Cytochrome C oxidase active site mimics: New ligands for copper and an unexpected oxidative C-C bond formations

Article abstract of DOI:10.3987/com-06-s(w)46

Ligands that mimic the unusual active site polypeptide of cytochrome c oxidase have been prepared in overall good yield; structures of the corresponding Cu(II) complexes are secured by single crystal X-ray analysis. The key step in the synthesis of the li

Full text of DOI:10.3987/com-06-s(w)46

HETEROCYCLES, Vol. 70, 2006
147
HETEROCYCLES, Vol. 70, 2006, pp. 147 - 152. © The Japan Institute of Heterocyclic Chemistry
Received, 11th September, 2006, Accepted, 23rd October, 2006, Published online, 24th October, 2006. COM-06-S(W)46
CYTOCHROME C OXIDASE ACTIVE SITE MIMICS: NEW LIGANDS
FOR COPPER AND AN UNEXPECTED OXIDATIVE C-C BOND
FORMATION‡
Yakira R. Landaverry,1 Kimberly N. White,1 Marilyn M. Olmstead,2 Ólöf
Einarsdóttir,1 and Joseph P. Konopelski1*
1Department of Chemistry and Biochemistry, University of California, Santa Cruz,
2
CA 95064, and Department of Chemistry, University of California, Davis,
California 95616, USA. Email: joek@chemistry.ucsc.edu
Abstract – Ligands that mimic the unusual active site polypeptide of cytochrome
c oxidase have been prepared in overall good yield; structures of the
corresponding Cu(II) complexes are secured by single crystal x-ray analysis. The
key step in the synthesis of the ligands is the coupling of a suitable
organolead(IV) phenol derivative with the ε-N of the histidine imidazole ring. In
the process of preparing crystals of a tridentate ester ligand an unusual oxidative
C-C bond-forming reaction occurs that affords the imidazotetrahydropyridine ring
system.
Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transport chain, catalyzing the
reduction of dioxygen to water. This redox reaction is coupled to proton translocation across the inner
mitochondrial membrane, creating an electrochemical proton gradient that serves as the driving force for
ATP synthesis.1 While CcO has been studied for decades, the detailed mechanism of dioxygen reduction
is still unknown.2 The active site of CcO, centered on the heme a3/CuB bimetallic center, is unique in the
copper binding site (Figure 1). Posttranslational modification3 of the 200 kDa enzyme produces a new
C-N bond between His240 and Tyr244,4 i and i+4 residues along an α-helical section of subunit 1.5 Thus, a
rigid binding site is constructed for CuB, which is ligated to two distal histidine residues together with
His240, in close proximity heme a3. The side chain cross-linked Tyr244 has been proposed to play an
important role in proton and electron transfer to the iron-bound oxygen molecule, but direct evidence has
been lacking.
‡
This paper is dedicated to Professor Steve Weinreb for his many contributions to heterocyclic
chemistry

