI, but only by a small amount
(Δν(C≡N) = −8 cm−1) [59]. The shift to lower energy in all
these cases could be due to the presence of a more strongly
donating axial base trans to the isonitrile ligand. This will
most likely decrease the σ-donation to the Fe(II) from the
2104
2098
2112
(DIMPI)]
[(F8)FeII-NO]
[(PPy)FeII-NO]
[(PIm)FeII-NO]
[(PImH)FeII-NO]
408
417
423
425
1688
1648
1650
1650
2.09/2.01/1.99
2.08/2.00/1.97
2.07/2.00/1.97
2.07/1.99/1.95
the structure of (3)-DIMPI (Fig. 5, left) that these two aryl
rings and the imidazolyl ring seem to lie in the plane of
the sheet. This imidazole orientation could also lead to a
close interaction of F2…H7pyrrole (C7) in the solution state
(see 19F-NMR spectroscopy, below), whereas in solid-state
structure, this distance seems too long for this interaction to
occur (Table 2).
Similarly, for (4)-DIMPI, we observe close to the linear
CH…F interaction between a methyl group H atom on the
DIMPI ligand (H66) and F6 from the o-F porphyrinate aryl
substituent, shown by the green dotted line on the lower left
side of Fig. 5, right; here, the CH…F distance and angle are
2.54 Å and 176°, respectively (Table 2; Fig. 5-right, also
see Fig. S8). The other methyl group on the DIMPI ligand
likewise forms an H-bond, with the F2 atom on the aryl
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J Biol Inorg Chem
One of the two absorptions, −110.8 or −110.6 corresponds
to a single o-fluorine atom (F2) also coupling to a pyrrole
CH7 atom, but by symmetry, this is in a different chemical
environments then for the interactions discussed just above
for F5 and F3. (d) Then, the other upfield absorption is a
unique interaction of F1 with the DIMPI methyl group H
atoms (CH64…F1).
Similarly, for (4)-DIMPI, we observe four ortho
F-atom resonances, three doublets, and one multiplet in
19F-NMR spectroscopy (Fig. 7). The peaks at −111.6 and
−110.8 ppm both integrate to two fluorine atoms, which
we propose are due to (a) fluorine atoms F6 and F2 cou-
pling with the DIMPI methyl H atoms H66A and H67C
(see Table 2; Fig. 5), (b) while the other generally upfield
doublet represents two fluorine atom being in the same
chemical environments due to the coupling of F atoms F3
and F4 from the same porphyrinate phenyl ring, forming
H-bonds with the pyrrole H atoms H8 and H12. If (or prob-
ably when) the aryl ring with F3 and F4 tilts the other way,
then symmetry-related switching of H-bonding to pyrrole
H atoms may (or probably does) occur, but the result is the
same type of CH…F H-bonding. Furthermore, the doublet
resonance at −110.5 ppm should correspond to F5 coupling
to the pyrrole H atom H17. This leaves the multiplet peak
at −110.9 ppm which likely arises due to the H-bonding
interaction and magnetic coupling of F1 to two H atoms,
the imidazole H atom H40 and the dangling pyridine H
atom H35 (see Table 2; Fig. 5). During the dynamic behav-
ior expected in solution, the dangling pyridyl ring may
break its H35…F1 interaction, and twist over to the other
side (to the left in Fig. 5, right), as the imidazole bound to
Fe also twists to maintain the very strong imidazole N–H to
pyridine nitrogen hydrogen bond); the pyridyl H35 would
now H-bond to F5 (instead of F1); furthermore, F5 may
then gain an H-bond to H40. All these motions would leave
the 19F-NMR shifts and couplings unchanged.
Fig. 7 19F-NMR of (1)-DIMPI (top), (3)-DIMPI (middle), and (4)-
DIMPI (bottom) complexes in THF at room temperature
isocyanide ligand while increasing backbonding to the
ligand C≡N π* orbitals from the Fe(II) dπ orbitals.
