Formation and spectroscopic characterization of the dioxygen adduct of a heme-Cu complex possessing a cross-linked tyrosine-histidine mimic: modeling the active site of cytochrome c oxidase.

Article abstract of DOI:10.1039/b311538k

A binucleating porphyrin with covalently appended copper chelates having a cross-linked imidazole-phenol group as the novel active site model of cytochrome c oxidase has been prepared, and the dioxygen adduct of its iron(II)-copper(I) complex was spectros

Full text of DOI:10.1039/b311538k

Formation and spectroscopic characterization of the dioxygen adduct of
a heme–Cu complex possessing a cross-linked tyrosine–histidine mimic:
modeling the active site of cytochrome c oxidase†
Jin-Gang Liu, Yoshinori Naruta,* Fumito Tani, Takefumi Chishiro and Yoshimitsu Tachi
Institute for Materials Chemistry and Engineering, Kyushu University, Higashi-ku, Fukuoka 812-8581,
Japan. E-mail: naruta@ms.ifoc.kyushu-u.ac.jp; Fax: +81-(0)92-642-2715; Tel: +81-(0)92-642-2731
Received (in Cambridge, UK) 19th September 2003, Accepted 21st October 2003
First published as an Advance Article on the web 24th November 2003
A binucleating porphyrin with covalently appended copper
chelates having a cross-linked imidazole–phenol group as the
novel active site model of cytochrome c oxidase has been
to regenerate the phenolic hydroxyl group and the hydroxyl free
ligand LOH (8)‡ is obtained in a moderate yield (60%).
Stepwise metalation of the porphyrins, 7 and 8, begins with
addition of excess FeBr2 in THF at reflux, followed by extraction
with an aqueous Na2EDTA solution, yielding the corresponding
mononuclear FeII porphyrins, LOMOMFeII and LOHFeII, respec-
tively. Addition of copper salt [Cu(CH3CN)4]+ CF3SO32 gives the
desired FeII/CuI complexes with similar UV–vis spectra to those of
their mononuclear FeII complexes, [LOMOMFeIICuI]+ (9), ESI–MS
m/z = 1330.5 (M+); [LOHFeIICuI]+ (10), ESI–MS m/z = 1286.5
(M+).
Both 9 and 10 react with O2 at 2 30 °C in CH3CN to give the
dioxygen adducts [LOMOMFeIII–O2–CuII]+ (11) and [LOHFeIII–O2–
CuII]+ (12), respectively. The formation of the corresponding
peroxo species was evidenced by the following observations: (1)
upon exposure of the reduced form 9 or 10 to O2, its UV–vis spectra
show distinctive changes with clear isosbestic points. The Soret
band shifts from 429 nm to 421 nm, and the Q-band at 533 nm
disappears (Fig. 1), which indicates the formation of a dioxygen
adduct as described for those of our previously isolated peroxo-
bridged Fe–O2–Cu species.8 (2) ESI mass spectra of the dioxygen
adducts show a distribution of peaks centered at 1362.5 (M+) for 11,
and 1318.5 (M+) for 12. The observed isotope distribution of peaks
agrees very well with the simulated pattern based on the ratio of
FeII–CuI : O2 = 1 : 1. The expected increase in mass of 4 is
observed when 11 (m/z, M+, 1366.5) or 12 (m/z, M+, 1322.5) forms
from 18O2. (3) The resonance Raman spectra of 11 shows an isotope
dependent peak at 801 cm21 which shifts to 755 cm21 with 18O-
labeled dioxygen, and 12 displays a similar isotope sensitive band
at 799 (16O2) and 752 cm21 (18O2), respectively (Fig. 2). The
observed isotopic shifts are in good agreement with the value
prepared, and the dioxygen adduct of its iron(II)–copper(
complex was spectroscopically characterized.
I
)
Cytochrome c oxidase (CcO), the terminal enzyme of the
respiratory chain, catalyzes the 4e/4H+ reduction of dioxygen to
water without generating toxic reactive intermediates, conserving