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Our work in this area stems from an interest in understanding the intricacies of the biological O2 reduction
reaction. For this purpose we successfully constructed compound (1) as a model system of the active site
His-Tyr system and explored some of the basic properties of the new biaryl construction.6 Key to the
success of this synthetic work was the use of aryllead(IV) reagents for the formation of the desired C-N
bond under mild conditions.7 However, to explore the role of the copper-polypeptide interaction required
the construction of a more elaborate set of compounds that would function as ligands for the metal. To
that end we prepared compounds (2-5), which incorporate one (2 and 3) and two (4 and 5) pyridine units.
It was expected that these donor residues would act as additional ligating elements (together with the
cross-linked imidazole and sp3 nitrogens) for copper, allowing us to explore the relationship of the metal
to the phenol (tyrosine) functionality.
His290
NH
OH
OH
His240
Tyr244
N
N
N
N
N
His291
N
NHAc
N
N
N
Cu
N
R
2
N
NH
CO Me
2
R
1
OH
2 R1 = CH2OMe; R2 = Me
3 R1 = CO2Me; R2 = Me
N
1
Fe
4 R1 = CH2OMe; R2 = CH2C5H4N
5 R1 = CO2Me; R2 = CH2C5H4N
N
N
Figure 1. Schematic depiction of the heme a3/CuB active site of cytochrome c oxidase and CcO active
site mimics
Our first publication in this area describes the synthesis of compounds (2 and 3) together with solid state
structures of the corresponding copper complexes and physical data that relates to the question of the
copper/tyrosine interaction through the imidazole ring of the histidine residue.8 In this publication we
present the synthesis of compounds (4 and 5), and the solid state structure of the Cu(II) complex of 4. In
addition, we present evidence of a novel oxidative coupling reaction of 3 which could have implications
to the biosynthesis of the key His/Tyr C-N bond formation of CcO.
Initial investigations into the alkylation of histidine with 2-(chloromethyl)pyridine for the preparation of 4
and 5 were not successful; reactions resulted in low yields of material that was difficult to purify.
However, the use of reductive amination as the cornerstone reaction led to the successful synthesis of the
desired compounds, as shown in Scheme 1. The synthesis of the tetradentate ether was completed in six
steps with an overall yield of 27%. Known histidine derivative (6)9 was reacted with two equivalents of
N-(benzyloxycarbonyloxy)succinimide to give the corresponding biscarbamate which, without isolation,
was selectively deprotected with propylamine to yield 7. 10 Compound (7) was coupled to
triacetoxy[(2-methoxymethoxy)phenyl]lead (8) to afford compound (9) in 75% yield.7 Subsequent
deprotection under transfer hydrogenation conditions provided the free amine,11 which was subjected to
direct hydride-induced reductive amination with 2-pyridine carboxaldehyde.12 Finally, stirring the

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149
reaction product at room temperature in a CH2Cl2 solution saturated with HCl(g) afforded the free phenol
product (4) as a multiple hydrochloride salt.
Following the synthesis of 4, the production of methyl ester 5 was straightforward. Commercial
Z-His-OMe (10) was subjected to aryllead(IV) coupling with 8 to give 11, which afforded 5 following the
reaction sequence developed for the preparation of 4 (reductive removal of the Z-protection group,
exhaustive reductive amination with 2-pyridine carboxaldehyde and removal of the phenol protection
group).
·2 HCl
HN
N
HN
N
1. Z-OSu, Et3N, CH2Cl2
2. C3H7NH2
NH
NHZ
OMe
2
70% (2 steps)
OMe
7
6
OMOM
Pb(OAc)
3
OMOM
N
OH
HN
N
N
NHZ
8
1. 1,4-cyclohexadiene, Pd/C
2. C5H4NCHO, NaBH(OAc)3
N
N
N
N
NHZ
N
3. HCl(g)
R
R
9 R = CH2OMe, 75%
11 R = CO2Me, 90%
4 R = CH2OMe, 51% (3 steps)
5 R = CO2Me, 53% (3 steps)
R
7 R = CH2OMe
10 R = CO2Me
Scheme 1. Synthesis of ligands 4 and 5.
O
N
O
Cl
Cu
N
Me
N
HO
imidazole
N
Figure 2. Single crystal X-Ray determination of Cu(II)-(4) (left) and Cu(II)-(12), with chemical structure
of Cu(II)-12.
Blue crystals of the copper(II) complex of 4 were grown by slow evaporation of a methanol/acetonitrile
mixture of the ligand and Cu(ClO4)2 plus added chloride ion; the resulting structure was determined by
single crystal x-ray analysis is shown in Figure 2. While it was anticipated that the Cu(II) atom would be
5-coordinate with chloride as one ligand, the coordination of the methyl ether unit rather than the
imidazole ring came as a surprise. The ether is only loosely associated with the metal (2.43 Å); it appears

