Synthetic models of the active site of cytochrome c oxidase: Influence of tridentate or tetradentate copper chelates bearing a his-tyr linkage mimic on dioxygen adduct formation by heme/cu complexes

Article abstract of DOI:10.1002/chem.200601884

Two synthetic models of the active site of cytochrome c oxidase-[(L ^N4-OH)Cu^I-Fe^II(TMP)]⁺ (1a) and [(L^N3-OH)Cu^I-Fe^II(TMP)]+ (2a)-have been designed and synthesized. These models

Full text of DOI:10.1002/chem.200601884

FULL PAPER
DOI: 10.1002/chem.200601884
Synthetic Models of the Active Site of Cytochrome c Oxidase: Influence of
À
Tridentate or Tetradentate Copper Chelates Bearing a His Tyr Linkage
Mimic on Dioxygen Adduct Formation by Heme/Cu Complexes
Jin-Gang Liu, Yoshinori Naruta,* and Fumito Tani[a]
Abstract: Two synthetic models of the
active site of cytochrome c oxidase–-
N
tion to superoxide (ꢀ30%) increases
further when the crosslinked phenol
moiety in 2a is deprotonated to
(18O2)}, while
[(LN4-OH)CuI-FeII
[(LN3-OH)CuI-FeII
A
G
produce
the
bridged
A
peroxide
{nOÀ :
O
been designed and synthesized. These
models each contain a heme and a co-
valently attached copper moiety sup-
ported either by a tetradentate N4-
copper chelate or by a tridentate N3-
copper chelate including a moiety that
acts as a mimic of the crosslinked His-
Tyr component of cytochrome c ox-
idase. Low-temperature oxygenation
reactions of these models have been in-
vestigated by spectroscopic methods in-
cluding UV/Vis, resonance Raman,
ESI-MS, and EPR spectroscopy. Oxy-
genation of the tetradentate model 1a
in MeCN and in other solvents produ-
ces a low-temperature-stable dioxygen-
bridged peroxide [(LN4-OH)CuII-O2-FeIII-
[(LN3-OH)CuII-O2-FeIII
812 (16O2)/765 cmÀ1 (18O2)} as the main
dioxygen intermediate. The weak re-
ducibility and decreased O2 reactivity
of the tricoordinated CuI site in 2a are
responsible for the solvent-dependent
formation of dioxygen adducts. The ini-
tial binding of dioxygen to the copper
site en route to the formation of a
bridged heme-O2-Cu intermediate by
model 2a is suggested and the depro-
tonated crosslinked His-Tyr moiety
might contribute to enhancement of
the O2 affinity of the CuI site at an
early stage of the dioxygen-binding
process.
Introduction
heme a3-CuB binuclear center, with an iron–copper separa-
tion of about 5.0 Š. CuB is coordinated by three histidine
residues. The current consensus with regard to the process
of O2-binding and reduction during the CcO catalytic cycle
is that the process occurs through an initial association of di-
oxygen with CuB, followed by transfer of O2 to heme a3, to
form the first spectroscopically observable intermediate:
compound A (FeIII-superoxo). The next detectable inter-
Cytochrome c oxidase (CcO), the terminal enzyme in the
respiratory chain, catalyzes the thermodynamically favora-
ble four-electron reduction of dioxygen to water and con-
comitantly pumps four protons across the mitochondrial or
bacterial membrane, generating the electrochemical proton
[1]
gradient used by ATPsynthase for the formation of ATP.
À
The reduction of dioxygen to water takes place at the
mediate has already undergone O O bond cleavage to pro-
duce the ferryl-oxo (FeIV=O) species (compound P) and
II
À
À
CuB OH. The heterolytic O O bond cleaving process and
other details remain to be clarified. A bridging peroxo spe-
cies (FeIII-O-O-CuII) has been proposed as a possible inter-
mediate in the catalytic cycle,[2] but has yet to be observed
during the course of the enzymatic reaction. Since X-ray
crystallographic analysis revealed an unprecedented cross-
link between one of the CuB-coordinated histidine ligands
[a] Dr. J.-G. Liu, Prof. Dr. Y. Naruta, Dr. F. Tani
Institute for Materials Chemistry and Engineering
Kyushu University, Higashi-ku, Fukuoka, 812-8581 (Japan)
Fax : (+81)92-642-2731
E-mail: naruta@ms.ifoc.kyushu-u.ac.jp
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2007, 13, 6365 – 6378
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6365

and a tyrosine residue (Figure 1),[3] intense efforts to eluci-
date the role(s) of this novel His-Tyr crosslink during the
catalytic cycle of O2 reduction have been undertaken. Al-
To refine our model of the CcO tridentate (His)2CuBACHTREUNG(His-
Tyr) site further and to obtain further insights into the di-
oxygen reduction mechanism, we prepared [(LN3-OH)CuI-FeII-
AHCTREUNG
(TMP)]+ (2a, Figure 2), which represents a new example of
Figure 1. Dioxygen metabolic site structure of bovine heart cytochrome c
oxidase.[3b]
Figure 2. Chemical structures of the model compounds.
though several functional/structural roles for the crosslinked
His-Tyr-CuB moiety have been proposed, its exact role re-
mains elusive.[4] Recent investigations suggest that the pro-
cesses of binding and fission of dioxygen are responsible for
driving internal proton rearrangements, such as deprotona-
tion of the crosslinked tyrosine.[5]
a covalently tethered heme/Cu CcO model bearing a triden-
tate N3-copper chelate with a moiety acting as a crosslinked
His-Tyr mimic. The oxygenation reaction of the N3-copper
chelate heme/copper model compound produces a heme su-
peroxide intermediate in nitrile solvents, while in CH2Cl2/
7% EtCN the coexistence of a heme superoxide and a
bridged heme-O2-Cu peroxide species in equivalent amounts
is observed. Additionally, deprotonation of the crosslinked
phenol moiety results in an increased ratio of peroxide to
superoxide. The process of solvent-dependent formation of
dioxygen adducts by model 2a differs from that of its tetra-
The not fully understood mechanism of dioxygen reduc-
tion by CcO has inspired many chemists to model aspects of
its active binding site and related chemical properties. Nu-
merous heme/copper systems designed as synthetic function-
al models have been reported and investigations of their re-
actions with O2 have provided fundamental insights into the
reactivity of the enigmatic heme a3/CuB binuclear center in
CcO.[6] However, the majority of the reported biomimetic
models lack a crosslinked His-Tyr mimic moiety, which ap-
pears to play important roles in the process of CcO-cata-
lyzed dioxygen reduction. Examples of binucleating ligands
bearing a Tyr mimic and synthetic analogues of the His-Tyr
crosslinkage and their Cu and Zn complexes have recently
been reported,[7–9] but O2 reaction studies that combine a
CuB site mimic with a heme component are still very rare.
Karlin and co-workers reported an oxygenation reaction of
an untethered heme/copper assembly in which a crosslinked
imidazole–phenol was employed as a part of the tetraden-
tate copper ligand.[10] In our preliminary communication, we
reported the first example of a covalently appended tetra-
dentate copper ligand incorporating a crosslinked imida-
dentate
counterpart
1a
([(LN4-OH)CuI-FeII(TMP)]+;
ACHTREUNG
Figure 2), which only forms a bridged peroxide in a variety
of solvents. In this report we describe the details of the syn-
theses of these model compounds and the formation of the
corresponding low-temperature dioxygen adducts in differ-
ent solvents as observed by UV/Vis, resonance Raman, ESI-
MS, and EPR spectroscopy.
Results and Discussion
Design and syntheses: The copper complex [Cu(OH)TME-
DA]2Cl2 (TMEDA=N,N,N’,N’-tetramethylethylenediamine),
À
zole–phenol derivative with
a
heme functional group
previously developed for C C bond oxidative homocoupling
(Figure 2, 1a).[11] The resultant heme/copper compound
reacts with dioxygen to form a peroxide species that is
stable at low temperature. In a very recent paper[12] we de-
of naphthols or alkynes,[13] had been reported to catalyze the
À
formation of C N bonds between commercially available ar-
ylboronic acids and imidazole derivatives efficiently at room
temperature.[14] This system has the advantage of employing
mild, abasic conditions, but it has only been applied for a
limited number of substrates. When performed on a 4-sub-
stituted imidazole, the reaction has afforded mixtures of re-
gioisomers that have not been isolated. Here we extend this
approach to the preparation of N-arylimidazoles as building
scribed
a
fully integrated CcO model (LN4-OHCuI-
FeIITMPIm) possessing a His-Tyr crosslink mimic and a
heme a3 axial imidazole ligand. This model complex under-
goes a unique transformation from a heme-m-peroxo-CuII
species to a heme-superoxo/CuI intermediate in the course
of the oxygenation reaction at low temperature.
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Chem. Eur. J. 2007, 13, 6365 – 6378

Cytochrome c Oxidase Models
FULL PAPER
blocks of new model com-
pounds, incorporating the N-(2’-
hydroxyphenyl)imidazole
moiety as the CuB site mimic.
Scheme 1 shows the prepara-
tion of the tripodal copper
moiety for the model com-
pounds. The methoxymethyl-
protected (MOM-protected) 2-
hydroxyphenylboronic acid
3
was prepared in 65% overall
yield by sequential reactions in-
volving metallation (nBuLi/
Et2O/À708C) of MOM-protect-
ed
2-bromo-4-methylphenol
and treatment with BACHTRE(UNG OMe)3,
followed by acidic workup.
