Model studies of the CuB site of cytochrome c oxidase utilizing a Zn(ii) complex containing an imidazole-phenol cross-linked ligand

Article abstract of DOI:10.1039/b516090a

Cytochrome c oxidase, the enzyme complex responsible for the four-electron reduction of O₂ to H₂O, contains an unusual histidine-tyrosine cross-link in its bimetallic heme a₃-Cu _B active site. We have synthesise

Full text of DOI:10.1039/b516090a

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Model studies of the CuB site of cytochrome c oxidase utilizing a Zn(II)
complex containing an imidazole–phenol cross-linked ligand†‡
Russell P. Pesavento,§ Derek A. Pratt,¶ Jerry Jeffers and Wilfred A. van der Donk*
Received 14th November 2005, Accepted 29th March 2006
First published as an Advance Article on the web 20th April 2006
DOI: 10.1039/b516090a
Cytochrome c oxidase, the enzyme complex responsible for the four-electron reduction of O2 to
H2O, contains an unusual histidine–tyrosine cross-link in its bimetallic heme a3-CuB active site.
We have synthesised an unhindered, tripodal chelating ligand, BPAIP, containing the unusual
ortho-imidazole–phenol linkage, which mimics the coordination environment of the CuB center.
The ligand was used to investigate the physicochemical (pKa, oxidation potential) and coordination
properties of the imidazole–phenol linkage when bound to a dication. Zn(II) coordination lowers the
pKa of the phenol by ∼0.6 log units, and increases the potential of the phenolate/phenoxyl radical
couple by approximately 50 mV. These results are consistent with inductive withdrawal of electron
density from the phenolic ring. Spectroscopic data and theoretical calculations (DFT) were used to
establish that the cationic complex [Zn(BPAIP)Br]+ has an axially distorted trigonal bipyramidal
structure, with three coordinating nitrogen ligands (two pyridine and one imidazole) occupying the
equatorial plane and the bromide and the tertiary amine nitrogen of the tripod in the axial positions.
Interestingly, the Zn–Namine bonding interaction is weak or absent in [Zn(BPAIP)Br]+ and the complex
gains stability in basic solutions, as indicated by 1H NMR spectroscopy. These observations are
supported by theoretical calculations (DFT), which suggest that the electron-donating capacity of the
equatorial imidazole ligand can be varied by modulation of the protonation and/or redox state of the
cross-linked phenol. Deprotonation of the phenol makes the equatorial imidazole a stronger r-donor,
resulting in an increased Zn–Nimd interaction and thereby leading to distortion of the axial ligand axis
toward a more tetrahedral geometry.
residue through a C6(tyrosine)–Ne(histidine) linkage.7 The cross-
linked histidine is one of three histidines ligated to CuB (Fig. 1A).
The exact role of the His–Tyr linkage remains unclear; in addition
to a potential structural capacity,8,9 the linkage may serve a
functional role.10–14
Introduction
Cytochrome c Oxidase (CcO) is the final enzyme complex in the
electron transport chain embedded in the inner membrane of
mitochondria, or bacterial membranes in organisms that respire
aerobically. CcO catalyzes the thermodynamically favourable four-
electron reduction of molecular oxygen to water, concomitant
with the pumping of four protons across the membrane, which
ultimately affords the energy for the biosynthesis of ATP. In
the mixed valence form of the enzyme, three of the required
four electrons are proposed to come from the redox-active metal
centers (FeII, CuI) in the bimetallic heme a3-CuB active site. It has
been proposed that the fourth electron originates from a highly
conserved tyrosine residue near the CuB site.1–4 This would place
CcO in a group of proteins utilizing tyrosyl radicals in catalysis.5,6
A high resolution X-ray structure of CcO from bovine heart
mitochondria, reported by Yoshikawa and coworkers in 1998,
indicated that this tyrosine is covalently bound to a histidine
Department of Chemistry, University of Illinois at Urbana-Champaign,
Urbana, IL, 61801. E-mail: vddonk@uiuc.edu; Fax: +1-217-244-8533;
Tel: +1-217-244-5360
† This report is dedicated to the memory of Jerry Jeffers.
‡ Electronic supplementary information (ESI) available: Tables S1–S5 and
Fig. S1–S3. See DOI: 10.1039/b516090a
§ Current address: Department of Chemistry, Harvard University, Cam-
Fig. 1 (A) The oxidised CuB site of CcO (R = H or OFea3; bovine
numbering). (B) The Zn(II)–BPAIP system used in the present work
(X = halide or hydroxide).
bridge, MA., USA.
¶ Current address: Department of Chemistry, Queen’s University,
Kingston, ON, Canada.
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Our initial model studies of the cross-link using 2-imidazol-1-yl-
4-methylphenol10 indicated that the introduction of an imidazole
at the ortho position lowers the phenolic pKa by 1.6 log units
and increases the phenolate oxidation potential by 66 mV. Fur-
thermore, oxidation of the phenol led to a marked decrease in
the basicity of the imidazole imino nitrogen by about 1 log unit.
Similar model studies15–18 have subsequently corroborated the
pKa lowering effect under a range of conditions (1.1–2.2 log
units). However, the effects of divalent metal coordination to the
cross-link have not been thoroughly addressed. A recent study17
suggested that Cu(II) coordination to an imidazole cross-linked to
phenol actually increases the pKa of the latter to the same value
as 4-methylphenol (p-cresol, 10.2). These results were rationalised
on the basis of theoretical calculations, which predicted a large
angle between the planes of the phenol and imidazole rings (66◦),
which would diminish electronic interactions between adjacent p-
systems. However, it is important to note that these calculations
were carried out on a conformation wherein the phenolic O–H was
intramolecularly H-bonded to the imidazole. In the enzyme active
site, the angle between the planes of the phenol and imidazole rings
is 44◦, consistent with a non-H-bonded structure (vide infra).7
X-Ray crystallographic studies indicate that the geometry of
the CuB site in CcO is four-coordinate and composed of a
distorted square planar Cu(II) with three coordinating histidine
residues and some residual electron density bridging the CuB–
Fea3 site, most likely a peroxide or hydroxide (Fig. 1A). This
picture is supported by spectroscopic data,19,20 which are consistent
with a four-coordinate Cu(II) with three histidine ligands and
an oxygen donor. Several synthetic models of the CuB site
incorporating the imidazole–phenol cross-link into a chelating
formation of a phenoxyl radical results in a significant decrease
in the basicity of the coordinating imidazole imino nitrogen.25 To
date, no experimental or theoretical studies have focused on the
influence of phenolate formation on the coordination chemistry of
an imidazole–phenol ligand bound to a divalent metal ion via the
imidazole. The present study was directed towards understanding
the role of a metal ion in perturbing the physicochemical properties
(pKa, oxidation potential) of the cross-linked phenol, and its
corresponding aqueous coordination chemistry as a function of
pH, in complexes that maintain the dihedral angle observed in the
enzyme.
