CO and O2 binding studies of new model complexes for CcO

Article abstract of DOI:10.1016/j.poly.2013.01.050

A very efficient, simple synthesis of a porphyrin mimic for cytochrome c oxidase is presented. This complex contains a covalently attached copper binding site and a phenyl ring that mimics the Tyr part of the enzyme. Structural studies revealed that the copper binding tridentate ligand is situated on top of the porphyrin plane and the hydroxyl of the phenyl ring is oriented towards the center of the molecule. Moreover, Fe at the reduced form of the model is capable of binding CO, as confirmed by FT-IR and upon photolysis CO is bound to Cu site. Finally, electrochemical studies using rotating ring-disk electrode showed that the complexes reduce oxygen via two mechanisms (2e^- and 4e^-) and have low stability.

Full text of DOI:10.1016/j.poly.2013.01.050

Polyhedron 54 (2013) 47–53
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Polyhedron
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CO and O2 binding studies of new model complexes for CcO
⇑
Kalliopi Ladomenou, Georgios Charalambidis, Athanassios G. Coutsolelos
Laboratory of Bioinorganic Chemistry, Chemistry Department, University of Crete, Voutes Campus, P.O. Box 2208, 71003 Heraklion, Crete, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A very efficient, simple synthesis of a porphyrin mimic for cytochrome c oxidase is presented. This com-
plex contains a covalently attached copper binding site and a phenyl ring that mimics the Tyr part of the
enzyme. Structural studies revealed that the copper binding tridentate ligand is situated on top of the
porphyrin plane and the hydroxyl of the phenyl ring is oriented towards the center of the molecule.
Moreover, Fe at the reduced form of the model is capable of binding CO, as confirmed by FT-IR and upon
photolysis CO is bound to Cu site. Finally, electrochemical studies using rotating ring-disk electrode
showed that the complexes reduce oxygen via two mechanisms (2eÀ and 4eÀ) and have low stability.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 27 November 2012
Accepted 30 January 2013
Available online 16 February 2013
Keywords:
Enzyme models
Porphyrin
Oxidase
Dioxygen
Metalloenzymes
Copper
Iron
1. Introduction
activation reaction, the Tyr role and the characterization of tran-
sient species. Therefore, the not fully understood mechanism of
Cytochrome c oxidase (CcO) is the terminal electron acceptor of
the mitochondrial electron transport chain and is responsible for
the four-electron reduction of dioxygen to water [1–4]. The energy
released in this reaction is used to produce a proton gradient across
the inner mitochondrial membrane, which provides the driving
force for the ATP synthesis [5]. X-ray crystallographic analysis re-
vealed that the catalytic active site of the enzyme consists of a
heme (heme a3), a tricoordinated copper ion (CuB) and a tyrosine
residue (Y244) which is covalently connected to one of the copper
bound histidines (H240) (Fig. 1) [2,6–9]. The reduction of dioxygen
to water takes place at the heme a3–CuB binuclear center, with an
iron–copper distance of about 5 Å. During the catalytic process
dioxygen is bound to CuB, followed by transfer of O2 to heme a3,
to form the first intermediate (FeIII-superoxo). After cleavage of
O–O bond, species such as ferryl-oxo (FeIV@O) and CuBII–OH are
formed. Moreover, the above O–O cleavage must be clarified. Also,
a bridging peroxo species (FeIII–O–O–CuII) has been proposed as a
possible intermediate, but still has not been observed at the
enzymatic reaction [10–14]. In addition the crosslinked Tyr is
thought to participate in catalysis by providing the fourth electron
needed to cleave the O–O bond in a net hydrogen abstraction [15].
Regarding all the above information, several points have not yet
been clarified such as, the participation of CuB in the oxygen
dioxygen reduction by CcO has inspired many chemists to model
aspects of its active site and related chemical properties. A series
of synthetic models have been developed and studied by Collman
[16–20], Karlin [21–25], Naruta and others [26,27]. Following our
previous efforts to understand the specific functions of cytochrome
c oxidase [28–30], we have successfully prepared and studied a
novel enzyme mimic. This model contains a copper binding site,
situated above the porphyrin ring and a tyrosine mimic, covalently
attached onto the porphyrin.