Compound 4, in which the hy-
droxy group is protected by a
tert-butyldiphenylsilyl
group
(TBDPS), was readily prepared
in a high yield of 92% from 4-
(hydroxymethyl)imidazole hy-
drochloride by treatment with
tert-butylACHTREUNG(chloro)diphenylsilane
and imidazole in DMF. The
Scheme 1. Preparation of the tripodal copper ligand for the model compounds. a) cat. [Cu(OH)TMEDA]2Cl2,
CH2Cl2, O2, RT, 75%; b) nBuN+FÀ, THF, RT, 93%; c) MnO2, CHCl3, reflux, 85%; d) 1) Et3N, MeOH,
2) NaBH4, 65%; e) 1) K2CO3, CH3CN, RT, 61%, 2) KOH, THF, RT, 86%; f) 1) Et3N, CH3CN, RT, 45%,
2) KOH, THF, RT, 90%.
preparation of 5a was per-
formed by coupling of
3
(1.1 equiv) with 4-(tert-butyldi-
phenylsilanyloxymethyl)-1H-imidazole (4, 1.0 equiv) in the
presence of [Cu(OH)TMEDA]2Cl2 (10 mol%) and oxygen
in CH2Cl2 at room temperature over 48 h. The resulting two
isomers 5a and 5b were readily separable by silica gel
column chromatography in yields of 75 and 7%, respective-
ly; the regioselectivity of this reaction is about 10:1. It is
noteworthy that when 1H-imidazole-4-carbaldehyde was
employed in the coupling reaction, none of the desired
product was obtained, while a change in the protecting
group in 4 from TBDPS (tBuPh2Si) to TBDMS (tBuMe2Si)
decreased both the reaction yield (57%) and the regioselec-
tivity (3:1). The steric hindrance and introduction of a sub-
stituent at the 4-position in imidazole appear to have a re-
markable effect on yield and regioselectivity. Since ortho-
methoxyphenylboronic acids have been found to depress
cross-coupling yields severely,[14] we chose MOM to protect
the phenolic hydroxy group. This choice of protecting group
was also based on the following requirements: 1) it should
tolerate basic or mildly acidic conditions during further re-
action steps; 2) it should be compact enough to avoid possi-
ble steric hindrance during the N-arylation of imidazoles,
and 3) it should be removable under mild conditions at the
final step.
subsequently reduced in situ with NaBH4 to give 9 in a yield
of 65%. The tripodal ligands 13 and 15 were obtained as fol-
lows: the amine 9 was first treated with methyl 6-(chlorome-
thyl)nicotinate (10) or methyl m-(chloromethyl)benzoate
(11) in the presence of a base such as K2CO3 or Et3N in
CH3CN, and the resultant products were then hydrolyzed in
KOH solution to provide moderate overall yields. The tripo-
dal ligands thus prepared are important building blocks
used in the assembly of CcO active site models.
The syntheses of the binucleating ligands 17 (Scheme 2)
and 19 (Scheme 3) were completed in subsequent steps. The
condensation reactions between 13 or 15 and the porphyrin
16 (TMPNH2: 2-[10,15,20-tris(2,4,6-trimethylphenyl)por-
phyrin-5-yl]phenylamine) were performed in the presence of
Et3N/2-(chloromethyl)pyridium iodide in CH2Cl2 to give the
covalent conjugates 17 (63%) and 19 (45%), respectively.
The MOM group in 17 was removed with bromotrimethylsi-
lane in CH2Cl2 at À308C to produce the binucleating free
hydroxy ligand 18 (LN4-OHTMP) in a moderate yield of 60%
(Scheme 2), and addition of an excess of FeBr2 to the por-
phyrin 18 in THF at reflux, followed by extraction with de-
gassed aqueous Na2EDTA solution, produced the corre-
sponding mononuclear FeII porphyrin. Further addition of
The isolated N-arylimidazole 5a was converted into 7 by
sequential deprotection of compound 5a in THF and oxida-
tion with freshly prepared MnO2 in chloroform. Treatment
of 7 with 2-aminomethylimidazole (8) in methanol generat-
ed the corresponding Schiff base intermediate, which was
(CH3CN)4]+ (TfOÀ) pro-
one equivalent of copper salt [CuACHTREUNG
vided the desired model compound 1a.
Compound 2a was prepared through a similar stepwise
metallation reaction with a minor alteration (Scheme 3): the
removal of the MOM group was carried out after insertion
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6367

Y. Naruta et al.
II
TPA)Cu -O2-FeIII(TMP)]+,
ACHTREUNG
which has absorption maxima
at 420 nm, 557 nm, and 612 nm
in MeCN and the structure of
which has been determined by
X-ray crystallographic analy-
sis.[15] Application of a vacuum
does not affect the UV/Vis fea-
tures of the oxy form, which in-
dicates that the formation of
the dioxygen adduct is irreversi-
ble and stable at À308C. The
dioxygen adduct is also stable
in solvent mixtures such as
MeCN/THF
and
MeCN/
CH2Cl2, but its thermal stability
decreases in CH2Cl2 and tolu-
ene.
A dioxygen adduct is also
generated on oxygenation of
the MOM-protected model 1b
Scheme 2. a) 2-(Chloromethyl)pyridinium iodide, Et3N, CH2Cl2, RT, 63%; b) Me3SiBr, CH2Cl2, À308C, 60%;
c) FeBr2, THF, Na2EDTA; d) [CuI
(CH3CN)4]·(CF3SO3), RT, 94% (1a), 95% (1b).
A
ACHTREUNG
of iron into the porphyrin. This
alteration raises the yield to
80%. Compound 2b, contain-
ing the deprotonated cross-
linked
imidazole–phenol
moiety, was prepared by treat-
ment of the dithionite reduced
THF solution of compound 21
with
a
degassed aqueous
Na2CO3 solution.
Oxygenation of [(LN4-OH)CuI-
FeII(TMP)]+
ACHTREUNG ACHTREUNG(1a): Oxygenation
of 1a in MeCN at À308C re-
sulted in significant UV/Vis
spectroscopic changes (Fig-
ure S1, Supporting Informa-
tion). The reduced form has ab-
sorption maxima at 429 nm (e=
82500mÀ1 cmÀ1), 533 nm (e=
9700mÀ1 cmÀ1), and 569 nm (e=
4000mÀ1 cmÀ1), and the intro-
duction of O2 produces a stable
dioxygen adduct with new spec-
tral features at 421 nm (e=
57000mÀ1 cmÀ1), 558 nm (e=
8000mÀ1 cmÀ1) and 614 nm (e=
3800mÀ1 cmÀ1). The UV/Vis
spectrum of the dioxygen
adduct resembles that of the di-
oxygen-bridged heme-peroxo-
Scheme 3. a) 2-(Chloromethyl)pyridinium iodide, Et3N, CH2Cl2, RT, 45%; b) FeBr2, THF, Na2EDTA, 88%;
c) Me3SiBr, CH2Cl2, À308C, 80%; d) Na2S2O4, THF/toluene, RT; d’) Na2S2O4, THF/toluene, 0.1m Na2CO3
copper
complex
[(5Me- (aq.), RT; e) [CuI
ACHTER(NGU CH3CN)4]·ACHTRE(UNG CF3SO3), RT, 94% (2a), 93% (2b), 95% (2c).
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Cytochrome c Oxidase Models
FULL PAPER
Table 1. Resonance Raman data for heme-peroxo-copper complexes.
Complexes nOÀ
in MeCN, exhibiting UV/Vis
features and thermal stability
similar to those of 1a (Fig-
ure S2, Supporting Informa-
tion).
References
O
nACHTERNGU ACHTREUGN
(16O2)/n(18O2)
tetradentate N4-copper chelate
A
G
799/752
801/755
790/746
803/759
793/751
788/744
808/762
815/769
813/769
this work
this work
[15]
[16c]
[16d]
[16b]
[16a]
[10]
A
E
Resonance Raman (rR) spec-
troscopy was used to character-
ize the dioxygen adduct under
the same experimental condi-
tions as used for UV/Vis spec-
troscopic measurements. The
rR spectrum of the dioxygen
adduct of 1a measured in
MeCN at À308C indicates that
it is a peroxo complex with a
high-spin ferric heme configura-
tion. In the high-frequency
region of the rR spectrum, the
spin-state maker band (n2) ap-
pears at 1552 cmÀ1, while the
peak at 1359 cmÀ1 is attributa-
A
G
A
G
G
[(6L)CuII-O2-FeIII
[(TMPA)Cu -O2-FeIII
A
R
ACHTREUNG
E
G
[10]
[12]
[12]
A
N
803, 787/752
804, 788/752
G
N
tridentate N3-copper chelate
A
N
810/764
812/765
747/707
this work
this work
[21a]
[21b]
[21c]
R
ACHTREUNG
[(2L)CuII-O2-FeIII
[(LMe2N)CuII-O2-FeIII
[(DHIm)
(TACN)CuII-O2-FeIII
]
+
U
G
767/726, 752/707
758/740
+
C
G
]
ble to the oxidation-state maker band (n4) of the porphyrin.
An oxygen isotope-sensitive band at 799 cmÀ1 that shifts by
À47 cmÀ1 with 18O2 substitution is observed in the low-fre-
quency region (Figure S3, Supporting Information).
The rR spectrum of the dioxygen adduct of 1b shows a
similar isotope-sensitive band at 801 cmÀ1, shifting to
755 cmÀ1 upon 18O2 substitution (Figure S4, Supporting In-
À
formation). The values of the O O vibrations are compara-
ble to those seen in previously reported heme-peroxo-
copper models possessing a tetradentate Cu-chelate moiety
(Table 1).[10,12,15,16] The fact that the O O vibration frequen-
À
cies of the peroxides formed by the free hydroxy model 1a
and the protected hydroxy model 1b are almost identical
suggests that interactions of the phenol moiety with the per-
oxide group are insignificant if they do occur.
Figure 3. ESI-MS spectrum of [(LN4-OH)CuII-O2-FeIII(TMP)]+. The solid
ACHTREUNG
line shows the observed spectrum and the bars represent the simulated
isotope distribution.
ESI mass spectrometry is very useful for the study and
characterization of the relatively stable peroxides described
here. The mass spectrum of the reduced form 1a exhibits a
parent ion at 1286.5 ([MÀTfO]+)—1330.5 ([MÀTfO]+) for
1b—and this shifts to peaks centered at 1318.5 ([MÀTfO]+)
—1362.5 ([MÀTfO]+) for 1b—when 16O2 is introduced to
the solution. The observed isotope distribution of peaks
agrees very well with the simulated pattern based on the
ratio of FeII-CuI/O2 1:1 (Figure 3 and Figure S5, Supporting
Information). The expected increase of 4 amu is observed
when 18O2 is substituted. Frozen solutions (CH3CN, 77 K) of
the dioxygen adducts of 1a and 1b are EPR silent, which in-
dicates the occurrence of antiferromagnetic coupling be-
tween the two paramagnetic metals and provides further
evidence of formation of a dioxygen-bridged peroxide.
From evidence obtained from UV/Vis, rR, ESI mass, and
EPR experiments, we conclude that the oxygenation reac-
tions of the tetradentate N4-copper chelate heme/Cu
models (1a and 1b) in MeCN generate dioxygen-bridged
II
scopic properties to those of [(5MeTPA)Cu -O2-FeIII-
AHCTREUNG
(TMP)]+, which adopts a m-h2:h1 dioxygen binding mode (h2
to Fe and h1 to Cu), we infer that the same dioxygen binding
mode exists for the peroxo complexes [(LN4-OR)CuII-O2-FeIII-
AHCTREUNG
(TMP)]+ (1a: R=H; 1b: R=MOM) described here.