In this report, we focus on a Zn(II) complex with a sterically
unencumbered chelating ligand that incorporates a phenol sub-
stituted at the ortho position, with imidazole to model the CuB
site (Fig. 1B). The Lewis acidity and ionic radius of Zn(II) are
comparable to Cu(II), while the lack of redox chemistry simpli-
fies electrochemical data. In addition, the diamagnetic electron
configuration (3d10) of Zn(II) permits 1H NMR characterization,
which was vital to the present study. Because of the difficulties
encountered in crystallising Zn(II) and Cu(II) complexes, DFT
calculations were used to provide insights into the coordination
geometry. These calculations, in combination with a range of
experimental studies, suggest that the tertiary amine nitrogen
either does not coordinate or coordinates only weakly to Zn(II),
and that phenol deprotonation increases the stability of these
complexes. Furthermore, Zn(II) coordination modestly lowers the
pKa of the cross-linked phenol when the dihedral angle between
the imidazole and phenol is close to that found in the enzyme.
Experimental
ligand have been reported recently with21,22 and without17,23,24
a
tethered heme functional group to mimic the heme a3 of the
bimetallic active site. Crystal structures of both four-coordinate
Cu(I) and five-coordinate Cu(II) complexes with such ligands have
been reported by Karlin and co-workers.23 The authors noted
little difference in the spectroscopy of the complexes containing
the unique imidazole–phenol cross-link, as compared to similar
copper containing complexes lacking the cross-linkage.
All materials were obtained from either Aldrich, Acros or Sigma
and used without further purification unless otherwise noted. 1H
and 13C NMR spectra were collected on Varian Unity 400 or
500 MHz spectrometers. CH2Cl2, CH3CN, and THF were distilled
over CaH2 or Na (THF) under N2 prior to use. Mass spectral
analyses were carried out by the Mass Spectrometry Laboratory
at the University of Illinois at Urbana–Champaign (UIUC).
The lack of an observed effect upon Cu complexation may
be attributed to the dihedral angle between the planes of the
phenol and imidazole rings resulting from the orientation of the
phenolic O–H. In the crystal structures of the Karlin complexes
the phenolic O–H is intramolecularly H-bonded to the imidazole
N due to tert-butyl groups in the ortho and para positions,
resulting in a large dihedral angle (>70◦) between the planes
of the aromatic rings.23 These tert-butyl groups moderate the
reaction of the complexes with O224 and also help in dissolving the
complexes for crystallization studies. As will be demonstrated in
this work, complexes lacking these groups are not intramolecularly
H-bonded and reproduce the dihedral angles seen in the enzyme
quite well, but their poor solubility has precluded the generation
of diffracting single crystals.
Theoretical calculations by Colbran and Paddon–Row25 that
utilize models of the CuB site suggest that the cross-linked
histidine–tyrosine ligand stabilises the Cu(II) state relative to the
Cu(I) state by 102 mV (2.3 kcal mol−1). These results were obtained
from calculations carried out on the non-intramolecularly H-
bonded conformation. Furthermore, their computational data
are consistent with our previous experimental observation10 that
Synthesis
4-Imidazoleacetic acid methyl ester (1)26
A modification to the literature procedure was employed for the
synthesis of 1. A round-bottomed flask was charged with sodium
4-imidazoleacetate semihydrate (1.00 g, 6.36 mmol) and anhydrous
MeOH (100 mL) and the mixture was stirred vigorously. A
solution of 2 M HCl in Et2O (19 mL) was added by syringe and
the reaction was allowed to proceed for 20 h. The solvent was
removed under reduced pressure and a saturated aqueous solution
of NaHCO3 (150 mL) was added to the residue. The aqueous layer
was extracted repeatedly with EtOAc (ca. 350 mL) and the organic
fractions were combined and dried over Na2SO4. The solvent was
removed under reduced pressure to yield a pale yellow oil (0.63 g,
70%).
o-Methoxyphenyllead triacetate (2)
The title compound was prepared according to a literature
procedure.27
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[1-(2-Methoxyphenyl)-1H-imidazol-4-yl]acetic acid methyl
ester (3)
to warm to rt. The solution was stirred for 3 h, diluted with
CH2Cl2, and a saturated solution of NaHCO3 was added. The
aqueous layer was extracted with CH2Cl2 and the organic fractions
were collected, dried over Na2SO4 and filtered through Celite. The
solvent was removed under reduced pressure to yield a brown oil
(0.137 g, 93%). IR (neat): 2995, 2537, 1964, 1593, 1512, 1344, 1256,
1169, 1020, 966, 899, 804, 757 (cm−1); dH(400 MHz, CDCl3) 7.71
(1H, d, J = 1.2, Im–H), 7.35 (1H, ddd, J = 8.2, 1.69, 0.8 Hz, Ph–H)
7.26 (1H, dd, J = 7.7, 1.8 Hz, Ph–H), 7.07–7.00 (3H, m, Ar–H),
4.56 (2H, t, J = 6.7 Hz, (–CH2–)), 3.85 (3H, s, (–CH3)), 3.09 (2H,
t, J = 6.2 Hz, (–CH2–)) 2.93 (3H, s, (–CH3)); dC(400 MHz, CDCl3)
152.65 (Cq), 137.48 (CH), 136.43 (Cq), 129.40 (CH), 126.27 (Cq),
125.62 (CH), 121.24 (CH), 118.27 (CH), 112.51 (CH), 69.65 (CH2),
56.01 (CH3), 37.31 (CH3), 29.50 (CH2); (m/z) HRMS (ESI+):
calculated 297.0909 for C13H17N2O4S (M+) found 297.0909.
To an oven-dried round-bottomed flask was added 1 (3.13 mmol,
0.440 g), 2 (3.13 mmol, 1.52 g), and catalytic Cu(OAc)2 (0.31 mmol,
0.056 g). The reaction mixture was flushed with N2 and CH2Cl2
(25 mL) was added and the reaction was allowed to stir at rt for
6 h. To the blue–green reaction mixture was added a saturated
solution of Na2S·9H2O in MeOH (10 mL). Upon addition, a
black precipitate formed that was removed by filtration through
Celite and the filter cake was washed with CH2Cl2 (ca. 350 mL).