2. Experimental
2.1. General
1H NMR spectra were recorded unless otherwise specified, as
deuteriochloroform solutions using the solvent peak as internal
standard on a Bruker AMX-500 MHz spectrometer. UV–Vis spectra
were recorded on a Shimadzu Multispec-1501 instrument. High-
resolution mass spectra were performed on a Bruker ultrafleXtreme
MALDI-TOF/TOF spectrometer using trans-2-[3-(4-tert-Butyl-
phenyl)-2-methyl-2-propenylidene] malononitrile as a matrix. Ele-
mental analyses were carried out using a Carlo-Erba EA 1110, CHNS
Eager 200 analyzer. Thin layer chromatography was performed on
silica gel 60 F254 plates. Chromatography refers to flash chromatog-
raphy and was carried out on SiO2 (silica gel 60, SDS, 70–230 mesh
ASTM). All dry solvents used were dried by the appropriate tech-
nique. Organic extracts were dried over magnesium sulfate unless
indicated otherwise. Evaporation of the solvents was accomplished
⇑
Correponding author. Tel.: +30 2810 545045 (office)/545036 (lab), mobile: +30
6944 979667; fax: +30 2810 545161.
E-mail address: coutsole@chemistry.uoc.gr (A.G. Coutsolelos).
URLs: http://www.biosolenuti.gr/index.html, http://www.chemistry.uoc.gr/
coutsolelos/ (A.G. Coutsolelos).
0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.01.050

48
K. Ladomenou et al. / Polyhedron 54 (2013) 47–53
Fig. 1. (A) CcO-active center, (B) model compound 1FeCu (the Cu counter anions are not represented).
on a rotary evaporator. Synthesis and characterization of all free
base porphyrins has been reported elsewhere [20].
CH2Cl2 was added and the mixture was stirred under argon at
À78 °C for 30 min and at 0 °C for 1 h. The reaction was quenched
with MeOH (8 mL) and H2O (16 mL) and stirred for 20 min at
0 °C. The mixture was washed with saturated aqueous NaHCO3
(2 Â 20 mL) and water (2 Â 20 mL). The organic phase was dried,
and the solvent was removed under reduced pressure. The crude
material was purified by column chromatography on a silica gel
column (0–6% methanol in dichloromethane). Compound 1 was
eluted with CH2Cl2/MeOH 50:3 and obtained as a purple solid
(31 mg, 93%). Rf (CH2Cl2/MeOH 9:1): 0.49. 1H NMR (500 MHz,
CDCl3): d = 9.66 (s, 1H, H32), 9.48 (s, 1H, H22), 8.96 (d, J = 8.5 Hz,
1H, H4), 8.91 (d, J = 8 Hz, 1H, H18), 8.85 (d, J = 4.5 Hz, 2H, Hpyr),
8.80 (d, J = 5 Hz, 2H, Hpyr), 8.68 (d, J = 5 Hz, 2H, Hpyr), 8.61 (d,
J = 4.5 Hz, 2H, Hpyr), 8.19 (dd, J1 = 7 Hz, J2 = 1 Hz, 1H, H1), 8.03 (d,
J = 7.5 Hz, 1H, H15), 7.90 (t, J = 7.5 Hz, 1H, H3), 7.85 (t, J = 7.5 Hz,
1H, H17), 7.57 (t, J = 7.25 Hz, 1H, H2), 7.51 (t, J = 7.5 Hz, 1H, H16),
7.46 (d, J = 4 Hz, 2H, H31), 7.19, (s, 2H, H10), 7.12 (s, 2H, H12), 6.94
(d, J = 7 Hz, 1H, H35), 6.75 (t, J = 7 Hz, 2H, H29), 6.57 (t, J = 6 Hz,
2H, H30), 6.43 (t, J = 7.5 Hz, 1H, H37), 5.97 (t, J = 7 Hz, 1H, H36),
5.46 (d, J = 8 Hz, 1H, H38), 5.28 (d, J = 7.5 Hz, 2H, H28), 2.65 (s, 2H,
2.2. Synthesis of
acetamidophenyl]-
a
-5-[2-(bis-(2-(pyridin-2-yl)ethyl)amino)-
-15-[2-((2-methoxy)benzamide) phenyl]-10,20-
a
bis-(2,4,6-trimethyl-phenyl)-porphyrin (3)
2-Methoxy benzoic acid (34 mg, 0.22 mmol) and 1,3 dicyclohex-
ylcarbodiimide (DCC) (48 mg, 0.23 mmol) were added to a solution
of porphyrin 2 (80 mg, 0.08 mmol) in CH2Cl2 (4 mL). The resulting
mixture was stirred for 48 h at 5 °C, after which time CH2Cl2
(20 mL) was added and washed with water (4 Â 30 mL). The organic
layer dried, filtered, concentrated and the residue was chromato-
graphed on a silica gel column (0–6% ethanol in dichloromethane).