Oxygenation of [(LN3-OH)CuI-FeII(TMP)]+
ACTHERNGU ACHTREU(GN 2a): The UV/Vis
spectroscopic changes that occur during the oxygenation re-
action of 2a in propionitrile at À808C are shown in
Figure 4. Exposure of 2a to dioxygen gives rise to distinctive
spectral changes in the Soret band, with shifting from
427 nm (e=72000mÀ1 cmÀ1) to 414 nm (e=56800mÀ1 cmÀ1)
and the appearance of a shoulder at 400 nm, while the Q-
band at 531 nm becomes flat (e=8900 to 4000mÀ1 cmÀ1). RR
spectroscopic investigation of this oxygenation reaction
under the same experimental conditions indicates a lack of
the characteristic oxygen isotope-sensitive band in the
region near 800 cmÀ1 or above. However, an oxygen iso-
peroxo complexes [(LN4-OR)CuII-O2-FeIII
(TMP)]+ (1a: R=
ACHTREUNG
H; 1b: R=MOM). Given the similarities of the spectro-
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6369

Y. Naruta et al.
a nitrile solution results in the formation of a heme superox-
ide adduct, in contrast to the formation of a dioxygen-bridg-
ed peroxide [799 (16O2)/752 cmÀ1 (18O2)] observed with the
tetradentate counterpart 1a in acetonitrile solution as de-
scribed above.
When the oxygenation reaction of compound 2a is carried
out in CH2Cl2/7% EtCN at À1008C, it gives rise to electron-
ic absorption spectral changes, with the Soret band shifting
from 428 nm (e=85000mÀ1 cmÀ1) to 419 nm (e=
73000mÀ1 cmÀ1) and the Q-band shifting from 532 nm (e=
7500mÀ1 cmÀ1) to 543 nm (e=6300mÀ1 cmÀ1) (Figure 6). The
Figure 4. UV/visible spectra changes of 2a upon oxygenation in EtCN at
À808C. Inset: Resonance Raman spectra of oxy-2a formed from 16O2 (a)
and 18O2 (b). c) Difference spectrum [a) minus b)]. Conditions: EtCN,
À808C, excitation at 413 nm, 25 mW; * solvent.
tope-sensitive band is observed at 576 (16O2)/551 cmÀ1 (18O2)
(Figure 4, inset) with a n4 band appearing at 1366 cmÀ1. The
observed isotopic frequency shift [Dobsd(16O2/18O2)=25 cmÀ1]
matches well with the expected value for the nFe mode in
À
O2
the diatomic harmonic approximation. The reported CcO
(NMePr)]+ (570 cmÀ1),[17]
À
model compound PorFe O2/[Cu
oxymyoglobin (570 cmÀ1),[18] oxy-CcO (572 cmÀ1),[19] and
other well characterized examples of heme superoxide com-
À
plexes manifest similar Fe O2 [nFe O ] stretching vibra-
À
2
Figure 6. UV/visible spectra of the reduced form (dashed) and the oxy
form (solid) of 2a in CH2Cl2/7% EtCN solution at À1008C. Inset: Reso-
nance Raman spectra of oxy-2a formed from 16O2 (a) and 18O2 (b). c) Dif-
ference spectra [a) minus b)]. Conditions: CH2Cl2/7% EtCN, À1008C,
excitation at 413 nm, 25 mW; * solvent.
tions,[20] so the Raman band at 576 cmÀ1 is assigned to the
À
Fe O2 stretching mode of a heme superoxide species. The
frozen sample of this dioxygen adduct displays a copper(II)
EPR signal of a shape and intensity similar to that of the
oxidized form at room temperature (Figure 5c), which sug-
gests that during the course of the oxygenation reaction at
À808C the copper(I) site is directly oxidized to copper(II).
Consequently, the oxygenation reaction of compound 2a in
rR spectrum of this oxy form exhibits two groups of isotope-
sensitive bands: one at 810 cmÀ1 (16O2)/764 cmÀ1 (18O2), and
the other at 576 cmÀ1 (16O2)/550 cmÀ1 (18O2) (Figure 6, inset).
The 810 cmÀ1 band is attributable to the O O stretching
À
[nO ] mode, and its value is similar to those in the reported
2
dioxygen adducts in the peroxy state with N4-copper chelate
heme/Cu complexes (Table 1) and contrasts with those of
N3-copper chelate heme-peroxo-Cu complexes, which ex-
[21]
hibit lower O O stretching vibrations. The 576 cmÀ1 iso-
À
À
tope-sensitive band is attributable to the Fe O2 stretching
vibration of a heme superoxide and shows the same value as
that observed in EtCN solution. This rules out assignment
as the nFe of the peroxide because of splitting of the oxida-
À
O
tion state marker band (n4) of the porphyrin at 1360 and
1366 cmÀ1 in the rR spectra (Figure 7). The ratio of the per-
oxide to superoxide as inferred from the intensity of the oxi-
dation marker band is about 1:1. The EPR spectrum of the
frozen sample of the oxy form reveals a CuII EPR signal of
an intensity corresponding to ꢀ50% of that of its oxidized
form at room temperature (Figure 5b), consistently with the
results obtained from the rR experiments. From this it can
Figure 5. ESR spectra of frozen samples (77 K) of a) oxy-2b, and b) oxy-
2a generated in CH2Cl2/7% EtCN at À1008C. c) Oxy-2a generated in
EtCN at À808C. d) Oxy-2a at RT.
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Cytochrome c Oxidase Models
FULL PAPER
Figure 7. High-frequency resonance Raman spectra of oxy-2b (c) and
oxy-2a (g) generated in CH2Cl2/7% EtCN at À1008C with 16O2. Oxy-
2a generated in EtCN (a) at À808C with 16O2; conditions: 413 nm ex-
citation, 25 mW.
Figure 9. Resonance Raman spectra of oxy-2b formed from 16O2 (a) and
18O2 (b). c) Difference spectrum [a) minus b)]. Conditions: CH2Cl2/7%
EtCN, À1008C, excitation at 413 nm, 25 mW.
be inferred that the oxygenation reaction of 2a in CH2Cl2/
7% EtCN at low temperature produces mixtures of [heme-
peroxo-copper(II)] and [heme-superoxo, copper(II)] inter-
mediates in equivalent amounts.
region, a new isotope-sensitive band at 491 (16O2)/463 cmÀ1
(18O2) is observable in addition to the 577 (16O2)/553 cmÀ1
18
À
( O2) band arising from the Fe O2 stretching vibration of a
Deprotonation of the crosslinked phenol moiety of com-
pound 2a by treatment with Na2CO3 solution yields com-
pound 2b. Notably, its oxygenation reaction results in the
formation of peroxide as the major dioxygen adduct with
the proportion of the superoxide species decreasing signifi-
cantly (peroxo/superoxo ꢀ70:30). Oxygenation of 2b in
CH2Cl2/7% EtCN solution at À1008C produces UV/visible
spectral changes similar to those seen with 2a, while a small
but apparent difference is observable in the Q-band region,
which shows new bands at 546 nm (e=5400mÀ1 cmÀ1) and
570 nm (sh) with decreased absorption coefficients
(Figure 8). The rR spectrum of the oxy form shows obvious
heme superoxide. Although not well defined from the back-
ground, this isotope-sensitive band is indeed present in dif-
ference spectra from repeated experiments. The band at
491 cmÀ1 (D18O2 =À28 cmÀ1) resembles that of the reported
nCu mode in binuclear copper peroxide adducts [nCu
:
À
O
À
O
488 cmÀ1 (D18O2 =À24 cmÀ1)],[22] whereas its value is lower
than that of the reported nFe in heme-O2-Cu peroxides
À
O
[nFe O: 529 cmÀ1 (D18O2 =À21 cmÀ1)].[10] As a result, it may
À
tentatively be assigned to the nCu mode of the newly
À
O
formed peroxide. The main porphyrin oxidation marker
band (n4) appears at 1360 cmÀ1 with a small shoulder at
1366 cmÀ1 (Figure 7), which suggests that the dioxygen ad-
ducts are dominated by the peroxide species in the solution.
The spin state marker band (n2) of the porphyrin appears at
1554 cmÀ1, consistent with the presence of five-coordinate
high-spin ferric heme. This peroxide species has poorer ther-
mal stability than its tetradentate counterpart [(LN4-OH)CuII-
À
Raman enhancement of the O O stretching vibration,
which appears at 812 cmÀ1 with 16O2 and shifts to 765 cmÀ1
upon substitution with 18O2 (Figure 9). In the low-frequency
O2-FeIII(TMP)]+ (1a-O2), requiring temperatures below
ACHTREUNG
À
À758C in CH2Cl2/7% EtCN solution and its O O stretching
frequency shifts by 13 cmÀ1. Only a weak CuII signal is ob-
servable in the EPR spectrum of the frozen sample of the
oxy form (Figure 5a), providing further evidence for the ex-
istence of a dioxygen-bridged peroxide species as the main
adduct. The EPR results also rule out the presence of a
side-on h2-peroxo dioxygen heme/CuII adduct because the
side-on h2-peroxo dioxygen heme adduct usually displays a
strong EPR marker signal at g=4.2.[23]
From all of these spectroscopic observations, we conclude
that the oxygenation reaction of the deprotonated-hydroxy
model compound 2b in CH2Cl2/7% EtCN solution yields a
bridged peroxide [(LN3-ONa)CuII-O2-FeIII(TMP)]+ as the main
ACHTREUNG
component and a heme superoxide as a minor component.
À
It is noteworthy that the frequency of the O O stretching
Figure 8. UV/visible spectra of oxy-2a (a) and oxy-2b (c) generated
in CH2Cl2/7% EtCN solution at À1008C.
vibration (812 cmÀ1) of this newly formed peroxide is distin-
Chem. Eur. J. 2007, 13, 6365 – 6378
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6371

Y. Naruta et al.
guishable from those of the tridentate copper chelate heme-
peroxo-Cu complexes, which exhibit lower nOÀ values (740–
O
770 cmÀ1) (Table 1), while its value resembles that of the
heme-peroxo-Cu complexes with tetradentate copper che-
late [nOÀO: ꢀ800 cmÀ1]. As the inclination of CuII forms is to
form five-coordinate complexes, tetradentate ligation of CuI
limits the binding of dioxygen to an end-on fashion, while
tridentate ligation of CuI favors the side-on binding of di-
oxygen.[24] It is most likely that the dioxygen binding mode
for [(LN3-ONa)CuII-O2-FeIII(TMP)]+ adopts a different m-h1:h2
ACHTREUNG
pattern with h1 to Fe and h2 to Cu. Nevertheless, these ex-
perimental results cannot rule out another outcome in
which dioxygen adopts the same binding configuration as in
N4-copper chelate heme-peroxo-Cu complexes (m-h1:h2 with
h2 to Fe and h1 to Cu), with the copper(II) site coordinated
by an additional solvent molecule such as EtCN (Figure 10).