The organic fractions were collected and washed once with H2O
(100 mL) and dried over Na2SO4. The solvent was removed under
reduced pressure and the yellow residue was purified by column
chromatography (SiO2, 19 :1 CH2Cl2 : MeOH, Rf = 0.27) to
yield an orange–yellow oil (0.875 g, 65%). IR (neat): 2950, 2836,
1740, 1605, 1512, 1440, 1259, 1150, 1016, 757, 705, 643 (cm−1);
dH(500 MHz, CDCl3) 7.73 (1H, d, J = 1.3 Hz, Im–H), 7.34 (1H,
ddd, J = 8.0, 1.7, 0.8 Hz, Ph–H), 7.27 (1H, dd, J = 7.8, 1.7 Hz,
Ph–H), 7.17–7.16 (1H, m, Im–H), 7.06–7.00 (2H, m, Ph–H), 3.85
(3H, s, (–OCH3)), 3.74 (3H, s, (–OCH3)) 3.72 (2H, d, J = 0.7 Hz,
(–CH2)); dC(500 MHz, CDCl3) 171.85 (Cq), 152.54 (Cq), 137.44
(CH), 134.51 (Cq), 128.96 (CH), 126.49 (Cq), 125.47 (CH), 121.08
(CH), 118.34 (CH), 112.39 (CH), 55.88 (CH3), 52.10 (CH3), 34.36
(CH2); (m/z) HRMS (EI+): calculated 247.1083 for C13H15N2O3
(M+), found 247.1078.
Bis(2-(2-pyridyl)ethyl)amine (6)
The title compound was prepared according to a literature
procedure.28
{2-[1-(2-Methoxyphenyl)-1H-imidazol-4-yl]ethyl}-
bis(2-pyridin-2-ylethyl)amine (BPAIA, 7)
An oven-dried flask was charged with 5 (0.132 mmol, 0.039 g),
NaI (0.53 mmol, 0.079 g) and acetone (3 mL). The yellow–orange
mixture was allowed to stir at rt for 24 h, then diluted with a
saturated solution of NaHCO3 (ca. 100 mL) and extracted with
CH2Cl2 (3 × 75 mL). The organic fractions were combined,
dried over anhydrous Na2SO4 and the solvent was removed
under reduced pressure. The crude material (0.11 mmol, 0.036
g) was immediately added to an oven-dried round-bottomed flask
containing 6 (0.11 mmol, 0.024 g) and crushed anhydrous K2CO3
(0.32 mmol, 0.044 g). The reaction vessel was flushed with N2,
diluted with CH3CN (1 mL) and the reaction was stirred for 48 h.
A solution of 7 M NaOH (50 mL) was then added to the reaction
mixture and the product was extracted with CH2Cl2 (3 × 100 mL).
The combined organic extracts were dried over anhydrous Na2SO4,
the solvent was removed under reduced pressure and the resulting
brown oil purified by column chromatography (basic alumina,
98/2 EtOAc/MeOH, Rf = 0.43) to yield a gold-coloured oil
(0.018 g, 40%). IR (neat): 2924, 2852, 1724, 1595, 1569, 1517, 1476,
1434, 1269, 1124, 1026, 752, 638 (cm−1); dH(500 MHz, CDCl3) 8.48
(2H, ddd, J = 5.8, 0.9, 0.8 Hz, Pyr–H) 7.68 (1H, d, J = 1.3 Hz,
Im–H) 7.50 (2H, td, J = 7.7, 1.9 Hz, Pyr–H), 7.32 (1H, ddd, J =
8.0, 1.5, 0.7 Hz, Ph–H), 7.21 (1H, dd, J = 7.8, 1.7 Hz, Ph–H),
7.10–6.98 (6H, m, Ar–H), 6.87–6.85 (1H, m, Im–H), 3.82 (3H, s,
(–OCH3)), 3.10–2.93 (10H, m, 5(–CH2–)), 2.79 (2H, t, J = 7.6 Hz,
(–CH2–)) dC(500 MHz, CDCl3) 160.97 (Cq), 152.62 (Cq), 149.35
(CH), 140.64 (Cq), 137.14 (CH), 136.14 (CH), 128.73 (CH), 126.84
(Cq), 125.55 (CH), 123.66 (CH), 121.21 (CH) 121.18 (CH), 116.89
(CH), 112.42 (CH), 55.99 (CH2), 54.18 (CH3), 53.84 (CH2), 36.16
(CH2), 26.43 (CH2); (m/z) HRMS (FAB+): calculated 428.2450
for C26H30N5O (M+), found 428.2449.
2-[1-(2-Methoxyphenyl)-1H-imidazol-4-yl]ethanol (4)
An oven-dried round-bottomed flask was charged with LiAlH4
(1.30 mmol, 0.049 g) and THF (8 mL). The suspension was placed
under N2 and cooled to 0 ◦C prior to the dropwise addition of
a solution of 3 (0.32 mmol, 0.080 g) in THF. A green colour
developed within 20 min, after which the reaction was quenched
with EtOAc (10 mL). The mixture was filtered through Celite, the
filter cake washed with EtOAc (ca. 50 mL), and the solvent was
removed from the filtrate under reduced pressure to yield a white
solid. The crude material was extracted several times with CHCl3
(total volume 50–75 mL) and the solvent was removed under
reduced pressure to yield a white, crystalline material (0.069 g,
97%). Mp = 117–119 ◦C; IR (KBr): 3172, 2914, 1600, 1512, 1466,
1388, 1248, 1181, 1124, 1052, 834, 757, 638, (cm−1); dH(400 MHz,
CDCl3) 7.72 (1H, d, J = 1.2 Hz, Im–H), 7.35 (1H, ddd, J = 7.8,
1.7, 0.9 Hz, Ph–H), 7.29 (1H, dd, J = 7.8, 1.7 Hz, Ph–H), 7.08–7.01
(2H, m, Ph–H), 7.02–7.00 (1H, m, Im–H), 3.96 (2H, t, J = 5.7 Hz,
(–CH2–)), 3.86 (3H, s, (–OCH3)), 3.53 (1H, br s, (–OH)), 2.87 (2H,
t, J = 5.4 Hz, –CH2–); dC(500 MHz, CDCl3) 152.66 (Cq), 140.03
(Cq), 137.39 (CH), 129.05 (CH), 126.63 (Cq), 125.58 (CH), 121.22
(CH), 117.14 (CH), 112.49 (CH), 62.51 (CH2), 55.99 (CH3), 30.80
(CH2); (m/z) HRMS (ESI+): calculated 219.1134 for C12H15N2O2
(M+), found 219.1134.