Compound 3 was eluted with CH2Cl2/EtOH 50:3 and obtained as a
purple solid (77 mg, 85%). Rf (CH2Cl2/MeOH 9:1): 0.50. 1H NMR
(500 MHz, CDCl3): d = 9.89 (s, 1H, H32), 9.65 (s, 1H, H22), 9.06 (d,
J = 8.5 Hz, 1H, H4), 8.86 (d, J = 8 Hz, 1H, H18), 8.82 (m, 4H, Hpyr),
8.70 (d, J = 4.5 Hz, 2H, Hpyr), 8.67 (d, J = 4.5 Hz, 2H, Hpyr), 8.05 (m,
1H, H35), 7.87 (m, 4H, H1, H3, H15, H17), 7.79 (d, J = 4 Hz, 2H, H31),
7.47 (m, 2H, H2, H16), 7.22 (s, 2H, H10), 7.14 (s, 2H, H12), 6.72 (m,
2H, H36, H37), 6.36 (t, J = 7 Hz, 2H, H30), 6.28 (t, J = 7 Hz, 2H, H29),
5.30 (m, 1H, H38), 4.59 (d, J = 7 Hz, 2H, H28), 2.72 (s, 2H, H24), 2.59
(s, 6H, Hp-meth), 1.68 (s, 6H, Ho-meth), 1.54 (m, 4H, H25), 1.44 (s, 6H,
H
24), 2.57 (s, 6H, Hp-meth), 1.60 (s, 6H, Ho-meth), 1.37 (s, 6H, Ho-meth),
1.25 (m, 4H, H ), 0.52 (m, 4H, H26), –2.40 (s, 2H, HNHpyr). 13C NMR
25
(125 MHz, CDCl3): d 169.8 (C23), 165.7 (C33), 157.7 (C39), 157.5
(C27), 148.2 (C31), 140.0 (C13), 139.8 (C5), 139.1 (C19), 138.9 (C9),
138.4 (C11), 137.9 (C8), 136.5 (C29), 135.3 (C1), 134.9 (C15), 133.1
(C37), 132.4 (C6), 131.4 (C20), 130.2 (C17), 130.1 (C3), 129.0 (C35),
128.3 (C10), 128.1 (C12), 123.3 (C2), 123.1 (C16), 122.5 (C28), 122.0
(C4), 121.3 (C30), 120.0 (C18), 119.3 (C36), 119.1 (C14), 117.8 (C34),
116.6 (C38), 114.8 (C7), 114.3 (C21), 58.9 (C24), 54.0 (C25), 34.0
(C26), 21.9 (Co-meth), 21.8 (Co-meth), 21.6 (Cp-meth). UV–Vis (CHCl3):
H
o-meth), 0.77 (m, 4H, H26), 0.22 (s, 3H, H40), À2.38 (s, 2H, HNHpyr).
13C NMR (125 MHz, CDCl3): d 169.6 (C23), 163.4 (C33), 157.6 (C27),
156.2 (C39), 148.3 (C31), 139.8 (C13), 139.5 (C5), 138.6 (C9), 138.5
(C19), 138.2 (C11), 137.6 (C8), 135.7 (C29), 135.4 (C1), 135.2 (C15),
132.8 (C37), 132.1 (C35), 131.7 (C6), 131.5 (C20), 129.9 (C3), 129.8
(C17), 128.1 (C10), 127.9 (C12), 123.0 (C16), 122.6 (C2), 122.0 (C28),
121.6 (C4), 120.9 (C36), 120.8 (C30), 120.7 (C34), 120.5 (C18), 119.2
(C14), 115.1 (C21), 114.5 (C7), 110.5 (C38), 58.2 (C24), 54.2 (C25), 52.5
(C40), 34.3 (C26), 21.7 (Co-meth), 21.5 (Co-meth), 21.3 (Cp-meth). UV–Vis
kmax (e
, mMÀ1 cmÀ1): 420 (340.5), 515 (17.0), 548 (4.7), 589 (4.8),
645 (2.3). Anal. Calc. for C73H65N9O3: C, 78.54; H, 5.87; N, 11.29.