Oxygenation of the MOM-protected compound 2c occurs
in a similar manner to that of 2a. In EtCN solvent at
À808C, the oxygenation reaction produces a heme superox-
ide as the only observable dioxygen adduct, whereas in
CH2Cl2/7% EtCN solution (Figure S6 and Figure S7, Sup-
porting Information) it yields both the heme superoxide
[nFe : 578 (16O2)/552 cmÀ1 (18O2)] and the peroxide species
À
O2
[nO : 812 (16O2)/765 cmÀ1 (18O2)] in proportions of about 55
Scheme 4. Reaction behavior of the untethered heme superoxide
2
[(NH2TMP)FeIII-(O2À)] with the copper(I) components [CuILN3-OH
[CuILN4-OH]+.
]
and
+
and 45%, respectively.
changed (Figure 11a). Upon
addition of one equivalent of
+
[CuILN4-OH
]
to the heme su-
peroxide solution, however, a
new species is generated, with
UV/Vis features (EtCN, lmax
=
421, 567, 616 nm, Figure 11b)
very similar to those of the
peroxide [(LN4-OH)CuII-O2-FeIII-
AHCTREUNG
(TMP)]+ formed with the teth-
ered model 1a (MeCN, lmax
=
421, 558, 614 nm). These re-
sults clearly show that the tri-
dentate chelated copper(I)
cannot reduce the corresponding
Figure 10. Possible dioxygen binding modes for the peroxide [(LN3-ONa)CuII-O2-FeIII(TMP)]+.
ACHTREUNG
Reactions between untethered heme superoxide and CuI
complexes: The experimental results demonstrate that the
oxygenation of the N3-copper chelate CcO model 2a differs
significantly from that of its tetradentate counterpart 1a. To
increase our understanding of the unusual oxygenation be-
havior of 2a further, we studied some preliminary reactions
complex [CuILN3-OH
]
+
heme superoxide and that the formation of the untethered
heme-peroxo-copper species is only observed with the tetra-
dentate-ligated copper(I) complex [CuILN4-OH]+. This is in-
terpreted in terms of a tridentate chelated copper(I) com-
plex usually being a poor reductant, while a tetradentate
ligand makes its copper(I) complex a stronger reductant, fa-
cilitating binding and reaction with the heme superoxide, as
is further manifested by the CuII/CuI redox potentials of
À
of untethered heme superoxide [(NH2TMP)FeIII
À
A
which is formed from the oxygenation of the lower part of
+
1a or 2a, with copper(I) components [CuILN4-OH
]
and
+
I
N4-
[CuILN3-OH]+, which correspond to the copper moieties of
the tethered models 1a and 2a, respectively (Scheme 4).
Oxygenation of (NH2TMP)FeII in EtCN at À808C produced
a superoxide with electronic absorption maxima at 415, 543,
these two complexes ([CuILN3-OH] : E = =À0.32 V; [Cu L
1
2
OH
+
+
1
] : E = =À0.47 V vs Fc/Fc in MeCN). Thus, the triden-
2
tate-chelated copper(I) complex [CuILN3-OH]+, the upper
part of 2a, does not react with the heme superoxide
À
and 582 nm. After removal of excess dioxygen, one equiva-
[(NH2TMP)FeIII-(O2 )], a moiety of the dioxygen adduct
+
lent of the copper(I) complex [CuILN3-OH
]
was added to the
formed with the heme part of 2a. Meanwhile, the complex
solution, during which the absorption maxima remain un-
readily reacts with dioxygen. It is notable that the oxygena-
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Chem. Eur. J. 2007, 13, 6365 – 6378

Cytochrome c Oxidase Models
FULL PAPER
À
transient species [LN3-OHCuI···
ACHTREUNG
duced, in which the copper portion [CuILN3-OH
]
cannot
+
react further with the corresponding superoxide because of
its weak reducibility. Consequently, copper(I) is directly oxi-
dized to copper(II) by the existing excess amounts of dioxy-
gen, until it finally produces the heme superoxide, with the
copper part oxidized to CuII. On the other hand, when the
oxygenation takes place in CH2Cl2/7% EtCN solution, bind-
ing of O2 both to the copper(I) site and to the lower ferrous
heme proceeds competitively and statistically generates two
À
transient species: [LN3-OHCuI···
A
-
À
CuII-(O2 )···FeIITMP]+. The former species ultimately pro-
duces the heme superoxide, leaving the cupric component
unbound, and the latter species—in which the side-on-
bound CuII
A
À
rous heme—eventually yields the bridged peroxide. Depro-
tonation of the crosslinked phenol moiety yields a monoa-
nionic ligand that endows the copper(I) complex with en-
hanced O2 reactivity. As a result, the relative proportion of
the second transient species increases and the formation of
a bridged heme-O2-Cu adduct rises as observed experimen-
tally. In the case of 1a, initial O2 binding may occur either
at the CuILN4-OH site or at the ferrous heme moiety, to gener-
À
ate the resultant transient species [LN4-OHCuI···
A
FeIIITMP]+ and [LN4-OHCuII-(O2 )···FeIITMP]+, either of
which will progress to the formation of the dioxygen-bridged
peroxide product.
À
With regard to the possible roles of the crosslinked His-
Tyr moiety, our very recent study on model complexes of
the CuB site of CcO reveals that the imidazole–phenol cova-
lent linkage has only a minor effect on the electronic struc-
ture of the phenoxyl radical. It appears that its role is more
likely to be structural rather than its acting as a provider of
the fourth electron in the dioxygen reduction process.[9a]
Since it has been proposed that association of dioxygen with
Figure 11. a) UV/visible spectrum of the superoxo compound
II
À
[(NH2TMP)Fe O2] (a) and the resultant spectrum after addition of
CuILN3-OH (c). b) UV/visible spectrum of the superoxo compound
II
À
[(NH2TMP)Fe O2] (a) and the resultant spectrum after addition of
I
CuB prior to binding to heme a3 might occur during the
CuILN4-OH (c).
early stage of the oxygenation reaction with reduced CcO,[26]
this model study leads us to propose that the deprotonated
crosslinked His-Tyr, identified by Yoshikawa et al.[3a] in the
fully oxidized state of the enzyme, may serve to enhance the
+
tion reaction of [CuILN3-OH
]
in EtCN is strongly suppressed
relative to that in CH2Cl2, which would usually be expected
when dioxygen binds to a tri-
dentate-ligated copper(I) com-
plex.[21a–b,25]
Possible mechanism for dioxy-
gen binding to model com-
pounds: From these experimen-
tal observations of the tethered
and untethered systems, step-
wise binding of dioxygen to the
N3-copper chelate CcO model
2a is proposed (Scheme 5).
When oxygenation of 2a is car-
ried out in EtCN, binding of O2
+
to the [CuILN3-OH
]
site is signif-
icantly suppressed by the sol-
vent EtCN molecules and
a
Scheme 5. Proposed stepwise binding of dioxygen to the model compounds.
Chem. Eur. J. 2007, 13, 6365 – 6378
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6373

Y. Naruta et al.
I
spectrometer. Scanning was in 0.1 Dalton steps with a 10 ms dwell time
per step. The orifice voltage was controlled from À20 to +180 V, depen-
dent on the intensity of TIC. High-resolution mass (HR-MS) spectra
were recorded on a JEOL LMS-HX-110 spectrometer with 3-nitrobenzyl
alcohol (NBA) as a matrix. The UV/Vis electronic spectra were recorded
on a Hamamatsu PMA-11 CCD spectrophotometer with a Photal MC-
2530 (D2/W2) as a light source. The temperature was controlled with a
NESLAB ULT-95 low=temperature circulator. Infrared spectra were re-
corded on a BIO-RAD FTS-6000 spectrometer. EPR spectra were ob-
tained at 77 K in 5.0 mm diameter ESR quartz tubes on a JEOL JES-
TE 300 spectrometer. The magnetic field was calibrated from the hyper-
fine coupling constants (hfccs) of MnII ion doped in MgO powder
(86.9 Gauss). The UV/Vis electronic spectra were recorded on a Hama-
matsu PMA-11 CCD spectrophotometer with a Photal MC-2530 (D2/W2)
as a light source. The temperature was controlled with a NESLAB ULT-
95 low-temperature circulator.
O2 affinity of CuB site at an early stage of the O2 binding
process.
Conclusion
Heterobinucleating ligands composed of a porphyrin togeth-
er with either a tetradentate N4-copper chelate or a triden-
tate N3-copper chelate with a crosslinked His-Tyr mimic,
and their corresponding heme-copper complexes [(LN4-
OH)CuI-FeII(TMP)]+ (1a) and [(LN3-OH)CuI-FeII(TMP)]+
ACHTREUNG ACHTREUGN
(2a), respectively, have been designed and synthesized as
model compounds of the active site of cytochrome c oxidase.
These two models showed distinct oxygenation reactions at
low temperature. Oxygenation of the tetradentate model 1a
both in MeCN and in other solvents produced a low-temper-
ature-stable dioxygen-bridged peroxide [(LN4-OH)CuII-O2-
Resonance Raman (rR) spectra were obtained on a SpectraPro-300i
spectrometer (Acton Research) with a 2400-groove grating, a Beam-
lok 2060 Kr ion laser (Spectra-physics), a holographic Supernotch filter
(Kaiser Optical Systems), and an LN-1100PB CCD detector (Princeton
Instruments) cooled with liquid N2. Spectra were collected in spinning
FeIII
(TMP)]+ with an O O stretching vibration at 799 cm
.