Diamagnetic 1H-NMR spectra were observed for all
Fe(II)-DIMPI (S = 0, d6) complexes. The pyrrole hydro-
gens of the Fe(II)-DIMPI porphyrinates resonate between
8.5 and 9.5 ppm compared with the starting reduced high-
spin five-coordinated paramagnetic Fe(II) (d6) species rang-
ing in δpyrole = 12–58 ppm [37]. We also observed a singlet
δ 2.65 ppm for the DIMPI o-methyl protons and a multiplet
for the aromatic protons of bound DIMPI at δ 7.2–7.5 ppm;
both the sets of peaks are slightly shifted downfield from
what is observed for free DIMPI.
In the 19F-NMR for bis-isocyanide complex, [(F8)FeII-
(DIMPI)2]/(1)-DIMPI, we observe one sharp singlet peak
at −109.0 ppm, as shown in Fig. 7. Therefore, unlike
what we have suggested (and described above) for what
is observed in the X-ray crystal structures for the super-
structured hemes [(3)-DIMPI and (4)-DIMPI], there are no
observed F-atom interactions or coupling to porphyrin or
isocyanide H atoms.
On the other hand, we observed CH…F coupling for
(3)-DIMPI and (4)-DIMPI (Fig. 7) with peaks for the
ortho F-atom resonances on the porphryinate aryl groups
occurring between −110 and −112 ppm [50]. In the case
of (3)-DIMPI, doublet peaks, proposed to be due to 19F
resonances coupled to an S = 1/2 H atom, are observed at
−111.7 and −111.0 ppm, and both these appear to inte-
grate to two F atoms. Our suggested assignments are as fol-
lows: (a) one of the two upfield (more negative delta value)
doublets corresponds to F4 and F6 coupling to imidazole H
atoms H39 and H41, which spatially line up rather well (see
Table 2; Fig. 5). (b) The other upfield doublet represents 2
o-fluorine H-bonds with pyrrole H atoms, possibly F3 and
F5 with pyrrole H atoms H8 and H2, and these also seem
to be in very closely matching chemical environments. (c)
As mentioned, for (1)-DIMPI, we do not observe any
DIMPI ligand H atom coupling to F atoms of the F8 heme.
In addition, we do not observe any such couplings for the
compound (THF)(F8)FeII-CO (unpublished observation).
We suggest that when there is a tethered axial ligand, such
as in PIm or PImH, there are constraints in the movement or
rotation of the axial ligand, which thereby allow for these
weaker imidazole-H…F interactions to be observed. Fur-
ther studies may be warranted.
Stable heme–Fe–nitrosyl formation
In this study, we have also investigated the reactivity of
[(F8)FeII], [(PPy)FeII], and [(PIm)FeII], and the newly syn-
thesized [(PImH)FeII] towards nitric oxide (NO). All iron
(II) complexes form an NO-adduct at room temperature by
bubbling NO(g) through the solution of each of the reduced
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J Biol Inorg Chem
Fig. 8 X-band EPR at 8 K in THF for ferrous-heme-NO complexes.
[(F8)FeII-NO] (red, 5C species), while (2), (3), and (4) form 6C spe-
cies. [(PPy)FeII-NO] (orange, 6C), [(PIm)FeII-NO] (green, 6C), and
[(PImH)FeII-NO] (blue, 6C). These spectra were analyzed further
using an EPR simulation computer program, and the results of those
fits, giving g values and hyperfine coupling constants, are given in the
ESI (Fig. S12)
Scheme 4 Six coordinate ferrous-heme mononitrosyl complexes of
(PPy)FeII, (PIm)FeII, and (PImH)FeII
iron complexes, as shown in Scheme 4. We have system-
atically characterized Fe(II)–NO complexes using UV–Vis,
1
FT-IR, H-NMR, 19F-NMR spectroscopy, and low-temper-
ature EPR spectroscopy, to access the binding properties of
our covalently tethered N-donor ligands.
complex with a Soret band λmax at 408 nm in the UV–vis
region at room temperature (Fig. 3), along with a charac-
teristic N–O stretching band ν(N–O) at 1680 cm−1 in its
IR spectrum [45, 62]. An additional evidence for the 5C
nitrosyl complex can be seen in its EPR spectrum, which
displays g values at 2.09, 2.02, and 1.99, with three hyper-
fine splittings at g(min) (Fig. 8) [45, 62]. In the 19F-NMR, a
broad o-phenyl fluorine signal is observed at −106.0 ppm.