Methanesulfonic acid 2-[1-(2-methoxyphenyl)-1H-imidazol-
4-yl]ethyl ester (5)
Compound 4 (0.49 mmol, 0.108 g), Et3N (0.74 mmol, 0.075 g)
and CHCl3 (0.5 mL) were added to a round-bottomed flask under
a N2 atmosphere and cooled to 0 ◦C. After stirring for 10 min,
methanesulfonyl chloride (0.74 mmol, 0.084 g) dissolved in CHCl3
(0.5 mL) was added dropwise to the reaction flask and allowed
{2-[1-(2-Hydroxyphenyl)-H-imidazol-4yl]ethyl}-bis(2-pyridin-
2-ylethyl)ammonium bromide (BPAIP·HBr(x), 8)
Compound 7 (0.070 mmol, 0.030 g) and freshly distilled CH2Cl2
(4 mL) were added to a flame-dried Schlenk flask under N2. The
3328 | Dalton Trans., 2006, 3326–3337
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flask was cooled to −78 ◦C and a solution of BBr3 (2 M in CH2Cl2,
0.28 mmol, 0.070 g) was added dropwise and the reaction stirred
for 30 min. The mixture was then gradually warmed to rt and
stirred for an additional 2 h, during which a small amount of
red–orange precipitate formed. The solvent was removed under
reduced pressure, the material was left under vacuum for 2–3 h to
remove excess BBr3, and the resulting orange powder was treated
with 0.5 mL of MeOH and left under a diethyl ether atmosphere
overnight to afford white crystals, which were washed with Et2O,
dried and stored under vacuum (0.035 g, quant.). Mp = 163–
175 ◦C; IR (KBr): 3500–2400, 1637, 1622, 1605, 1545, 1503, 1467,
1299, 1179, 778 (cm−1); dH(400 MHz, D2O) 8.86 (1H, d, J = 1.5 Hz,
Im–H), 8.51 (2H, dd, J = 5.8, 1.0 Hz, Pyr–H) 8.33 (2H, td, J =
8.0, 1.7 Hz, Pyr–H) 7.83 (2H, d, J = 8.2 Hz, Pyr–H) 7.76 (2H, ddd,
J = 5.9, 5.9, 1.0 Hz, Pyr–H) 7.53–7.50 (1H, m, Imd–H), 7.27 (2H,
d, J = 7.7 Hz, Ph–H) 6.98 (1H, dt, J = 7.9, 1.0 Hz, Ph–H) 6.92
(1H, td J = 8.0, 1.2 Hz, Ph–H) 3.70–3.62 (4H, m, 2(–CH2)) 3.67
(2H, t, J = 8.2 Hz (–CH2)) 3.51–3.41 (4H, m, 2(–CH2)) 3.23 (2H,
t, J = 7.9 Hz, (–CH2)); dC(500 MHz, D2O) 150.77 (ArC), 149.39
(ArC), 146.85 (ArC), 141.99 (ArC), 135.73 (ArC), 131.57 (ArC), 128.15
(ArC), 127.38 (ArC), 125.80 (ArC), 125.59 (ArC), 122.23 (ArC), 120.75
(ArC), 121.73 (ArC), 117.05 (ArC), 51.00 (CH2), 50.91 (CH2), 27.73
(CH2), 19.54 (CH2); (m/z) HRMS (FAB+): calculated 414.2292
for C26H30N5O (M+), found 414.2292.
was stirred for an additional 45 min, after which the mixture
was diluted with MeOH (2–3 mL) and then filtered. The pale
yellow filtrate was concentrated under reduced pressure to yield
a pale yellow oil, which was washed repeatedly with Et2O and
then placed under vacuum to yield a tar-like residue that solidified
upon standing (0.049 g, 58%). dH(400 MHz, d7-DMF) 8.53 (3H,
d, J = 4.2, Pyr–H), 7.71 (3H, td, J = 7.6, 1.7 Hz, Pyr–H), 7.29–
7.21 (6H, m, Pyr–H), 3.10–3.00 (6H, m, 3(–CH2–)), 3.00–2.93
(6H, m, 3(–CH2–)); (m/z) HRMS (ESI+): calculated 475.0476 for
C21H24N4ZnBr (M+), found 475.0468.
[Zn(TEPA)](OTf)2 (13)
Zn(OSO2CF3)2 (0.040 g, 0.110 mmol) was dissolved in anhydrous
MeOH (3 mL) and added to a MeOH solution (2 mL) of com-
pound 9 (0.040 g, 0.120 mmol). The solution remained clear, and
was stirred for an additional 45 min. The solvent was removed
and the resultant oil was washed three times with Et2O (3–5 mL)
and the excess solvent was removed under vacuum. Clear, fan-like
crystals developed upon standing. (0.073 g, Quant) dH(400 MHz,
CD3COCD3) 9.02 (3H, ddd, J = 5.5, 0.8, 0.8 Hz, Pyr–H), 8.32
(3H, td, J = 7.9, 1.6 Hz, Pyr–H), 7.84 (3H, d, J = 8.0 Hz, Pyr–
H), 7.75 (3H, ddd, J = 5.6, 5.6,1.3 Hz, Pyr–H), 3.54–3.49 (6H,
m, 3(–CH2–)), 3.39–3.34 (6H, m, 3(–CH2–)); (m/z) HRMS (ESI+)
calculated 395.1214 for C21H23N4Zn (M+), found 395.1317.
Tris(2-(2-pyridyl)ethyl)amine (TEPA, 9)
Structure determination of BPAIP·4HBr (8)
The title compound was prepared according to a literature
procedure.29
Single crystals of 8 were grown from the slow diffusion of a
MeOH/pentane mixture. The crystals were mounted on glass
fibers with Paratone-N oil (Exxon), cooled to −75 ◦C in a
cold nitrogen gas stream, and the diffraction data was col-
lected on a Siemens Platform/CCD diffractometer with graphite-
monochromated MoKaradiation. The structure was subsequently
solved using direct methods (SHELXTL).30 For further details
refer to the ESI‡ and ref. 53.