À1
À
cells (2.0 cm diameter, 1500 rpm) at an excitation wavelength lex
=
Oxygenation of the tridentate model 2a in EtCN solution,
413.1 nm, 908 scattering geometry, and 5 min data accumulation. Peak
frequencies were calibrated relative to indene and CCl4 standards (accu-
rate to Æ1 cmÀ1). During each Raman experiment, UV/Vis spectra were
simultaneously collected on a PMA-11 CCD spectrophotometer (Hama-
matsu) with a Photal MC-2530 (D2/W2) light source (Otsuka Electronic
Co.).
however, generated a heme superoxide species [(TMP)FeIII-
À
(O2 )···CuII
(LN3-OH)] with the copper moiety oxidized to cop-
per(II). Moreover, the coexistence of a heme superoxide
[(TMP)FeIII
(O2 )···CuII
(LN3-OH)] and
a
bridged peroxide
À
[(LN3-OH)CuII-O2-FeIII
ACHTREUNG
The cyclic voltammetry (CV) measurements were performed on a BAS
100B/W electrochemical analyzer in anhydrous MeCN containing [Bu4N]-
AHCTREU[NG ClO4] (0.1m) as a supporting electrolyte. A glassy carbon working elec-
trode was polished with a polishing alumina suspension and rinsed with
MeCN before use. A Pt wire was used as counter electrode. A nonaqu-
eous Ag/Ag+ electrode was used as reference electrode and the poten-
tials were determined by use of the ferrocene/ferricenium (Fc/Fc+)
couple as a reference. Electrochemical measurements were carried out at
258C under an atmospheric pressure of N2 in a glovebox (M. Braun
150B-G-II).
was observed when the oxygenation reaction of 2a was car-
ried out in CH2Cl2/7% EtCN. An increase in the ratio of
the peroxide (ꢀ70%) to superoxide (ꢀ30%) was found
when the crosslinked phenol moiety of 2a was deprotonat-
ed, producing a bridged peroxide complex [(LN3-ONa)CuII-O2-
ACHTREUNG
FeIII(TMP)]+ as the main dioxygen intermediate, with the
nOÀ frequency at 812 cmÀ1. Observations from the reactions
O
of the untethered copper moieties of [CuILN4-OH
]
and
+
[CuILN3-OH
]
with the untethered heme superoxide
+
Oxygenation reaction: Dry O2 gas was introduced through molecular
sieves by O2 line into a degassed solution of the model compound
(ꢀ10À4 m) in a 0.2 cm UV cuvette. The spectral changes were monitored
À
[(NH2TMP)FeIII-(O2 )] demonstrated that the tridentate
copper(I) component [CuILN3-OH
]
did not react with the
+
with
a UV/Vis spectrometer. Oxygenation of the sample solution
(ꢀ10À5 m) for ESI-MS measurement was carried out in the same way in a
1.0 cm UV cuvette, which was capped with a three-way stopcock sealed
with rubber septa and kept in a handmade cell box for UV/Vis measure-
ments, a Teflon capillary tube being immersed into the resultant solution
for ESI-MS measurement. The solution was introduced into the ion-
spray ionization port of the spectrometer by application of appropriate
N2 pressure. The sample for rR measurement was prepared by injection
of dry O2 gas (10 mL) by syringe into the solution of the model com-
pound (ꢀ10À4 m) in a cylindrical Raman cell at low temperature. The
sample for EPR measurement was prepared as follows: in a glovebox, a
solution of the model compound (ꢀ10À3 m) was transferred to an EPR
tube. The tube was capped with a rubber septum and moved out of the
glovebox and kept in a cold bath (liq. N2/ethanol) at À100 to À808C (for
1a and 1b, at À308C). Dry O2 gas was introduced by syringe and the re-
sultant oxygenated solution was allowed to stand at this temperature for
20 min, after which the solution was frozen in liquid N2 and excess O2
was removed under vacuum.
corresponding heme superoxide, whereas the tetradentate
counterpart tended to react and yield the resultant dioxy-
gen-bridged peroxide, which can also be generated with the
tethered model 1a. The different oxygenation behavior ex-
hibited by the tri- and tetradentate copper chelate heme/
copper models is explained by the tricoordinated copper(I)
site having a weak reducibility and decreased O2 reactivity
relative to those of the tetracoordinated complex. A step-
wise process of binding of dioxygen to the N3-copper che-
late heme/copper model 2a has been proposed on the basis
of the experimental observations with the tethered and un-
tethered systems, with initial association of dioxygen to the
copper site occurring en route to the formation of the dioxy-
gen-bridged heme-peroxo-Cu intermediate.
Reactions of CuILN3-OH and CuILN4-OH with the heme superoxide
[(NH2TMP)FeIII-(O2À)]: The complexes of CuILN3-OH and CuILN4-OH were
prepared in situ in a glovebox by mixing compound LN3-OH (or LN4-OH
)
and [CuI (TfOÀ) in 1:1 ratio in oxygen-free and dry EtCN, and
ACTHERNGU(CH3CN)4]ACHTREUGN
Experimental Section
the resultant solutions of the CuI complexes were stirred for 30 min at
RT and transferred to a volumetric flask as a stock solution. Oxygenation
of the [(NH2TMP)FeII] in EtCN at À808C to generate the heme superox-
ide [(NH2TMP)FeIII-(O2À)] was carried out in the same way as described
above and excess O2 was removed from the UV cuvette by evacuation of
1
Instruments and methods: H NMR and 13C NMR spectra were recorded
on a JEOL LMX-GX 400 (400 MHz) spectrometer. Chemical shifts were
referenced to the residual solvent signal. Electrospray ionization mass
(ESI-MS) spectra were obtained on a Perkin–Elmer Sciex API-300 mass
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Chem. Eur. J. 2007, 13, 6365 – 6378

Cytochrome c Oxidase Models
FULL PAPER
the headspace and backfilling with N2 (”5). One equivalent amount of
CuI complex was then added to the resultant solution and its UV/Vis
spectral changes were recorded.
afforded the crude compound, which was purified by silica gel column
chromatography (CHCl3/MeOH 15:1) to give the product as a white
solid (6.2 g, 92%). 1H NMR (400 MHz, CDCl3): d=7.85 (s, 1H), 6.94 (s,
1H), 4.72 (s, 2H), 0.90 (s, 9H), 0.07 ppm (s, 6H); 13C NMR (100 MHz,
CDCl3): d=135.42, 134.91, 134.86, 133.32, 129.60, 127.60, 59.26, 26.84,
19.25 ppm; ESI-MS: m/z: 337.2 [M+H]+; HR-MS (FAB, NBA): m/z:
calcd for C20H25N2OSi: 337.1736; found: 337.1736 [M+H]+.
Materials: All reagents and solvents were obtained commercially and
were used without further purification unless otherwise noted. Triethyla-
mine was dried with KOH and distilled before use. Chloroform was dis-
tilled over K2CO3. Acetone was distilled twice from anhydrous calcium
sulfate and stored for no longer than two weeks before use. N,N-Dime-
thylformamide (DMF) was distilled from CaH2 under reduced pressure
and kept over 4 Š molecular sieves. Diethyl ether (Et2O) was predried
over CaCl2 and distilled from Na/benzophenone under N2. Tetrahydrofur-
an (THF) was predried over KOH and distilled from Na/benzophenone
under N2. Dichloromethane (CH2Cl2) was stirred with concentrated
H2SO4 for several days and dried over K2CO3, and was then distilled
from CaH2 under N2. Synthetic-grade acetonitrile (MeCN) was distilled
from P2O5 and redistilled from CaH2. Dry MeCN for spectroscopic meas-
urements was further purified as follows: the double-distilled synthetic-
grade MeCN was degassed three times by the freeze-thaw method, and
was stirred over KO2 (Aldrich) for ca. 1 h and then passed through acti-
vated alumina (Wako), which had previously been dried at 2208C under
vacuum for 10 h. Subsequently, it was again degassed three times by
freeze-thaw technique. Propionitrile (EtCN) was treated as the same way
as MeCN. The other degassed solvents were also treated by the freeze–
thaw method. Preparation and handling of air-sensitive materials were
carried out under N2 in a glovebox (M. Braun 150B-G-II) fitted with a
circulating purifier (O2, H2O < 1 ppm).
4-(tert-Butyldiphenylsilanyloxymethyl)-1-(2-methoxymethoxy-5-methyl-
phenyl)-1H-imidazole (5):
A mixture of arylboronic acid 3 (2.2 g,
11 mmol), Im-TBDPS 4 (3.4 g, 10 mmol), and [Cu(OH)TMEDA]2Cl2
(0.46 g, 1 mmol) in dry dichloromethane (60 mL) was vigorously stirred
under dioxygen at room temperature for 48 h. The reaction mixture was
filtered, the filtrate was concentrated, and the residue was purified by
silica gel column chromatography (C-200, ethyl acetate/hexane 2:1) to
give the N-arylimidazole 5a (3.6 g, 75%). 1H NMR (400 MHz, CDCl3):
d=7.86 (s, 1H), 7.72 (d, J=8.0 Hz, 4H), 7.43–7.36 (m, 6H), 7.20 (d, J=
8.0 Hz, 1H), 7.15 (d, J=8.0 Hz, 1H), 7.11 (s, 1H), 7.08 (s, 1H), 5.12 (s,
2H), 4.86 (s, 2H), 3.37 (s, 3H), 2.36 (s, 3H), 1.11 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3): d=147.51, 141.73, 136.75, 135.13, 133.25, 131.76,
129.17, 128.72, 127.22, 126.95, 125.52, 116.90, 116.19, 94.91, 60.81, 55.77,
26.56, 20.11, 18.97 ppm; ESI-MS: m/z: 487.2 [M+H]+; HR-MS (FAB,
NBA): m/z: calcd for C29H35N2O3Si: 487.2417; found: 487.2410 [M+H]+.
Compound 5b (0.3 g, 7%) was also obtained. 1H NMR (400 MHz,
CDCl3): d=7.63 (s, 1H), 7.50 (d, J=8.0 Hz, 4H), 7.39 (t, 2H), 7.34 (t,
4H), 7.22 (d, J=8.0 Hz, 1H), 7.19 (s, 1H), 7.16 (d, J=8.0 Hz, 1H), 7.05
(s, 1H), 4.95 (s, 2H), 4.51 (s, 2H), 3.20 (s, 3H), 2.31 (s, 3H), 0.91 ppm (s,
9H); 13C NMR (100 MHz, CDCl3): d=149.70, 138.65, 135.13, 132.71,
131.86, 131.50, 130.17, 129.35, 129.02, 127.76, 127.36, 125.63, 115.74, 94.81,
60.06, 55.56, 26.34, 20.13, 18.83 ppm; ESI-MS: m/z: 487.2 [M+H]+; HR-
MS (FAB, NBA): m/z: calcd for C29H35N2O3Si: 487.2417; found: 487.2413
[M+H]+.