In the case of [(PPy)FeII-NO], the Soret band absorp-
tion is observed at 417 nm (Fig. 3), which lies in between
that known for 5C and 6C iron-nitrosyl complexes. This
indicates that in solution at room temperature, the proxi-
mal pyridine is weakly bound to the iron center to give 6C
species. Further evidence comes from the low-temperature
EPR spectrum of [(PPy)FeII-NO], shown in Fig. 8, Fig. S12
and Table 3, which clearly resembles the spectra of other
6C low-spin heme-Fe(II)-nitrosyl complexes [46], lowering
the temperature allows for stronger binding of the pyridyl
group, as would be expected. The spectrum shows small,
unresolved hyperfine splitting at g(mid) due to the pres-
ence of the proximal pyridine ligand (g = 2.07, 2.01, 1.98).
The lack of UV–Vis spectral features at 400 and 470 nm, as
well as EPR data for [(PPy)FeII-NO] confirms that in solu-
tion, it forms a 6C species, but where the pyridyl group is
weakly bound to the iron center at room temperature.
In-depth studies have been done by Lehnert and co-
workers [14, 46] using synthetic heme porphyrins with
tethered N-donor ligands which indicate a direct correlation
between the coordination geometry of the iron center and
the observed spectroscopic properties obtained from UV–
Vis, IR, and EPR. For five-coordinate (5C) heme nitros-
yls, the Soret band (UV–Vis) is typically about 405 nm,
whereas in six-coordinate (6C) porphyrinoids, where the
proximal ligand (N-donor) is bound to the Fe center, the
Soret λmax shifts to ~426 nm. Similarly, in IR spectroscopy,
5C and 6C ferrous-heme mononitrosyl species have distinct
N–O stretching modes. For 5C complexes, the N–O stretch
typically lies between 1675 and 1700 cm−1, whereas for 6C
complexes, the N–O stretch occurs at ~1630 cm−1. Low-
temperature EPR spectroscopy studies conducted by sev-
eral authors reveal interesting differences between the 5C
and 6C iron(II) porphyrin NO adducts [60, 61]. Hyperfine
lines resulting from the bound nitrogen of NO are observed
at the lowest g value (g min) in 5C ferrous-heme nitros-
yls. The coordination of the proximal nitrogen atom in 6C
ferrous-heme nitrosyls causes a broadening in the EPR
spectrum at g-mid resulting from the hyperfine lines of the
bound NO and the trans-N donor ligand. Based on spectro-
scopic data available from the literature, our [(F8)FeII-NO]
complex forms a typical 5C ferrous-heme mononitrosyl
On the other hand, complexes [(PIm)FeII-NO] and
[(PImH)FeII-NO] form very stable 6C iron(II) nitrosyl
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J Biol Inorg Chem
peaks were very sharp, and displayed visible H–F coupling
interactions.
Summary
In summary, we have described the synthesis of a new por-
phyrin with a covalently tethered histamine type ligand,
which binds to the iron center. These new ferric [(PImH)FeIII-
Cl] and ferrous [(PImH)FeII] hemes have been thoroughly
characterized by various spectroscopic methods. Using this
newly designed heme and a family of other covalently teth-
ered axial ligands (pyridine/imidazole) synthesized in our
lab, we have explored their reactivity towards 2,6-dimethyl-
phenyl isocyanide (DIMPI) and nitric oxide (NO). Towards
this aim, we have generated and characterized the six coor-
dinate ferrous-heme complexes; [(PPy)FeII(DIMPI)], [(PIm)
FeII(DIMPI)], and [(PImH)FeII(DIMPI)], which have UV–
Vis, IR, and EPR properties that are clearly distinguish-
able from those of [(F8)FeII(DIMPI)2]. The X-ray structures
reveal a significant contribution from H-bonding between
porphryinate meso-phenyl ortho-fluorine atoms, and these
have been described. These are emphasized in large part,
because 19F-NMR spectroscopy clearly indicates that most
if not all of these interactions are maintained in solution.