Found: C, 78.51; H, 5.96; N, 11.33%. HRMS (MALDI-TOF): m/z calc
for C73H66N9O3, 1116.5289 [M+H]+; found: 1116.5293.
(CH2Cl2): kmax (loge
, mMÀ1 cmÀ1): 420 (335.2), 515 (16.9), 548
(4.6), 589 (4.8), 645 (2.2). Anal. Calc. for C74H67N9O3: C, 78.63; H,
5.97; N, 11.15. Found: C, 78.58; H, 5.95; N, 11.19%. HRMS (MALDI-
TOF): m/z calc for
1130.5449.
C
74H68N9O3, 1130.5445 [M+H]+; found:
2.4. Synthesis of 2-methoxy-N-phenylbenzamide (6)
A mixture of aniline (0.24 mL, 2.6 mmol), 2-methoxy benzoic
acid (0.40 g, 2.6 mmol) and 1-hydroxy-1H-benzotriazole (HOBt)
(0.43 g, 3.2 mmol) in THF (10 mL), was cooled at À10 °C. A solution
of 1,3 dicyclohexylcarbodiimide (DCC) (0.65 g, 3.2 mmol) in THF
(3 mL) was added to the above solution and the mixture was stir-
red at À10 °C for 1 h. The resulting mixture was then stirred at
room temperature for 12 h and filtered. The filtrate was collected
2.3. Synthesis of
acetamidophenyl]-
bis-(2,4,6-trimethyl-phenyl)-porphyrin (1)
a
-5-[2-(bis-(2-(pyridin-2-yl)ethyl)amino)-
-15-[2-((2-hydroxyl)benzamide) phenyl]-10,20-
a
To a solution of porphyrin 3 (34 mg, 0.03 mmol) in dry CH2Cl2
(30 mL) a solution of BBr3 (1.0 M, 0.35 ml, 0.35 mmol) in dry

K. Ladomenou et al. / Polyhedron 54 (2013) 47–53
49
and the solvent was removed under reduced pressure. The residue
was purified by column chromatography, and the desired product
6 was eluted with CH2Cl2 to give a white solid (0.57 g, 95%). Rf
(CH2Cl2): 0.62. 1H NMR (500 MHz, CDCl3): k 9.80 (s, 1H, H5), 8.30
(dd, J1 = 7.75 Hz, J2 = 1.5 Hz, 1H, H8), 7.68 (d, J = 8 Hz, 2H, H3),
7.50 (td, J1 = 7.75 Hz, J2 = 1.5 Hz, 1H, H10), 7.36 (t, J = 8.00 Hz, 2H,
H2), 7.14 (m, 2H, H1, H9), 7.04 (d, J = 8.5 Hz, 1H, H11), 4.06 (s, 3H,
procedures excess of NaCl was used in order to confirm that a Cl
atom remains as the axial Fe ligand. Rf (CH2Cl2/MeOH 9:1) = 0.45.
UV–Vis (CH2Cl2): kmax
(e,
mMÀ1 cmÀ1): 419 (82.4). Found,
1169.4415. Anal. Calc. for C73H63N9O3ClFe: C, 72.72; H, 5.27; N,
10.46. Found: C, 72.79; H, 5.22; N, 10.41%. HRMS (MALDI-TOF):
m/z calc for C73H63N9O3Fe, 1169.4403 [MÀCl]+; found: 1169.4419.
H
13). 13C NMR (125 MHz, CDCl3): d 163.4 (C6), 157.4 (C12), 138.5
2.7. Synthesis of porphyrin 1FeCu
(C4), 133.4 (C10), 132.7 (C8), 129.1 (C2), 124.3 (C1), 122.0 (C7),
121.8 (C9), 120.6 (C3), 111.7 (C11), 56.4 (C13). Anal. Calc. for
To a solution of porphyrin 1Fe (10 mg, 8.5
lmol) in MeCN
C14H13NO2: C, 73.99; H, 5.77; N, 6.16. Found: C, 74.05; H, 6.01;
(5 ml) was added Cu acetate [9.0 mol, from a standard solution
l
N, 6.28%.