[Zn(BPAIP)Br]Br (10)
To a small round-bottomed flask was added 8 (0.01 g, 0.02 mmol)
and anhydrous MeOH (0.5 mL). ZnBr2 (0.006 g, 1.3 equiv.) was
dissolved in anhydrous MeOH (0.5 mL) and added dropwise to
the solution of 8, with no visual change in appearance. After
75 min of stirring at rt, the solvent was removed under reduced
pressure. The residue was washed with THF (8–10 mL) and then
CH2Cl2 (8–10 mL). The resulting tar-like residue was stored under
vacuum. (0.012 g, Quant). dH(500 MHz, D2O) 8.84 (1H, d, J =
1.5 Hz, Im–H), 8.48 (2H, d, J = 5.8 Hz, Pyr–H), 8.28 (2H, t, J =
8.0 Hz, Pyr–H), 7.79 (2H, d, J = 8.3 Hz, Pyr–H), 7.70 (2H, t,
J = 6.8 Hz, Pyr–H), 7.50–7.48 (1H, m, Im–H), 7.29–7.24 (2H, m,
Ph–H), 6.97 (1H, dd, J = 8.2, 0.9 Hz, Ph–H), 6.91 (1H, td, J =
8.1, 1.3 Hz, Ph–H), 3.68–3.60 (4H, m, 2(–CH2–)), 3.55 (2H, t, J =
7.7 Hz, (–CH2–)), 3.46–3.41 (4H, m, 2(–CH2–)), 3.20 (2H, t, J =
7.8 Hz, (–CH2–); dC(500 MHz, D2O) 151.50 (ArC), 149.36 (ArC),
146.31 (ArC), 142.20 (ArC), 135.64 (ArC), 131.53 (ArC), 128.52 (ArC),
127.19 (ArC), 125.55 (sh) (ArC), 125.53 (ArC), 122.22 (ArC), 120.77
(ArC), 120.61 (ArC), 117.05 (ArC), 50.99 (CH2), 50.93 (CH2), 27.99
(CH2), 19.71 (CH2); (m/z) HRMS (ESI+): calculated 556.0690 for
C25H27N5OZnBr (M+), found 556.0702.
CCDC reference number 290020.
For crystallographic data in CIF or other electronic format see
DOI: 10.1039/b516090a
Determination of the pKa of the imidazole–phenol group of the free
ligand and Zn complex
Spectrophotometric titrations were carried out on a Hewlett
Packard 8453 UV-Vis spectrophotometer at 0.19 mM analyte
concentration, and adjusted to an ionic strength of 0.5 M KCl.
The reported pKa values were extracted from the data obtained
for a series of buffered solutions of different pH and in which the
phenolate had a UV-Vis absorption maximum (300 nm) distinct
from the neutral phenol (250 nm). The data were fit to a two-state
model (phenol/phenolate).
Theoretical calculations
[Zn(TEPA)Br]Br (12)
Structures were optimised in the gas phase using gradient-
corrected density functional theory (DFT) with the (U)B3LYP
functional,31–33 6-31G(d) basis sets34 for the main group elements
(C, N, O, H, Br) and TZV basis sets35 for the transition
metal elements (Zn, Cu). Vibrational frequency calculations were
performed to confirm the identity of stationary points as minima
Compound 9 was added to a round-bottomed flask (0.050 g,
0.150 mmol) and dissolved in anhydrous MeOH (1.0 mL). ZnBr2
(0.034 g, 0.150 mmol) was dissolved in anhydrous MeOH (1.0 mL)
and added slowly to the MeOH solution containing 9. Within
seconds a white precipitate formed. The heterogeneous mixture
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on the potential energy surface and to obtain thermochemical
corrections to the free energy at 298.15 K. All calculations were
performed with the Gaussian-03 suite of programs36 compiled to
run on the National Computational Science Alliance IBM P690,
located on the UIUC campus.
Electrochemistry
Cyclic voltammetry was carried out using a CH Instruments
617A electrochemical analyzer. Experiments in aqueous solution
were performed at an ionic strength of 0.3 M (KNO3) following
degassing with Ar (20 min). A standard three electrode setup
was utilised, consisting of a glassy carbon working electrode,
platinum wire counter electrode, and a KCl saturated Ag/AgCl
reference electrode. The working electrode was polished prior to
each measurement. Voltammograms recorded in organic solvents
were obtained similarly to those acquired in aqueous solution. The
solvents (acetone and DMF) were freshly distilled over CaSO4
and stored over activated molecular sieves under N2 prior to
use. The anodic peak potentials given in the text are referenced
to the Ag/AgCl electrode used in the experiments. In buffered
solutions of pH 7.0 (25 mM NaH2PO4/Na2HPO4), the reversible
3−/4−
Fe(CN)6
couple was +230 mV versus the Ag/AgCl electrode.
In DMF, the reversible ferrocene/ferrocenium couple was +507
mV versus the Ag/AgCl electrode.
Results and discussion
I
Preparation and structure of the BPAIP chelating ligand
The chelating ligand 8 ({2-[1-(2-hydroxyphenyl)-H-imidazol-4-
yl]-ethyl}-bis-(2-pyridin-2-yl-ethyl)amine, BPAIP), containing the
imidazole–phenol cross-link, is similar to a related ligand recently
reported by Karlin and coworkers.23 The two key structural differ-
ences are that we chose ethylene linkers to tether the two pyridine
rings to the tertiary amine nitrogen in lieu of the methylene linkers
used by Karlin’s group, and as mentioned our imidazole–phenol
moiety does not contain ortho- and para-tert-butyl substituents
on the phenolic ring. The synthetic route to 8 is shown in
Scheme 1. Esterification of 4-imidazoleacetic acid yielded the
methyl ester 1,26 which was coupled, under mild conditions,37 with
the organolead reagent 2 in the presence of catalytic Cu(OAc)2 to
yield 3. Reduction of 3 to the alcohol 4 was followed by conversion
to the methylsulfonate 5. Intermediate 5 was treated with excess
NaI in acetone to yield an unstable alkyl iodide derivative, which
was used immediately in the alkylation of the secondary amine
6 in the presence of excess K2CO3 in CH3CN. Deprotection
of the methyl aryl ether of BPAIA (7) was achieved by the
addition of excess BBr3 to a Schlenk flask containing 7 at −78 ◦C
under strictly anaerobic conditions. As a result of the acidic
conditions utilised to cleave the aryl methyl ether bond, BPAIP
8 was commonly isolated as the HBr salt, with small amounts of
bis, tris and tetra HBr adducts. Alternatively, pure BPAIP·4HBr
could be isolated following deprotection by quenching the excess
BBr3 with MeOH and removing all volatiles from the reaction
flask. Recrystallization from a pentane/methanol mixture (slow
diffusion) yielded clear crystals that were suitable for X-ray
diffraction. The structure of BPAIP·4HBr is depicted in Fig. 2A.