We have also characterized several ferrous-heme mononi-
trosyl complexes by multinuclear NMR, UV–Vis, EPR, and
solid-state FT-IR spectroscopy. At room temperature, [(PIm)
FeII] and [(PImH)FeII] form very stable six-coordinate fer-
rous iron-NO complexes. [(F8)FeII] forms a five-coordinate
ferrous-heme nitrosyl complex, while [(PPy)FeII] appears to
be somewhere between 5 coordinate and 6 coordinate due
to its weakly binding axial pyridyl ligand.
Fig. 9 19F-NMR of (1)-NO (top), (3)-NO (middle), and (4)-NO (bot‑
tom) complexes in THF at room temperature
species. In the UV–vis region, the observed Soret bands
shift to 423 nm for (3)-NO and 425 nm for (4)-NO (Fig. 3)
which match very well with reported 6C iron(II) nitrosyl
complexes [46]. To further investigate the strength of the
proximal (imidazole) ligand binding to the iron center in
(3)-NO and (4)-NO, the EPR spectra of both complexes
were recorded. The observed g values are 2.07, 2.00, and
1.97 for (3)-NO and 2.06, 1.99 and 1.96 for (4)-NO (Fig. 8;
Fig. S12; Table 3). For both species, the hyperfine pattern
is on g(mid) and the hyperfine lines are not well resolved.
In addition, the stretching frequency, ν(N–O), for complexes
(3)-NO and (4)-NO is the same at 1650 cm−1. This lower
stretching frequency is due to the binding of the N-donor
ligand (Imidazole) trans to the NO, which weakens the Fe–
NO σ-bond [63]. Interestingly, these frequencies are higher
in energy compared to a similar ferrous-heme nitrosyl with
a free axial base, [Fe(To-F2PP)(MI)(NO)] (MI = methyl-
imidazole; ν(N–O) = 1624 cm−1). The trend observed is in
line with a previous study by Scheidt [64], further suggest-
ing that the tethered axial ligand bases bind to the heme
more weakly than would or does a freely added (or present)
base. The IR data match closely with reported work by
Lehnert and co-workers [46] and indicate that the benzyl-
imidazole linker and the histamine linker impede the bind-
ing of the proximal N-donor ligand when compared to the
free base, but still allow for the formation of very stable 6C
complexes at room temperature.
As mentioned, the impetus for synthesizing ferrous-
heme porphyrinates using the (PPy), (PIm), and (PImH
)
ligand systems is to utilize these porphyrins in our ongo-
ing research into modeling the active-site chemistry of
cytochrome c oxidase. An understanding of the nature of
reactions and structures, i.e., coordination numbers, liga-
tion preferences (e.g., pyridyl vs imidazolyl vs solvent
THF), and other bonding or structural aspects can help to
better understand the types of structures obtained in heme-
O2-copper chemistry, and also inform the design of hemes
utilized for such studies.
Acknowledgments This work was supported by the National Insti-
tutes of Health (R01 GM 060353 to K.D.K).
In 1H-NMR, the pyrrole hydrogen atoms resonate at 8.8
and 9.65 ppm for (3)-NO and (4)-NO, respectively. While
in 19F-NMR, the o-fluorine atoms resonate at −106, −107,
and −110 ppm for (3)-NO and −105 and −108 ppm for
(4)-NO, as shown in Fig. 9. Here, peaks are very broad
compared with all the Fe(II)-DIMPI complexes, where
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