of 50 mg of Cu(CH3COO)2 in 25 mL of MeCN], and the mixture
was stirred at 60 °C for 10 min. The solvent was then removed un-
der vacuum, and the resulting solid was dissolved in CHCl3. Upon
the addition of pentane, the complex precipitated. The solid was
filtered and washed with pentane, giving 1FeCu as brown-purple
solid (10.3 mg, 98%). Rf (CH2Cl2/MeOH 9:1): 0.28. UV–Vis (CH2Cl2):
2.5. Synthesis of 2-hydroxy-N-phenylbenzamide (5)
To a solution of compound 6 (100 mg, 0.44 mmol) in dry CH2Cl2
(50 mL) a solution of BBr3 (1.0 M, 2.0 ml, 2.0 mmol) in dry CH2Cl2
was added and the mixture was stirred under argon at À78 °C
for 30 min and at 0 °C for 1 h. The reaction was quenched with
MeOH (40 mL) and H2O (80 mL) and stirred for 20 min at 0 °C.
The mixture was washed with saturated aqueous NaHCO3
(2 Â 50 mL) and water (2 Â 50 mL). The organic phase was dried,
and the solvent was removed under reduced pressure. The crude
material was purified by column chromatography on a silica gel
column (0–6% ethanol in dichloromethane). Compound 5 was
eluted with CH2Cl2/EtOH 95:5 and obtained as a white solid
(88 mg, 94%). Rf (CH2Cl2/EtOH 9:1): 0.62. 1H NMR (500 MHz,
CDCl3): d = 11.98 (s, 1H, H13), 7.94 (s, 1H, H5), 7.58 (d, J = 7.5 Hz,
2H, H3), 7.53 (dd, J1 = 7.75 Hz, J2 = 1.0 Hz, 1H, H8), 7.45 (td,
J1 = 7.5 Hz, J2 = 1.0 Hz, 1H, H10), 7.40 (t, J = 7.5 Hz, 2H, H2), 7.21 (t,
J = 7.5 Hz, 1H, H1), 7.04 (d, J = 8.5 Hz, 1H, H11), 6.93 (t, J = 8.5 Hz,
1H, H9). 13C NMR (125 MHz, CDCl3): d 168.5 (C6), 162.1 (C12),
136.7 (C4), 134.9 (C10), 129.4 (C2), 125.6 (C1), 125.5 (C8), 121.3
(C3), 119.2 (C11), 119.1 (C9), 114.7 (C7). Anal. Calc. for C13H11NO2:
C, 73.23; H, 5.20; N, 6.57. Found: C, 73.45; H, 5.11; N, 6.48%.
kmax (e
, mMÀ1 cmÀ1): 419 (75.5). HRMS (MALDI-TOF): m/z calc for
C
73H63N9O3FeCu, 1232.3699 [MÀCl]+; found: 1232.3711.
2.8. Synthesis of the reduced form of porphyrin 1FeCu
In a glove-box, complex 1Fe (Fe+3) is dissolved in chloroform
and reduced with the addition of aqueous Na2S2O4. After separat-
ing the two phases and drying the organic phase the solvent is re-
moved. The remaining solid residue was dissolved in a mixture of
acetonitrile–ethanol and was added a solution of a copper salt
[Cu(MeCN)4PF6] dissolved in acetonitrile. The resulting mixture
was stirred for 10 min at room temperature. After the removal of
solvents the product is dissolved in THF and removed from the
glove box.
3. Results and discussion
3.1. Synthesis of compounds
Synthesis of compound 1 is based on porphyrin 2 that was pre-
viously synthesized by our group [28], in which there is a Cu tri-
dentate coordination site. The remaining synthetic steps are
presented in Scheme 1, and were done in order to attach a tyrosine
mimic moiety onto the porphyrin ring. Firstly, compound 2 was
coupled with 2-methoxy benzoic acid in the presence of DCC,
yielding porphyrin 3 (85%). The synthesis of model 1 was com-
pleted with deprotection of the hydroxyl group. An excess of boron
tribromide (BBr3) was added to a solution of 3 leading to the for-
mation of compound 1 in 93% yield.
2.6. Synthesis of porphyrin 1Fe
In a Schlenk flask and under an inert atmosphere, porphyrin 1
(40 mg, 0.036 mmol) and iron dibromide (FeBr2) (78 mg,
0.36 mmol) were added. Then, deoxygenated and anhydrous THF
inserted in the flask and the solution was stirred at 55 °C for
12 h. The color of the solution changed from purple to orange.