Syntheses: (1-Methyl-1H-imidazol-4-yl)methanol hydrochloride,[27] 2-
(aminomethyl)-1-methyl-1H-imidazole dihydrochloride (8),[28] 6-(chloro-
methyl)nicotinic acid methyl ester hydrochloride (10),[29] [Cu(OH)TME-
[13]
DA]2Cl2 and [CuI (TfOÀ)[30] were prepared by literature pro-
ACHTREUNG(CH3CN)4]ACHTREUGN
cedures. The preparation of (2-[10,15,20-tris-(2,4,6-trimethylphenyl)por-
phyrin-5-yl]phenylamine (TMPNH2, 16) and insertion of iron into the
[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-yl]methanol (6):
Tetrabutylammonium fluoride in THF (1.0m, 30 mL) was added to a so-
lution of 5a (4.90 g, 10 mmol) in THF (25 mL) and the mixture was
stirred at room temperature overnight. Water (40 mL) was added to the
solution, which was extracted with CHCl3 (3”50 mL). The combined or-
ganic layer was washed with water (3”80 mL) and dried over anhydrous
Na2SO4, the solvent was removed under reduced pressure, and the resul-
tant residue was purified by silica gel column chromatography with
CHCl3/CH3OH 15:1. The product was isolated as a pale yellow oil
(2.30 g, 93%). 1H NMR (400 MHz, CDCl3): d=7.86 (s, 1H), 7.15–7.20
(m, 3H), 7.10 (s, 1H), 5.13 (s, 2H), 4.70 (s, 2H), 3.42 (s, 3H), 2.34 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=147.76, 141.81, 137.41, 132.04,
129.15, 126.73, 125.80, 117.38, 116.25, 95.08, 57.55, 56.11, 20.34 ppm; ESI-
MS: m/z: 249.1 [M+H]+; HR-MS (FAB, NBA): m/z: calcd for
C13H17N2O3: 249.1239; found: 249.1240 [M+H]+.
ACHTREUNG
porphyrin ([FeII(TMPNH2)]+) were by established methods.[31]
2-Bromo-1-methoxymethoxy-4-methylbenzene: A mixture of 2-bromo-p-
cresol (7.5 g, 40 mmol) and K2CO3 (13.8 g, 100 mmol) in dry acetone-
ACHTREUNG(100 mL) was stirred at RT for 1 h, and chloromethyl methyl ether
(3.8 mL, 50 mmol) was added dropwise to the mixture. The mixture was
stirred at RT overnight. After filtration, water (80 mL) was added to the
filtrate and the mixture was extracted with ethyl acetate (3”80 mL). The
combined organic phase was washed with HCl (1.0m) and brine and then
dried over anhydrous Na2SO4. Solvent removal gave the methoxymethyl
derivative as a colorless oil (8.6 g, 93%). 1H NMR (400 MHz, CDCl3):
d=7.37 (s, 1H), 7.04 (s, 2H), 5.21 (s, 2H), 3.52 (s, 3H), 2.28 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=151.34, 133.48, 132.78, 128.76, 116.14,
112.49, 95.16, 56.19, 20.18 ppm.
2-Methoxymethoxy-5-methyl-phenylboronic acid (3): n-Butyllithium
(1.6m in hexane, 25.0 mL, 40 mmol) was added to a cold (À708C) solu-
tion of 2-bromo-1-methoxymethoxy-4-methylbenzene (6.93 g, 30 mmol)
in dry Et2O (300 mL). The resulting solution was stirred for 2 h and a
white precipitate appeared. The mixture was again cooled to À708C and
trimethyl borate (4.5 mL, 40 mmol) was slowly added. Stirring was con-
tinued for 6 h at room temperature, before hydrolysis with a hydrochloric
acid solution (1.0m) and stirring for further 0.5 h at room temperature.
The ethereal extract was washed with water (100 mL) and dried
(MgSO4). Removal of solvents under reduced pressure followed by pu-
rification by recrystallization from Et2O/hexane afforded the pure aryl-
boronic acid as colorless crystals (3.9 g, 70%). 1H NMR (400 MHz,
CDCl3): d=7. 66 (s, 1H), 7.23 (d, J=8.0 Hz, 1H), 7.03 (d, J=8.0 Hz,
1H), 5.99 (s, 2H), 5.27 (s, 2H), 3.50 (s, 3H), 2.31 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3): d=160.16, 137.02, 136.99, 133.20, 131.35, 113.24,
94.67, 56.39, 20.46 ppm.
1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazole-4-carbaldehyde
(7): A mixture of compound 6 (2.48 g, 10 mmol) and activated MnO2
(50 mmol) in chloroform was heated at reflux for 3 d. The reaction mix-
ture was allowed to cool to room temperature and filtered, and the fil-
trate was concentrated. The residue was purified by silica gel column
chromatography with CHCl3/CH3OH 15:1 to give the title compound
(2.1 g, 85%). 1H NMR (400 MHz, CDCl3): d=9.95 (s, 1H), 7.90 (s, 1H),
7.85 (s, 1H), 7.20 (dd, J=8.0 and 3.6 Hz, 2H), 7.12 (s, 1H), 5.14 (s, 2H),
3.37 (s, 3H), 2.35 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=186.09,
147.91, 141.83, 139.14, 132.38, 130.47, 125.80, 125.69, 125.59, 116.32, 95.24,
56.36, 20.43 ppm; ESI-MS: m/z: 247.1 [M+H]+; HR-MS (FAB, NBA):
m/z: calcd for C13H15N2O3: 247.1083; found: 247.1080 [M+H]+.
[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)amine (9): 2-(Aminomethyl)-1-methyl-
1H-imidazole dihydrochloride (8, 1.85 g, 10 mmol) and aldehyde
7
4-(tert-Butyldiphenylsilanyloxymethyl)-1H-imidazole (4): 4-(Hydroxyme-
thyl)imidazole hydrochloride (2.68 g, 20 mmol), TBDPSC (6.68 g,
24 mmol), and imidazole (3.44 g, 50 mmol) were dissolved in DMF
(10 mL) and the mixture was stirred under nitrogen at 508C for 24 h.
Water (60 mL) was added to the mixture, which was extracted with
CHCl3 (3”80 mL). The combined organic layer was washed several
times with water (3”100 mL) and dried (Na2SO4), and solvent removal
(2.46 g, 10 mmol) were dissolved in dry methanol (100 mL). Triethyl-
amine was added to neutralize the solution, which was kept at reflux
under nitrogen for 8 h and then allowed to cool to room temperature.
NaBH4 was added to the cooled mixture, which was stirred overnight.
Water was added to the solution, which was extracted with chloroform
(3”80 mL). The combined organic layer was washed with water and
dried over Na2SO4 and the residue was purified by silica gel column chro-
Chem. Eur. J. 2007, 13, 6365 – 6378
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6375

Y. Naruta et al.
matography (CHCl3/MeOH 15:1) to give the product (2.2 g, 65%).
1H NMR (400 MHz, CDCl3): d=7.72 (s, 1H), 7.16 (d, J=8.0 Hz, 1H),
7.12 (d, J=8.0 Hz, 2H), 7.08 (s, 1H), 6.93 (s, 1H), 6.82 (s, 1H), 5.11 (s,
2H), 3.96 (s, 2H), 3.85 (s, 2H), 3.70 (s, 3H), 3.38 (s, 3H), 2.32 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=147.72, 146.24, 139.90, 137.36,
137.20, 132.07, 129.01, 126.89, 125.79, 120.98, 117.45, 116.29, 95.13, 56.15,
46.34, 44.72, 32.68, 20.43 ppm; ESI-MS: m/z: 342.1 [M+H]+; HR-MS
(FAB, NBA): m/z: calcd for C18H24N5O2: 342.1930; found: 342.1937
[M+H]+.
MS (FAB, NBA): m/z: calcd for C26H30N5O4: 476.2298; found: 476.2300
[M+H]+.
6-{[[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)amino]methyl}-N-{2-[10,15,20-tris(2,4,6-
trimethylphenyl)porphyrin-5-yl]-phenyl}-nicotinamide (17): Triethyl-
amine (28 mL, 0.2 mmol) and 2-chloro-1-methylpyridinium iodide (51 mg,
0.2 mmol) were added to a solution of compound 16 (76 mg, 0.1 mmol)
and 13 (47 mg, 0.1 mmol) in dichloromethane. The mixture was stirred
for several days under nitrogen. The progress of the reaction was moni-
tored by TLC. After completion of the reaction, the solvent was removed
and the residue was subjected to flash column chromatography (MeOH/
CH2Cl2 8%). Yield: 76 mg, 63%. 1H NMR (400 MHz, CDCl3): d=8.87
(d, J=8.0 Hz, 1H), 8.75 (d, J=4.4 Hz, 2H), 8.67 (d, J=4.4 Hz, 2H), 8.63
(d, J=3.6 Hz, 4H), 8.03 (d, J=7.2 Hz, 1H), 7.86 (d, J=7.2 Hz, 1H), 7.83
(s, 1H), 7.54 (t, J=7.2 Hz, 1H), 7.48 (s, 1H), 7.24–7.26 (m, 6H), 7.21 (s,
2H), 7.04 (s, 1H), 6.87 (s, 1H), 6.83 (s, 1H), 6.79 (d, J=8.0 Hz, 1H), 6.63
(d, J=8.0 Hz, 1H), 6.47 (s, 1H), 6.15 (s, 1H), 4.91 (s, 2H), 3.36 (s, 3H),
3.29 (s, 2H), 3.25 (s, 2H), 3.18 (s, 3H), 2.93 (s, 2H), 2.61 (s, 3H), 2.59 (s,
6H), 2.24 (s, 3H), 1.88 (s, 3H), 1.82 (s, 6H), 1.80 (s, 3H), 1.75 (s, 6H),
À2.53 ppm (s, 2H); ESI-MS: m/z: 1214.6 [M+H]+; HR-MS (FAB, NBA):
m/z: calcd for C78H76N11O3: 1214.6133; found: 1214.6130 [M+H]+.