the released energy for the synthesis of ATP.1 The active site of O2
reduction is comprised of heme a3/CuB binuclear moiety in which
one of the copper-bound histidines is covalently cross-linked to a
tyrosine residue between the C6 of Tyr244 and the e-nitrogen of His
240 (in the bovine enzyme sequence).2 This unprecedented Tyr-His
cross-link is proposed either to function as an electron and a proton
donor to the dioxygen bound to heme a3 or to fix CuB in a certain
configuration and distance from heme a3 during the catalytic O2
reduction.3 A number of heme-based dinuclear Fe–Cu complexes
have been reported as model compounds in the hope of unraveling
the mechanism of O2 reduction in the active site of CcO.4 However,
the reported heme-based models are devoid of the Tyr-His cross-
linkage. There are a few recent reports5 about the syntheses and
physicochemical investigations of cross-linked phenol–imidazoles.
Very recently, Karlin and co-workers6 reported copper complexes
with imidazole–phenol cross-links as an initial synthetic model for
the CuB site in CcO. Herein we report the first example of
constructing a heme-containing model with covalently appended
copper chelates having a cross-linked imidazole–phenol group as a
novel CcO model compound.
The synthetic routes to the desired compounds are shown in
Scheme 1.‡ The aldehyde 3 is prepared firstly from the coupling of
methoxymethyl (MOM)-protected 2-hydroxyphenylboronic acid
(1) with 4(5)-(tert-butyldiphenylsilanyloxymethyl)-1H-imidazole
(2),7 followed by removing the silyl protecting group and then
oxidized by activated MnO2. It is noteworthy that none of the
desired product is obtained when 1H-imidazole-4-carbaldehyde is
employed for the coupling. The MOM-protected phenylboronic
acid (1) can be obtained in 65% overall yield by sequential
reactions involving metalation (n-BuLi/ether/270 °C) of MOM-
protected 2-bromo-4-methylphenol, and treatment with B(OMe)3,
followed by acidic work-up. Treatment of 3 with 2-aminomethyli-
midazole in methanol generates the corresponding Schiff base
intermediate, which is consequently reduced by NaBH4 in situ to
give 4 in a yield of 65%. The tripodal ligand 5 is isolated by reacting
the amine 4 with methyl 6-chloromethylnicotinate in the presence
of K2CO3 in CH3CN, and then hydrolyzing in a KOH solution. The
prepared tripodal ligand is an important building block in
assembling CcO active site models. The condensation reaction
between 5 and the porphyrin 6 (2-[10,15,20-tris-(2,4,6-trimethyl-
phenyl)-porphyrin-5-yl]-phenylamine) is performed in the pres-
ence of Et3N/2-chloromethylpyridinium iodide in CH2Cl2 to give
the covalent conjugate LOMOM (7) in 63% yield. Finally, the MOM
group is removed with bromotrimethylsilane in CH2Cl2 at 2 30 °C
Scheme 1 Reagents and conditions: (i) cat. [Cu(OH)TMEDA]2Cl2,
CH2Cl2, O2, rt, 75%; (ii) n-BuN+F2, THF, rt, 93%; (iii) MnO2, CHCl3,
reflux, 85%; (iv) a, 2-aminomethylimidazole, Et3N, MeOH, b, NaBH4,
65%; (v) methyl 6-chloromethylnicotinate, K2CO3, CH3CN, rt, 61%; (vi)
KOH, THF, rt, 86%; (vii) 2-chloromethylpyridinium iodide, 5, Et3N,
CH2Cl2, rt, 63%; (viii) Me3SiBr, CH2Cl2, 2 30 °C, 60%.
† Electronic supplementary information (ESI) available: experiment proce-
dures for preparing metal porphyrins and oxygenation reaction. See http://
www.rsc.org/suppdata/cc/b3/b311538k/
120
C h e m . C o m m u n . , 2 0 0 4 , 1 2 0 – 1 2 1
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