The completion of the reaction was confirmed by UV–Vis spectros-
copy and TLC chromatography. The solvent removed under vac-
uum and the residue dissolved in chloroform (30 ml) and washed
twice with 2 N HCl (1 Â 30 ml), then once with saturated NaHCO3
(30 ml), dried, filtered, and concentrated under vacuum. The
resulting crude product was purified by column chromatography
(0–6% MeOH in chloroform). The product was eluted at 2% MeOH
in chloroform and recrystallized in CH2Cl2/pentane, to obtain 1Fe,
as a brown-purple solid (33 mg, 79%). Throughout the purification
3.2. 1H NMR studies and synthesis of reference compounds
Next, we wanted to obtain more structural information about
the orientation of Cu binding site and the orientation of phenyl ring
that mimics the Tyr part of the enzyme in 1. In order to achieve
Scheme 1. Synthesis of porphyrin 1.

50
K. Ladomenou et al. / Polyhedron 54 (2013) 47–53
phenyl ring most likely adopts a favorable conformation for the
catalyzed reduction of dioxygen.
Table 1
Chemical shifts of compounds 1, 4 and 5.
Ha
7.46
8.37
Hb
6.57
6.96
Hc
6.75
7.43
Hd
5.28
7.02
He
6.94
Hf
5.97
Hg
6.43
Hh
5.46
3.3. Iron metalation reaction
1
4
5
D
In the next stage, we performed metalation reactions with iron
and copper of final derivative 1. For the metalation reaction of the
porphyrin core, we used iron dibromide (FeBr2), under anaerobic
conditions [30]. After completion of the reaction, we exposed the
solution to the atmosphere and the Fe was immediately oxidized
from the +2 to the more stable +3 oxidation state. During this oxi-
dation iron can be complexed with atmospheric oxygen and vari-
ous oxygenated species can be formed. The separation of the
above species is difficult, thus extractions with dilute solution of
HCl were carried out [31]. With this procedure a chlorine atom
was coordinated with iron and the dimer units were splitted. Con-
sequently, we obtained exclusively monomeric species, where iron
was pentacoordinated and had an axial chlorine ligand. The struc-
ture of iron porphyrin 1Fe is shown in Fig. 3. For the metalation
reaction we used a 10-fold excess of iron dibromide, therefore Fe
could also coordinate to tridentate ligand (Cu coordination site).
However, this coordination is weak and the iron is liberated from
that site, due to protonation of nitrogen atoms by the use of HCl
extractions [32]. The incorporation of iron is confirmed by UV–
Vis spectroscopy [33] and TLC chromatography. The Soret band
of the porphyrin ring was shifted to shorter wavelengths, charac-
teristic of iron binding with the four pyrrolic nitrogens. Also, fewer
7.53
6.93
7.45
7.04
ppm À0.91 À0.39 À0.68 À1.74 À0.59 À0.96 À1.02 À1.58
All chemical shifts are in parts per million; 1H NMR 500 MHz, 300 K.
that, compounds 4 and 5 were synthesised and their 1H NMR
chemical shifts were compared to 1 (Table 1 and Fig. 2). The syn-
thesis of 4 has been described previously [28], while preparation
of 5 is shown in Scheme 2. Unexpectedly, reaction of aniline with
2-methoxy benzoic acid in the presence of DCC, afforded com-
pound 6 in 95% yield. This is probably due to the presence of –
OMe group. The free hydroxyl product 5 was obtained reacting 6
with an excess of BBr3 in 94%. All 1H NMR spectra of compounds
1, 4, 5 are shown in Fig. 2. Protons Ha–Hd of compound 1 are shifted
upfield in comparison to BPEA 4, with values varying from
D
d = 1.74 ppm for Hd to Dd = 0.39 ppm for Hb. This upfield shift ob-
served is due to the porphyrin ring current, which means that the
tridentate ligand is situated inside the cone shield of the porphyrin.
On the other hand, comparison of 1H NMR chemical shifts of phe-
nol protons of 1 and compound 5, could give us some inside about
the orientation of the OH group. Therefore, the phenolic proton
would be a good candidate but is not reliable as it is mobile,
exchangeable and its resonance frequency is temperature-depen-
dent. In contrast, the chemical shifts of the other phenyl ring pro-
tons are indicative of their location in the shielding ring current of
the macrocycle. Comparing these protons we observed smaller
chemical shifts for compound 1 than reference 5, indicating that
are located above the porphyrin ring current. In addition, greater
Q bands and a decrease of extinction coefficient (e) was observed.