Scheme 1
Fig. 2 (A) The X-ray structure of BPAIP·4HBr. (B) View of the dihedral
angle (45.4◦) between the imidazolium and phenol rings of BPAIP
(bromide ions and hydrogen atoms omitted for clarity).
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Notable bonding distances include the covalent linkage between
halide, etc.). Thus, in order to provide a benchmark of the
approach and ensure that the coordination geometry of this type
of complex is well-reproduced, we first optimised the structure of
the crystallographically-characterised [Cu(TEPA)(Cl)]PF6.40 Two
coordination geometries of similar energy were found: a trigonal
bipyramidal structure and a square pyramidal structure (see ESI‡).
In both cases, the Cu(II) complex was five-coordinate, as evident
the imidazolium and phenolic ring systems, r(N1im–C6phenol) =
˚
1.436 0.014 A, and the imidazolium ring distances of r(N1im–
˚
˚
C2im) = 1.312 0.014 A and r(N1im–C5im) = 1.386 0.012 A.
The dihedral angle (ø) between the planes of the rings of the cross-
linked imidazolium and the phenol was 45.4◦.38 This value is in
close agreement with the value of 44◦ found in the CuB site of CcO
previously reported by Yoshikawa7 and the value of 42.2◦ in the
crystallographically-characterised model compound, 2-(imidazol-
1-yl)-4-methylphenol.39
˚
by a Cu–Namine bonding distance of 2.17 A in the square pyramidal
˚
geometry and 2.186 A in the trigonal bipyramidal geometry.
This is in good agreement with the X-ray data, in which a Cu–
˚
The complex of BPAIP with Zn(II) (10) was prepared in
quantitative yield by adding a dilute solution of ZnBr2 in MeOH
to 8 in MeOH at room temperature. Its identity as the bromide-
coordinated monocation (m/z 558.0668) was confirmed by high-
resolution mass spectrometry (HRMS), and ligand coordination
to Zn(II) was confirmed by changes in the 1H NMR spectra of the
free ligand and the Zn(II) complex (see ESI‡). The complex was
soluble in water and methanol, but despite extensive efforts we
have been unable to obtain single crystals of any Zn(II) (or Cu(II))
complex with 8.
Namine distance of 2.100 A is reported for the chloride coordinated
complex, which has distorted square pyramidal geometry. For the
analogous bromide complex, [Cu(TEPA)Br]+, which has not been
structurally characterised, the calculated Cu–Namine distances were
˚
slightly longer for both geometries, with distances of 2.201 A and
˚
2.213 A, respectively.
The optimised structure of [Zn(BPAIP)(X)]+ (X = Br, OH) is
best described as a distorted trigonal pyramid with two pyridine
ligands and the imidazole–phenol ligand defining the equatorial
plane (Fig. 3). The relevant bond lengths and angles are given
in Tables 1 and 2. In each case, the anionic ligand is bound in
the axial position, trans to the tertiary amine nitrogen. The Zn–
II Density Functional Theory (DFT) studies of relevant Zn(II)
models
˚
Namine distance of 10 (X = Br) is 2.756 A, outside the range of
typical Zn–Namine bond lengths reported in five-coordinate Zn(II)
41–45
˚
complexes with mixed ligand sets (2.079–2.325 A),
elongated Zn–Namine contacts (2.411–2.652 A) with varying degrees
of interaction have been reported.46–48 The equatorial Zn–NIm
bond (2.037 A) in 10 is slightly shorter than the two Zn–Npyr
bonds at 2.095 and 2.102 A. The average bond angle in the
although
In order to support and facilitate the interpretation of the spec-
troscopic studies on this complex (vide infra), Density Functional
Theory (DFT) was applied to determine its gas-phase structure
while varying the protonation and oxidation states of the phenolic
moiety. The focus of the calculations was on compound 10 as
both the monobromide (X = Br−) and monohydroxide (X =
OH−) complexes, [Zn(BPAIP)(X)]+, as well as the corresponding
Zn(II) complex with the more thoroughly studied ligand tris(2-(2-
pyridyl)ethyl)amine (TEPA, 9), for comparison.
˚
˚
˚
equatorial plane (N–Zn–N) is approximately 114◦, and the Zn(II)
is positioned 13.4◦ above the trigonal plane with axial bond
angles (N–Zn–Br) larger than 90◦, indicating distortion from
idealised trigonal pyramidal geometry. The calculated dihedral
angle between the planes of the phenol and imidazole rings is 45.9◦,
in good agreement with the X-ray structure of 8·4HBr (45.4◦),
To the best of our knowledge, no structural data exists for
Zn(II) complexes of 9 with an exogenous fifth ligand (solvent,
+
+
˚
Table 1 Relevant geometric features in [Zn(TEPA)Br] (11, X = Br) and [Zn(BPAIP)Br] (10, X = Br) derivatives. All bond lengths in A and angles in
degrees
10, X = Br
Phenol
11, X = Br
Phenolate
Phenoxyl
No phenola
Zn–Namine
Zn–Npyr1
Zn–Npyr2
Zn–Npyr3/Im
Zn–Br
2.571
2.117
2.113
2.118
2.468
2.756
2.095
2.102
2.037
2.450
2.965
2.11s7
2.124
1.965
2.433
2.725
2.089
2.101
2.048
2.468
2.732
2.088
2.104
2.054
2.457
N
N
N
amine–Zn–Npyr1
amine–Zn–Npyr2
amine–Zn–Npyr3/Im
85.2
85.1
84.9
179.6
118.8
118.7
95.0
120.3
95.1
94.7
9.9
82.7
83.9
81.9
176.4
114.8
128.3
95.6
112.1
99.7
79.4
79.6
79.8
175.5
114.5
123.0
97.3
112.8
99.2
83.2
84.6
82.3
175.6
115.4
129.0
95.1
111.5
99.7
83.0
84.4
82.3
175.6
115.0
129.5
95.1
111.1
100.0
96.0
Namine–Zn–Hal
Npyr1–Zn–Npyr2
N
pyr1–Zn–Npyr3/Im
Npyr1–Zn–Br
pyr2–Zn–Npyr3/Im
Npyr2–Zn–Hal
N
N
pyr3/Im–Zn–Hal
96.7
13.4
45.9
104.6
18.0
23.6
95.8
12.5
30.6
Improper dihedral (w)b
Dihedral (ø) c
16.3
n/a
n/a
a Phenol replaced by hydrogen. b The improper dihedral angle is the mean angle between the Zn(II) and the plane made up of the nitrogen atoms of the
three equatorial ligands. c The dihedral angle is defined by the planes of the phenol and imidazole rings.