6-{[[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)amino]methyl}nicotinic acid methyl ester
(12): 6-(Chloromethyl)nicotinic acid methyl ester hydrochloride (10,
1.22 g, 5.5 mmol) and triethylamine (1.53 mL, 11 mmol) were dissolved in
dry acetonitrile (30 mL). Amine 9 (1.71 g, 5 mmol) was added to the mix-
ture, which was stirred for several days at RT. The progress of the reac-
tion was monitored by TLC. After completion of the reaction, the mix-
ture was filtered and the precipitate was washed several times with di-
chloromethane. The combined organic layer was evaporated and the resi-
due was passed through a silica gel column (CHCl3/MeOH 15:1) to give
the product as a pale yellow oil (1.5 g, 61%). 1H NMR (400 MHz,
CDCl3): d=9.11 (s, 1H), 8.23 (d, J=8.0 Hz, 1H), 7.75 (s, 1H), 7.61 (d,
J=8.0 Hz, 1H), 7.23 (s, 1H), 7.16 (d, J=8.0 Hz, 1H), 7.11 (d, J=8.0 Hz,
2H), 6.96 (s, 1H), 6.80 (s, 1H), 5.11 (s, 2H), 3.99 (s, 2H), 3.95 (s, 3H),
3.93 (s, 3H), 3.76 (s, 2H), 3.73 (s, 2H), 3.37 (s, 3H), 2.34 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=165.58, 161.22, 150.78, 148.65, 144.56,
137.43, 137.30, 135.92, 132.11, 129.60, 126.53, 125.85, 126.48, 124.64,
122.08, 121.90, 119.76, 116.32, 95.18, 58.86, 56.04, 51.70, 49.54, 33.98,
20.41 ppm; ESI-MS: m/z: 491.2 [M+H]+; HR-MS (FAB, NBA): m/z:
calcd for C26H31N6O4: 491.2407; found: 491.2400 [M+H]+.
6-{[[1-(2-Hydroxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-methyl-
1H-imidazol-2-ylmethyl)-amino]methyl}-N-{2-[10,15,20-tris(2,4,6-trime-
thylphenyl)porphyrin-5-yl]phenyl}nicotinamide (LN4-OHTMP, 18): Trime-
thylsilyl bromide (0.13 mL, 1.0 mmol) was added to a cooled solution
(À408C) of the above methoxymethyl ether 17 (0.12 g, 0.1 mmol) in di-
chloromethane (6.0 mL) containing 4 Š molecular sieves. The solution
was stirred at À208C for 4 h and then for 4 h at 08C. The reaction mix-
ture was then poured into a solution of saturated sodium bicarbonate, ex-
tracted with dichloromethane, and dried over anhydrous magnesium sul-
fate. The solvent was removed by evaporation and the residue was puri-
fied by flash column chromatography (MeOH/CH2Cl2 8%). Yield:
70 mg, 60%. 1H NMR (400 MHz, CDCl3): d=8.84 (d, J=8.0 Hz, 1H),
8.75 (d, J=4.8 Hz, 2H), 8.67 (d, J=4.4 Hz, 2H), 8.63 (d, J=3.6 Hz, 4H),
8.03 (d, J=6.0 Hz, 1H), 7.85 (d, J=7.2 Hz, 1H), 7.83 (s, 1H), 7.76 (s,
1H), 7.54 (t, J=6.0 Hz, 1H), 7.24–7.26 (m, 6H), 7.20 (s, 2H), 7.14 (s,
1H), 6.82 (d, J=8.0 Hz, 1H), 6.77 (d, J=6.0 Hz, 1H), 6.70 (d, J=8.0 Hz,
1H), 6.59 (d, J=8.0 Hz, 1H,), 6.28 (s, 1H), 6.03 (s, 1H), 3.29 (s, 2H),
3.25 (s, 2H), 3.16 (s, 2H), 2.86 (s, 3H), 2.60 (s, 3H), 2.58 (s, 6H), 2.13 (s,
3H), 1.85 (s, 3H), 1.82 (s, 6H), 1.79 (s, 3H), 1.75 (s, 6H), À2.54 ppm (s,
2H); IR (KBr): n˜ =3411, 3318, 3026, 2916, 2855, 1697, 1683, 1674, 1652,
1599, 1578, 1558, 1520, 1472, 1457, 1446, 1399, 1377, 1344, 1284, 1257,
1217, 1188, 1131, 1070, 968, 804 cmÀ1; ESI-MS: m/z: 1170.6 [M+H]+;
HR-MS (FAB, NBA): m/z: calcd for C76H72N11O2: 1170.5870; found:
1170.5869 [M+H]+.
6-{[[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)amino]methyl}nicotinic acid (13): An
aqueous solution of KOH (1.0m) was added to a THF solution of the
above ester 12 (2.45 g, 5 mmol). After stirring for 24 h, the solution was
neutralized with aqueous HCl, and the solvent was removed under re-
duced pressure until dryness. The residue was then extracted with di-
chloromethane. Removal of the solvent gave the product as a pale yellow
solid (2.0 g, 86%). 1H NMR (400 MHz, CDCl3): d=10.65 (br, 1H), 9.03
(s, 1H), 8.10 (d, J=8.0 Hz, 1H), 7.98 (s, 1H), 7.45 (d, J=8.0 Hz, 1H),
7.43 (s, 1H), 7.17 (d, J=8.0 Hz, 2H), 7.12 (d, J=8.0 Hz, 2H), 6.94 (s,
1H), 5.14 (s, 2H), 4.30 (s, 2H), 3.98 (s, 2H), 3.93 (s, 2H), 3.87 (s, 3H),
3.38 (s, 3H), 2.33 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=167.97,
161.43, 150.26, 147.74, 144.70, 137.77, 137.49, 136.12, 132.30, 129.72,
126.86, 126.18, 125.84, 123.15, 122.82, 122.05, 120.10, 116.22, 95.21, 59.45,
56.33, 50.96, 48.81, 33.89, 20.45 ppm; ESI-MS: m/z: 477.3 [M+H]+; HR-
MS (FAB, NBA): m/z: calcd for C25H29N6O4: 477.2250; found: 477.2249
[M+H]+.
A
(TMP)]
U
A
ACHTREUNG(TfO)
(1b): The preparation of [(LN4-OR)CuII-FeIII
ACHTREUNG
4-{[[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl]-(1-
methyl-1H-imidazol-2-ylmethyl)amino]methyl}benzoic acid methyl ester
(14): This compound was prepared in a similar way to compound 12.
Yield: 45%. 1H NMR (400 MHz, CDCl3): d=7.89 (d, J=8.0 Hz, 2H),
7.68 (s, 1H), 7.36 (d, J=8.0 Hz, 2H), 7.10–7.02 (m, 4H), 6.80 (s, 1H),
6.69 (s, 1H), 5.03 (s, 2H), 3.81 (s, 3H), 3.70 (s, 2H), 3.65 (s, 2H), 3.58 (s,
2H), 3.53 (s, 3H), 3.28 (s, 3H), 2.26 ppm (s, 3H); 13C NMR (100 MHz,
CDCl3): d=166.70, 147.65, 145.08, 144.66, 137.72, 137.12, 132.03, 129.17,
129.02, 128.60, 128.41, 126.86, 126.73, 125.74, 121.32, 119.09, 116.27, 95.09,
57.38, 56.06, 51.80, 51.01, 50.14, 32.76, 20.31 ppm; ESI-MS: m/z: 490.2
[M+H]+; HR-MS (FAB, NBA): m/z: calcd for C27H32N5O4: 490.2454;
found: 490.2450 [M+H]+.
MOM) has been described previously.[11]
4-{[[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)amino]methyl}-N-{2-[10,15,20-tris(2,4,6-
trimethylphenyl)porphyrin-5-yl]phenyl}-benzamide (LN3-OMOMTMP; 19):
This compound was prepared in a similar way to compound 17. Yield:
45%. 1H NMR (400 MHz, CDCl3): d=8.94 (d, J=8.0 Hz, 1H), 8.77 (d,
J=4.8 Hz, 2H), 8.67 (d, J=4.8 Hz, 2H), 8.63 (d, J=2.8 Hz, 4H), 7.97 (d,
J=7.2 Hz, 1H), 7.94 (s, 1H), 7.84 (t, J=8.0 Hz, 1H), 7.52 (s, 1H), 7.49
(d, J=8.0 Hz, 1H), 7.21–7.27 (m, 6H), 7.10 (d, J=7.6 Hz, 2H), 7.04 (s,
2H), 6.89 (s, 1H), 6.79 (s, 1H), 6.60 (d, J=8.0 Hz, 2H), 6.52 (d, J=
7.6 Hz, 2H), 4.91 (s, 2H), 3.30 (s, 2H), 3.19 (s, 2H), 3.17 (s, 3H), 3.11 (s,
2H), 2.90 (s, 3H), 2.61 (s, 3H), 2.59 (s, 6H), 2.24 (s, 3H), 1.89 (s, 3H),
1.82 (s, 6H), 1.77 (s, 9H), À2.55 ppm (s, 2H); IR (KBr): n˜ =3419, 3322,
2921, 2855, 1717, 1676, 1610, 1578, 1559, 1516, 1457, 1446, 1384, 1345,
1304, 1248, 1157, 1139, 1081, 968, 804, 732 cmÀ1; ESI-MS: m/z: 1213.6
[M+H]+; HR-MS (FAB, NBA): m/z: calcd for C79H77N10O3: 1213.6180;
found: 1213.6179 [M+H]+.
4-{[[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)amino]methyl}benzoic acid (15): The
compound was prepared in a similar way to compound 13. Yield: 90%.
1H NMR (400 MHz, CDCl3): d=10.00 (br, 1H), 7.89 (d, J=8.0 Hz, 2H),
7.85 (s, 1H), 7.35 (d, J=8.0 Hz, 2H), 7.15–7.02 (m, 4H), 6.91 (s, 1H),
6.74 (s, 1H), 5.05 (s, 2H), 3.83 (s, 2H), 3.68 (s, 2H), 3.66 (s, 2H), 3.59 (s,
3H), 3.30 (s, 3H), 2.26 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
169.16, 147.67, 144.77, 142.76, 137.45, 136.83, 132.12, 130.75, 129.48,
129.40, 128.53, 126.41, 125.75, 124.58, 121.67, 119.62, 116.22, 95.10, 57.94,
56.16, 50.88, 49.25, 33.30, 20.33 ppm; ESI-MS: m/z: 476.2 [M+H]+; HR-
[(LN3-OMOMTMP)FeIII]Br (20): FeBr2 (25.9 mg, 120 mmol) was added to a
solution of the free base porphyrin 19 (36.4 mg, 30 mmol) in deoxygenat-
ed THF (30 mL) at reflux, and heating was continued for 3 h. After the
solution had cooled to RT, an aqueous EDTA solution (0.2m, 10 mL)
6376
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6365 – 6378

Cytochrome c Oxidase Models
FULL PAPER
was added and the mixture was stirred for 10 min. The organic phase was
separated and washed with water (3”30 mL) and with saturated NaBr
solution under air and was then dried over anhydrous Na2SO4. The sol-
vent was removed under vacuum and the resultant residue was purified
by flash column chromatography (silica gel, MeOH/CHCl3 1:15). Yield:
35 mg, 88%. UV/Vis (THF): lmax (e)=418 (54000), 507 (8200), 650 nm
(1800 molÀ1 m3 cmÀ1); ESR (THF, 77 K): g=5.54, 2.0; ESI-MS: m/z:
1266.5 [MÀBr]+; HR-MS (FAB, NBA): m/z: calcd for C79H74FeN10O3:
1266.5295; found: 1266.5326 [M-Br]+.