Moreover, in order to determine the electronic configuration of
1Fe, 1H NMR spectrum was obtained. At the 1H NMR spectrum
we observed a broad peak at 77.9 ppm (Scheme S9) that corre-
sponds to the pyrrolic protons. This signal is characteristic of
high-spin pentacoordinated Fe+3 porphyrin compounds.
shifts were observed for Hg and Hh, with
Dd = 1.02 ppm and
3.4. Copper metalation reaction
D
d = 1.58 ppm, respectively. This observation favors an orientation
of the phenyl ring with the hydroxyl oriented towards the center of
the molecule. Thus, in model 1, host ligand for Cu ion and the
In the next stage, metalation reaction with Cu was performed
(Fig. 3). The insertion of Cu is done at the iron porphyrin, due to
Fig. 2. 1H NMR of porphyrin 1 and ligands 4, 5 in CDCl3.

K. Ladomenou et al. / Polyhedron 54 (2013) 47–53
51
Scheme 2. Synthesis of ligand 5.
Fig. 3. More likely structures of metalated complexes (the Cu counter anions are
not represented).
harsh conditions used for Fe metalation. Otherwise copper metal
would be replaced by iron. For the introduction of copper, Cu+2
was used [31]. To a solution of porphyrin in MeCN/ethanol, equi-
molar quantity of [Cu(CO2CH3)2] in MeCN was added. The mixture
was stirred at 60 °C for 10 min and the product was obtained pure
after precipitation using pentane. The metalated Cu–Fe product
could not be submitted to further purification due to weak coordi-
nation of Cu ion. 1H NMR spectrum of this complex was obtained
and we observed once again the characteristic pyrrolic signal at
around 80 ppm, indicating that the electronic configuration of
the iron remains the same and is not influenced by the presence
of the copper ion. HRMS aided in the identification of the formed
complex and the results were in agreement with the target
compound.
Fig. 4. UV–Vis spectra in CHCl3 of model 1Fe. Oxidized form (orange line), reduced
form (purple line), bound with oxygen (red line). (Color online)
at 418 nm, that corresponds to the oxidized form of compound
1Fe (Fe+3).
3.6. Binding studies of CO to 1FeCu
In order to proceed to binding studies of CO to model 1FeCu,
both iron and copper must be at their reduced form (Cu+1 and
Fe+2). Towards this effort when a reducing agent (Na2S2O4) was
added, the copper was removed from its pyridine ligand. Therefore,
a different route was followed, reducing first the iron, and then
adding the copper into its reduced form, under anaerobic condi-
tions in a glove box. Then, the CO binding to the reduced form of
1FeCu complex was studied, using FT-IR and UV–Vis spectrosco-
pies. In a solution of 1FeCu in THF carbon monoxide is added
and the color of the solution changed from orange to red. This color
change is observed at UV–Vis spectrum where the Soret band
shifted from 433 to 424 nm, due to coordination of CO to iron
forming a stable low spin six-coordinated complex. The FT-IR spec-
trum of the above complex is shown is Fig. 6. A peak at 1964 cmÀ1
is attributed to the vibration of bound CO to the metal iron of the
porphyrin. At the enzyme of bovine cytochrome c oxidase the value
for v(Fe–CO) is 1965 cmÀ1, that is very close to the one observed
for 1FeCu [22]. At the natural enzyme CO coordinates only with
the heme iron and not with the copper, however during the photol-
ysis of the enzyme CO is released from the iron and is transferred
to the copper ion [34]. In this way the value of bovine CcO v(Cu–
CO) is determined at 2065 cmÀ1 [22]. Similar photolysis
experiments were performed in the case of model compound
1FeCu. The photolysis was done with a nanosecond laser pulse
3.5. Binding studies of O2 to 1Fe
UV–Vis spectroscopy was used in order to study iron metalated
model 1Fe. The complex of the oxidized form (Fe+3) in chloroform,
appears a Soret band at 417 nm (Fig. 4). After the addition of a
reductant (Na2S2O4) the Soret band shifted at 435 nm (Fig. 4).
The shift of the Soret band is due to the iron reduction (Fe+2) and
the simultaneous binding of an axial ligand. Since the solvent used
for the reduction (chloroform) does not bind to iron, possibly one
of the pyridine rings (Cu binding site) is bound to the iron. Due
to all the above results we can conclude that the copper ligand is
placed close to the center of the porphyrin ring, which is in accor-
dance with the structural NMR studies (Section 2.2). Then, the
solution of the reduced form of model 1Fe (Fe+2) is left in contact
with atmospheric oxygen and the UV–Vis spectrum was obtained.