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Table 2 Relevant geometric features in [Zn(TEPA)(OH)]+ (11, X = OH) and [Zn(BPAIP)(OH)]+ (10, X = OH) derivatives. All bond lengths in A and
˚
angles in ◦
11, X = OH
10, X = OH
Phenol
Phenolate
Phenoxyl
No phenola
Zn–Namine
Zn–Npyr1
Zn–Npyr2
Zn–Npyr3/Im
Zn–OH
2.375
2.185
2.097
2.206
1.906
2.572
2.137
2.095
2.058
1.911
2.887
2.128
2.132
1.981
1.890
2.540
2.092
2.137
2.071
1.914
2.517
2.094
2.141
2.076
1.913
N
N
amine–Zn–Npyr1
amine–Zn–Npyr2
86.5
91.2
89.1
174.9
110.6
139.6
89.4
109.6
93.0
92.1
2.8
85.4
88.9
84.2
175.7
109.6
138.7
93.7
110.1
95.4
93.7
8.7
81.9
80.9
80.7
169.9
118.3
118.0
96.1
116.8
91.5
85.9
89.7
84.3
174.0
108.9
139.8
93.8
109.9
96.0
92.1
8.0
86.3
90.1
84.5
173.8
108.7
141.1
93.3
109.0
95.9
91.9
7.6
Namine–Zn–Npyr3/Im
Namine–Zn–OH
Npyr1–Zn–Npyr2
N
N
Npyr2–Zn–Npyr3/Im
Npyr2–Zn–OH
pyr1–Zn–Npyr3/Im
pyr1–Zn–OH
N
pyr3/Im–Zn–OH
108.8
17.3
24.1
Improper dihedral (w) b
Dihedral (ø) c
n/a
46.7
28.7
n/a
a Phenol replaced by hydrogen. b The improper dihedral angle is the mean angle between the Zn(II) and the plane made up of the nitrogen atoms of the
three equatorial ligands. c The dihedral angle is defined by the planes of the phenol and imidazole rings.
The protonation and oxidation states of the imidazole–phenol
ligand were varied and compared to the parent structures in
order to examine the structural ramifications. While the overall
symmetry of the complex does not change upon formation of the
phenolate, phenoxyl radical or with an unsubstituted imidazole
ligand, some interesting trends are observed (Tables 1 and 2).
It is important to note that the dihedral angle between the
planes of the phenol and imidazole rings changes from 45.9◦ to
23.6◦ upon deprotonation of the cross-linked phenol and then
to 30.6◦ upon formation of the phenoxyl radical in the bromide
complex, with the same trend apparent in the analogous hydroxide
complex, 46.7◦ (phenol) to 24.1◦ (phenolate) and 28.7◦ (phenoxyl)
(Table 2). The reduction in the dihedral angle between phenol
and o-imidazole upon deprotonation has been documented in
previous theoretical models,49 but it should be noted that in these
previous models the phenolic hydrogen was H-bonded to the
imidazole Ne-atom, which yields a larger initial angle of 59◦ and
Fig. 3 Calculated structure of [Zn(BPAIP)Br]+ (10, X = Br): (a) axial
brings about a larger decrease (−32◦) upon deprotonation. We
chose to examine the non-intramolecularly H-bonded conformer
(despite its higher energy of ca. 1 kcal mol−1 in the gas phase)
since it should be the only relevant conformer in solution (or
the enzyme) due to stronger intermolecular H-bonding. This is
also the only conformer present in both the crystal structure
of 8·4HBr (Fig. 2) and the crystal structure of o-imidazole-p-
cresol.39
view; (b) equatorial view.
and similar to what is reported for the cross-link in the CuB site
of the enzyme (44◦).7 The analogous [Zn(TEPA)Br]+ complex 11
was calculated to be isostructural with 10, with similar equatorial
ligand bonding distances and a shorter Zn–Namine distance of
˚
2.571 A. This distance is still outside the typical bonding distances
found in trigonal bipyramidal complexes containing Zn(II). The
bulk of the discussion thus far has focused on the bromide
coordinated species of each Zn(II) complex as they are observed
from mass spectral data obtained from the complexes prepared
in MeOH. However, it is important to note that the same trends
in bond distances and angles apply to the analogous hydroxide
coordinated complexes that may be more relevant in aqueous
solution, with the main difference being closer Zn–Namine contacts
Interestingly, the DFT results indicate that formation of the
phenolate results in a stronger imidazole nitrogen–Zn(II) bonding
interaction. This is most evident by comparing the Zn–NIm bond
in the unsubstituted imidazole, imidazole–phenol, imidazole–
phenolate, and imidazole–phenoxyl radicals containing Zn(II)
complexes for both the monobromide (Table 1) and monohy-
droxide complexes (Table 2). The decreased Zn–NIm bond length
in the bromide complex coincides with a slight increase in the
˚
˚
˚
in the hydroxide complex (2.375 A for TEPA (9) and 2.572 A for
BPAIP, 8).
Zn–Npyr bonding distances (+0.02 A) versus the other phenol
derivatives.
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In addition, the Zn–Namine distance in the imidazole–phenolate
˚
is lengthened appreciably by over 0.2 A versus imidazole–phenol,
with a small decrease in the axial Zn–Br distance. The equatorial
bond angles in the Zn(II) complex remain virtually unchanged as
a function of the protonation or oxidation state of the phenol. In
contrast, the axial bond angles and distortions from the equatorial
plane listed in Table 1 suggest the Zn(II) cation is raised slightly
out of the trigonal plane of the two pyridine nitrogen atoms and
the imidazole nitrogen, with a larger improper dihedral angle (18◦)
in the phenolate when compared to the other phenol derivatives.
Again, the same trend also applies to the phenolate derivative
of the hydroxide complex, with a Zn–Namine distance increase
˚
of over 0.3 A compared to the parent phenol structure, and
an improper dihedral angle of 17.3◦ from 8.7◦ (Table 2). This
suggests that the nature of the distortion with increasing electron-
donating capacity of the equatorial imidazole is further away from
idealised trigonal bipyramidal geometry and towards tetrahedral
geometry in both structures, which is most common for four-
coordinate Zn(II) complexes. This is illustrated in Fig. 4A, where
the relationships between the key distortions are plotted for the
different Zn(II)–Br complexes. It is interesting to note that from the
correlation of Zn–Namine bond distance with w, we can derive the
length of the Zn–Namine bond in an idealised trigonal bipyramidal
˚
(w = 0) structure as being 2.216 A.