calculated from the harmonic oscillator approximation of the O–O
oxygenation reaction with its iron(II)–copper( ) complex has been
I
16
stretching vibration [D
( O2/18O2) = 46 cm21]. These ob-
preliminarily investigated by various spectroscopic methods.
This work was financially supported by the Grant-in-Aid for
COE Research (#08CE2005) and for Scientific Research on
Priority Areas (#09235225 and 11228207) from MEXT and for
Scientific Research (A) (#14204073) from JSPS. P&P project,
Green Chemistry, of Kyushu University partly supported this
research. J.-G. Liu gratefully acknowledges JSPS for providing
postdoctoral fellowship.
calcd
served n(O–O) values are similar to those of previous reported
dioxygen adducts in the peroxy state.4,8 (4) Both 11 and 12 are EPR
silent in a frozen solution (CH3CN, 77K), which indicates the
presence of the strong antiferromagnetic coupling between the two
metals.
The formed peroxo species are stable at 2 30 °C in CH3CN, and
on warming of the solution to room temperature (after removal of
excess O2 in vacuo), the dioxygen adducts [LOMOMFeIII–O2–CuII]+
(11) and [LOHFeIII–O2–CuII]+ (12) exhibit interesting differences.
For 11, the major decomposed product is the m-oxo complex
formulated as [LOMOMFeIII–O–CuII]+ [m/z, 1346.6 (M+)] with UV–
vis features [lmax = 440 nm (Soret)] similar to the reported m-oxo
analogues.8,9 By contrast, no m-oxo final species is observed for 12.
The final decomposed product demonstrates features like that of the
hydroxo ferric porphyrin derivatives.10 The EPR spectrum (MeCN,
77 K) of the product shows signals at g = 5.56 and 1.99
corresponding to a high spin iron(III) porphyrin, and signals at g∑ =
2.23 and g4 = 2.06, which are assigned to a S = 1/2 Cu(II) ion in
a tetragonal field.11 We tentatively formulate the product as
[LOHFeIII–OH, CuII]2+. The decomposition mechanism and further
product characterization are in progress.
Notes and references
‡ Synthetic details will be reported elsewhere. All new compounds were
fully characterized by spectroscopic methods. Stated yields refer to isolated
compounds and the purity was guaranteed by chromatography. Data for
LOH (8), 1H NMR (400 MHz, CDCl3) d 8.84 (d, J = 8.0, 1 H), 8.75 (d, J =
4.8, 2 H), 8.67 (d, J = 4.4, 2 H), 8.63 (d, J = 3.6, 4 H), 8.03 (d, J = 6.0,
1 H), 7.85 (d, J = 7.2, 1 H), 7.83 (s, 1 H), 7.76 (s, 1 H), 7.54 (t, 1 H), 7.24
~ 7.26 (m, 6 H), 7.20 (s, 2 H), 7.14 (s, 1 H), 6.82 (d, J = 8.0, 1 H), 6.77
(d, J = 6.0, 1 H), 6.70 (d, J = 8.0, 1 H), 6.59 (d, J = 8.0 Hz, 1 H,), 6.28
(s, 1 H), 6.03 (s, 1 H), 3.29 (s, 2 H), 3.25 (s, 2 H), 3.16 (s, 2 H), 2.86 (s, 3
H), 2.60 (s, 3 H), 2.58 (s, 6 H), 2.13 (s, 3 H), 1.85 (s, 3 H), 1.82 (s, 6 H), 1.79
(s, 3 H), 1.75 (s, 6 H), 2 2.54 (s, 2 H). IR (KBr) 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 cm21. HR-
MS (FAB, NBA) Found: 1170.5869. Calcd for C76H72N11O2: [M + H]+,
1170.5870.
In summary, a novel heme-based binucleating ligand incorpo-
rated with N-(2A-hydroxyphenyl)imidazole moiety as a CcO’s CuB
site mimic has been designed and successfully prepared. The
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Fig. 1 UV–visible spectral changes of 10, [LOHFeIICuI]+, to 12 upon
exposure to dioxygen in CH3CN at 230 °C.
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(CH3CN) lmax = 418, 560 nm. ESI–MS m/z = 651.9 (M2+).
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2.01, g∑ = 2.24 and g4 = 2.09, respectively.
Fig. 2 Resonance Raman spectra of 12 formed from 16O2 (A) and 18O2 (B).
The difference spectra A minus B is shown as trace C (3% toluene in
CH3CN, 230 °C, 413 nm excitation).
C h e m . C o m m u n . , 2 0 0 4 , 1 2 0 – 1 2 1
121