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that the depicted collection of 5-membered rings incorporating copper is preferable, at least in the solid
state, to the presence of a 6-membered ring that would necessarily be formed should the imidazole
nitrogen be involved in chelation. Studies on the solution structure of Cu(II)-(4) will be initiated shortly.
Gram quantities of crystalline ligand-metal complexes were required for the mechanistic studies related to
CcO; only crystalline material gave assurance of the required 1:1 ligand-to-metal ratio. Therefore, the
development of efficient methods for the large scale crystallization of these complexes became a focus of
keen interest. Since chloride is known to be a strong ligand for Cu(II), experiments were conducted to
determine the best sources of this halide.
Treatment of ligand (3) (tridentate ester) with CuCl2, Cu(ClO4)2/NaCl, Cu(ClO4)2/Et3NHCl and
Cu(ClO4)2/H4NCl all produced the desired material, which could be obtained as needle-like crystals by
slow evaporation from methanol.8 However, robust cubic crystals formed readily from the methanol
solution when Amberlite IRA-410 (Cl¯ form) was employed as the chloride source. Indeed, isolation of
these crystals and subjecting the mother liquors to further crystallization afforded the well-known
needle-like material that had already been identified as Cu(II)-(3).8 Single crystal x-ray analysis of the
cubic crystalline material afforded the structure shown in Figure 2, in which a new C-C bond is evident.
This new bond links C-6, the methylene generated in the reductive amination reaction, and C-12, the
imidazole carbon adjacent to the key C-N bond between the heterocycle and phenol (x-ray numbering), to
give Cu(II)-(12). The structure of this new fused ring system, a tetrahydroimidazopyridine, shows some
disorder around the phenol and original histidine methyl ester unit, which now exists as the free acid
complexed directly to the copper atom. As with all the structures observed in these studies, the central
copper atom is 5-coordinate, now relying on a single chloride together with the carboxylate of the
histidine residue and three nitrogen donors (histidine amine and pyridine from one molecule and
imidazole from the adjacent molecule in the crystal lattice).
Cu ion
N
OH
+
Cu(II)-(3)
Cu(II)-(12)
N
CH
3
CO R
2
N
N
13 R = H or CH3
Scheme 2. Proposed mechanism for the formation of Cu(II)-(12).
The cyclization of histidine with an aldehyde via the classic Pictet-Spengler reaction readily affords the
spinacine ring system.13 However, to invoke such a reaction mechanism requires an unforeseen oxidation
of tertiary amine Cu(II)-(2) to iminium ion (13), which is then subject to attack by the electron-rich
imidazole ring.

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Although we know of no ongoing studies on the biosynthesis of the His/Tyr side chain coupling reaction
in CcO, it is reasonable to speculate that an electron-poor phenol derivative is generated as an acceptor to
the electron-rich nitrogen of the imidazole ring held in close proximity by the i to i+4 α-helix relationship
of the two residues. Indeed, such a mechanism has been suggested for the biosynthesis of the Tyr/Cys
cross-link at the active site of galactose oxidase, in which a thioether bond (analogous to the C-N bond in
CcO) is formed in the final processing steps to the fully functional enzyme.14 In the suggested
mechanism the copper-phenolate species allows for oxidation and activation of the phenol ring toward
attack of the cysteine side chain sulfur atom.
By analogy, we can speculate that copper mediates the oxidation of the histidine amine to corresponding
iminium ion (13),15 which is in proper position to form the new fused bicyclic system. The reasons for
the uniqueness of the resin-bound chloride source to promote this reaction, the changes in the oxidation
state of the copper ion and the timing of the methyl ester to acid transformation are currently under study
and will be disclosed in due course.
ACKNOWLEDGMENTS
We wish to thank the NIH (CA 98878 (JPK), GM53788 (ÓE) and GM58903 (MBRS fellowship to
YRL)) for generous support of this work. Purchase of the 600 MHz NMR used in these studies was
supported by funds from the National Institutes of Health (S10RR019918) and National Science
Foundation (CHE-0342912).
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