(Fig. 4) The Soret band is shifted to 421 nm, value due to oxygen
binding, as observed previously [30]. Finally, successive UV–Vis
spectra were obtained (Fig. 5), showing a shift of the Soret band

52
K. Ladomenou et al. / Polyhedron 54 (2013) 47–53
Fig. 5. Successive UV–Vis spectra of model 1Fe in CHCl3.
Fig. 7. FT-IR difference spectra before and after the photolysis of 1FeCu.
(416 nm-close to the Soret band) and the intensity of the laser used
was about 10 mW. At the FT-IR difference spectrum obtained a
negative peak at 1964 cmÀ1 attributed to v(Fe–CO) and a positive
peak at 2082 cmÀ1 assigned to v(Cu–CO) (Fig. 7). The value of
v(Cu–CO) at the natural enzyme is smaller to the one observed at
the model compound 1FeCu. This variation is due to different ori-
entation of copper at the enzyme compared to the synthetic model.
carry out the electrochemical experiments, the molecule was ad-
sorbed on the disk electrode, which was a section of a high ordered
graphite rod, with the planes of graphite being perpendicular to
the surface of the electrode (edge-plane graphite electrode, EPGE).
The potential of this modified graphite electrode was scanned from
high to lower values so as to record the voltammogram for the
reduction of O2. The graphite electrode was surrounded by a plat-
inum ring electrode, the potential of which was set at fixed value
(1.0 V) such that H2O2 eventually produced at the disk was oxi-
dized to O2. Catalytic studies of compound 1FeCu are shown in
Fig. 8. First we studied complex 1FeCu (Fig. 8, curve a), the reduc-
tion of dioxygen started at 0.1 V versus SCE. From the comparison
with the 2eÀ reduction wave on a bare EPGE, the number of elec-
trons exchanged per O2 molecule was 3. H2O2 is detected through
its oxidation as soon as O2 is reduced, from the current increase at
the platinum electrode encompassing the graphite disk. This
means that the 2eÀ and 4eÀ reduction mechanisms occur simulta-
neously. From the study of 1Fe (Fig. 8, curve b) we observed that
the absence of Cu did not seem to affect the catalytic ability of
the complex. The number of the necessary electrons for the reduc-
tion per O2 molecule was 2.6 that are less compared to 1FeCu. In
this case more H2O2 is produced as shown from the increased cur-
rent on the platinum electrode (ring). Therefore, catalyst 1Fe
shows lower catalytic activity compared to 1FeCu, due to the
3.7. Electrochemical studies using rotating ring-disk electrode
Following, porphyrin 1FeCu was assessed as promising enzyme
mimic electrochemically using
a rotating ring-disk electrode
(RRDE) in the presence of molecular oxygen. The goal of this inves-
tigation was firstly to understand the role of copper in the catalytic
activity, and secondly to observe if the presence of the hydroxyl
group was essential for the 4eÀ reduction of dioxygen to water.
On the one hand, the main advantage of this technique is its ability
to detect H2O2 by a ring current at the anode (made of a platinum
ring). It is important to note that the H2O2 detected at the anode
was produced by the reduction of dioxygen at the cathode (com-
posed of a graphite disk) and had migrated significantly. On the
other hand, a disadvantage of this technique consists in the irre-
versible adsorption of the catalyst (the synthetic model) on the
surface of the electrode. Indeed, there is no direct way to observe
how the catalyst evolves during the catalytic process. In order to
Fig. 6. Region of FT-IR complex of 1FeCu with addition of CO where the value of
v(Fe–CO) is shown.
Fig. 8. RRDE voltammograms (a: 1FeCu, b: 1Fe, c and d: 2nd scan for 1FeCu and
1Fe, respectively; bottom: disk current, top: ring current).

K. Ladomenou et al. / Polyhedron 54 (2013) 47–53
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In conclusion, metalated porphyrin 1FeCu was effectively syn-
thesized, containing a covalently attached tridentate ligand site
and a tyrosine mimic. NMR spectroscopy was used in order to
investigate the orientation of Cu binding site and the orientation
of phenyl ring that mimics the Tyr. Furthermore, the compound
1Fe can bind oxygen, forming a non stable complex. Also, the re-
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