2H), 7.24 (t, J=7.2 Hz, 2H), 7.17 (t, J=7.2 Hz, 1H), 7.12 (s, 1H), 7.09 (s,
1H), 7.05 (s, 2H), 6.83 (s, 1H), 6.72 (s, 1H), 5.04 (s, 2H), 3.71 (s, 2H),
3.62 (s, 2H), 3.61 (s, 2H), 3.54 (s, 3H), 3.30 (s, 3H), 2.28 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=147.52, 145.22, 138.80, 137.88, 136.88,
131.88, 128.84, 128.70, 127.73, 126.84, 126.48, 126.44, 125.61, 121.14,
118.87, 116.17, 94.97, 57.56, 55.91, 50.66, 49.74, 32.64, 20.19 ppm; ESI-
MS: m/z: 432.2 [M+H]+; HR-MS (FAB, NBA): m/z: calcd for
C25H30N5O2: 432.2400; found: 432.2398 [M+H]+.
2-(4-{[Benzyl-(1-methyl-1H-imidazol-2-ylmethyl)amino]methyl}imidazol-
1-yl)-4-methylphenol (LN3-OH): Cleavage of the MOM group was carried
out in a similar way to that used to prepare 18. Yield: 50%. 1H NMR
(400 MHz, CDCl3): d=7.65 (s, 1H), 7.27 (d, J=7.2 Hz, 2H), 7.22 (t, J=
7.2 Hz, 1H), 7.17 (t, J=6.8 Hz, 2H), 7.13 (s, 1H), 6.98–6.93 (m, 3H), 6.75
(s, 1H), 6.64 (s, 1H), 3.65 (s, 2H), 3.56 (s, 4H), 3.49 (s, 3H), 2.24 ppm (s,
3H); 13C NMR (100 MHz, CDCl3): d=149.09, 145.52, 138.71, 137.09,
136.97, 129.24, 129.05, 128.64, 128.09, 126.89, 125.38, 125.26, 124.91,
121.47, 119.68, 118.04, 58.70, 51.35, 49.75, 33.17, 20.37 ppm; ESI-MS:
m/z: 388.2 [M+H]+; HR-MS (FAB, NBA): m/z: calcd for C23H26N5O:
388.2137; found: 388.2130 [M+H]+.
[(LN3-OHTMP)FeIII]Br (21): Trimethylsilyl bromide (0.11 mL, 0.8 mmol)
was added to a cooled solution (À408C) of [LN3-OMOM-FeIIITMP]Br (20,
54 mg, 40 mmol) in dichloromethane (10.0 mL) containing 4 Š molecular
sieves. The solution was stirred for 8 h at À308C, and the reaction mix-
ture was then poured into a solution of saturated sodium bicarbonate
(30 mL) and extracted with CHCl3 (3”40 mL). The organic phase was
combined, washed with saturated sodium bicarbonate and saturated
NaBr solution, and dried over anhydrous Na2SO4. The solvent was re-
moved by evaporation and the residue was purified by flash column chro-
matography (silica gel, MeOH/CHCl3 1:15). Yield: 44 mg, 80%. UV/Vis
(THF): lmax (e)=418 (62000), 504 (8200), 560 (4700), 650 nm
(3300 molÀ1 m3 cmÀ1); ESR (THF, 77 K): g=5.50, 2.0; ESI-MS: m/z:
1222.5 [MÀBr]+; HR-MS (FAB, NBA): m/z: calcd for C77H70FeN10O2:
1222.5033; found: 1222.5032 [MÀBr]+.
[1-(2-Methoxymethoxy-5-methylphenyl)-1H-imidazol-4-ylmethyl](1-
methyl-1H-imidazol-2-ylmethyl)-pyridin-2-ylmethylamine
(LN4-OMOM):
This compound was prepared in a similar way to LN3-OMOM with use of 2-
picolyl chloride hydrochloride. Yield: 41%. 1H NMR (400 MHz, CDCl3):
d=8.50 (d, J=4.8 Hz, 1H), 7.73 (s, 1H), 7.62 (t, J=5.6 Hz, 1H), 7.47 (d,
J=8.0 Hz, 1H), 7.18 (t, J=8.0 Hz, 1H), 7.13 (d, J=8.0 Hz, 2H), 7.09 (s,
1H), 7.07 (s, 1H), 6.86 (s, 1H), 6.74 (s, 1H), 5.08 (s, 2H), 3.82 (s, 2H),
3.77 (s, 2H), 3.69 (s, 2H), 3.60 (s, 3H), 3.34 (s, 3H), 2.31 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=158.86, 148.35, 147.43, 144.92, 137.35,
137.00, 135.95, 131.83, 128.84, 126.62, 126.31, 125.55, 123.03, 121.48,
121.10, 119.06, 116.07, 94.91, 59.35, 55.89, 51.05, 49.66, 32.63, 20.14 ppm;
ESI-MS: m/z: 433.2 [M+H]+; HR-MS (FAB, NBA): m/z: calcd for
C24H29N6O2: 433.2352; found: 432.2352 [M+H]+.
ACHTREUNG ACHTREUNG(TMP)]ACHTREUNG
[(LN3-OH)CuI-FeII (TfO) (2a): [LN3-OH-FeIIITMP]Br (21, 12.2 mg,
0.01 mmol) and sodium dithionite (35 mg, 0.20 mmol) were dissolved in a
mixture of degassed THF (4.0 mL), toluene (6.0 mL), and water
(2.0 mL). The solution was stirred at room temperature for 1 h and the
color of the solution changed from brown to red. The two phases were al-
lowed to separate and the aqueous layer was removed by pipette. The or-
ganic layer was washed with dioxygen-free water to remove inorganic
salts. The solvent was evaporated under vacuum and the residue was
dried for 12 h under continuous high-vacuum pumping. The deprotected
compound [LN3-OH-FeIITMP] was obtained in 94% yield (11.5 mg). UV/
Vis (EtCN/CH2Cl2 7%): lmax (e)=369 (18000), 436 (83000), 532 (6700),
561 nm (5600), 609 nm (2800 molÀ1 m3 cmÀ1); ESI-MS: m/z: 1223.6
[M+H]+.
4-Methyl-2-(4-{[(1-methyl-1H-imidazol-2-ylmethyl)-pyridin-2-ylmethyla-
mino]methyl}imidazol-1-yl)phenol (LN4-OH): This compound was prepared
in a similar way to LN3-OH. Yield: 45%. 1H NMR (400 MHz, CDCl3): d=
8.41 (d, J=5.2 Hz, 1H), 7.66 (s, 1H), 7.51 (t, J=8.0 Hz, 1H), 7.39 (d, J=
8.0 Hz, 1H), 7.16 (s, 1H), 7.05 (t, J=6.0 Hz, 1H), 6.95–6.87 (m, 3H), 6.74
(s, 1H), 6.65 (s, 1H), 3.75 (s, 2H), 3.70 (s, 2H), 3.60 (s, 2H), 3.49 (s, 3H),
2.20 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=158.85, 148.91,
148.42, 145.23, 137.07, 136.76, 136.50, 129.20, 128.61, 125.53, 125.24,
124.70, 123.50, 121.94, 121.52, 119.74, 117.74, 61.58, 51.60, 49.80, 33.07,
20.32 ppm; ESI-MS: m/z: 389.2 [M+H]+; HR-MS (FAB, NBA): m/z:
calcd for C22H25N6O: 389.2090; found: 389.2086 [M+H]+.
A degassed solution of an equimolar amount of [CuI
was injected into the solution of [LN3-OH-FeIITMP] by microsyringe to
give a solution of [LN3-OHCuI-FeIITMP](TfOÀ) (2a). The solvent was re-
(TfOÀ)
ACHRTUNEG(CH3CN)4]ACHTREUGN
AHCTREUNG
moved to dryness under reduced pressure and the solid was dried under
high vacuum. A quantitative yield of (2a) was obtained. UV/Vis (EtCN/
CH2Cl2 7%): lmax (e)=428 (85000), 532 (7500 molÀ1 m3 cmÀ1); ESI-MS:
m/z: 1285.3 [MÀTfO]+.
ACHTREUNG ACHTREUNG(TMP)]ACHTRE(UNG TfO) (2b): Compound 2b was prepared in a
[(LN3-ONa)CuI-FeII
similar way to that described for 2a.
[(LN3-ONa-TMP)FeII]: After reduction of compound 21 as described for
2a, the organic phase was washed with Na2CO3 solution (0.1m). The sol-
vent was evaporated under vacuum and the residue was dried for 12 h
under continuous high-vacuum pumping. The deprotonated compound
[LN3-ONa-FeIITMP] was obtained in 95% yield. UV/Vis (EtCN/CH2Cl2
7%): lmax (e)=368 (16500), 437 (82000), 533 (6000), 560 nm (5500),
610 nm (2700 molÀ1 m3 cmÀ1); ESI-MS: m/z (%): 1245.6 (60) [M+H]+.
Acknowledgements
This work was financially supported by a Grant-in-Aid for Scientific Re-
search (S) (17105003) from the Japan Society for the Promotion of Sci-
ence, as well as by grants from the Asahi Glass Foundation and the
Takeda Science Foundation. J.-G.L. gratefully acknowledges the JSPS for
providing a Research Fellowship for Foreign Researchers.
ACHTREUNG ACHTREUNG(TMP)]AHCTRE(UNG TfO) (2b): UV/Vis (EtCN/CH2Cl2 7%): lmax
[(LN3-ONa)CuI-FeII
(e)=428 (84000), 533 (6500 molÀ1 m3 cmÀ1). ESI-MS: m/z (%): 1307.3
(50) [MÀTfO]+.
ACHTREUNG ACHTREUNG(TMP)]ACHTRE(NGU TfO) (2c): MOM-protected compound 2c
[(LN3-OMOM)CuI-FeII
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Received: December 26, 2006
Published online: May 14, 2007
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