Gas phase binding free energies (DGbinding) were calculated to
assess the change in the energetics of the ligand–Zn(II) interaction
upon changing the electron-donating character of an equatorial
ligand (eqn 1). The calculated energetics are fully consistent with
the calculated geometries (Fig. 4B): the interaction is weakest in
the complex with TEPA (9), where a pyridine is the third equatorial
donor, and strongest in the complex with deprotonated BPAIP (8).
Both the Zn–Namine distance and the DGbinding are similar in the BPAI
(lacking the phenol), BPAIP and BPAIP (phenoxyl) complexes.
These results suggest an increased stability of the [Zn(BPAIP)(X)]+
Fig. 4 Correlations demonstrating the relationship between the Zn–Namine
distance in the [Zn(BPAIP)Br]+ complexes 10 (phenol, phenolate and
phenoxyl), [Zn(BPAI)Br]+ and [Zn(TEPA)Br]+ (11) and (A): the mean
deviation of Zn(II) from the plane defined by the three coordinating
heterocyclic nitrogens (᭹, solid line) and the Zn–NIm distance (᭺, dashed
symbols); (B): the gas-phase binding free energy changes (DGbinding) defined
as: [Zn(L)Br]+ → Zn2+ + L+ Br−.
1
at high pH, which is fully supported by H NMR studies (vide
infra).
[Zn(L)Br]+ → Zn2+ + L + Br−
(1)
In summary, the DFT calculations suggest that both complexes,
[Zn(BPAIP)(X)]+ (X = Br−, OH−), have comparable structural
changes that occur upon deprotonation of the imidazole–phenol.
An overall lengthening of the z-axis defined by the vertical plane
(Xanonic–Zn–Namine) takes place with simultaneous contraction of the
Zn–Nimd distance and an elevation of the Zn(II) above the trigonal
plane of equatorial donors. In each [Zn(BPAIP)(X)]+ complex,
the overall changes resulting from formation of the phenolate
are structural distortions away from trigonal pyramidal (ignoring
the Zn–Namine interaction) and toward tetrahedral geometry with
increased binding affinity, as compared to the parent (neutral
phenol) complex.
8 yielded a pKa of 8.65 0.08, virtually indistinguishable from
the values determined in our previous model studies performed
under similar conditions with either ortho-imidazole–phenol (pKa
of 8.69
0.04).10 On the other hand, titration of a Zn(II) complex of 8
resulted in a pKa of 8.01 0.07 (Fig. 5). In aqueous solution,
0.09)18 or ortho-imidazole-para-cresol (pKa of 8.60
Zn(II) lowers the pKa of a bound water to approximately 9–
10 (eqn 2).50,51 Thus, there exists the possibility of exchange
of the axial bromide for a hydroxide ligand in dilute aqueous
solution under basic pH conditions. Although the identity of
the coordinated anion in aqueous solution is not definitively
known (bromide, hydroxide, or chloride from buffer), based on
the DFT calculations the coordinated anion is not expected to
change the geometry substantially. The modest decrease in the
pKa of ∼0.6 log units (Fig. 5) upon Zn(II) coordination can
be attributed to an inductive effect, as the divalent metal ion
withdraws electron density from the imidazole, stabilizing the
phenolate anion. Importantly, and in contrast to a previous
III Zn(II)–BPAIP solution chemistry
A spectrophotometric titration was performed to study the
effect of an imidazole-bound Zn(II) on the pKa of the cross-
linked phenol. Upon deprotonation, the kmax of the cross-linked
phenol undergoes a characteristic red-shift of 50–60 nm that is
unmistakably associated with the phenolate.10,15–18 Monitoring the
pH dependent increase of the phenolate absorbance of free BPAIP
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Cyclic voltammetry (CV) was carried out on 10 in order
to qualitatively probe the effect of Zn(II) coordination on the
phenolate/phenoxyl radical couple of the cross-link. One broad
peak and a higher potential shoulder were observed for both 8 and
10 at pH 9.6, where both are deprotonated (Fig. 6). The broad peak
in 8 is the expected oxidation of the phenolate to the phenoxyl
radical (Epa = +558 mV), essentially identical to that of ortho-
imidazole–phenol (Epa = +556 mV) determined under the same
conditions.18 The corresponding peak in the voltammogram of 10
is shifted by approximately +50 mV to higher potential, consistent
with the electron withdrawing effect of the Zn(II) ion observed
in the UV-Vis titrations. The high potential shoulder observed in
both 8 and 10 occurs at about 0.7 V. Independent CV scans of other
tertiary amines, such as triethylamine (TEA) (pH 11.5, Epa = 692
mV), indicated that the origin of the shoulder peak in both 8 and
10 may be due to the oxidation of the tertiary amine of BPAIP. This
shoulder is not observed in the voltammogram of ortho-imidazole–
phenol under similar conditions, suggesting it is not due to follow
up chemistry upon oxidation of the phenolate. Additionally, an
independent voltammogram of tetraethylammonium bromide in
a pH 9.6 buffer solution shows no oxidation peaks within the same
window. These observations imply the tertiary amine nitrogen in
10 is not, or at most only weakly, coordinated to Zn(II) in solution
at basic pH, and as such can be oxidised electrochemically. This
is possibly due to a trans influence brought about in part by the
coordinated anion that lengthens the Zn–Namine bond, but more
importantly by the deprotonation of the phenol, which substan-
tially increases the Zn–N bond length as shown in Tables 1 and 2.
Fig. 5 Spectrophotometric titrations at 300 nm of BPAIP 8 (᭺, dashed
line) and [Zn(BPAIP)(X)]+ 10 (᭹, solid line) in 0.5 M KCl (X = halide,
OH−).
report,17 metal coordination does not increase the pKa back to
the value typically observed for an unsubstituted phenol when the
dihedral angle between the rings is similar to that in CcO.
pK =9−10
a
+
+
[Zn(H2O)6]2+
[Zn(H2O)5(OH)] + H
(2)
−−−−ꢀ
ꢁ−−−−
H
O
2
1H NMR spectra of 10 were recorded in buffered solutions
of pH 6.8, 8.2 and 9.6 to monitor changes as a function of the
protonation state of the phenol. At pH 6.8, well below the pKa
measured for the phenol, it appears that 10 is in equilibrium
(rapid exchange) as the proton signals from the aromatic rings
involved in coordination (imidazole, pyridine) are significantly
broadened compared to the free ligand 8 (Fig. S1‡). The three
signals corresponding to the phenolic ring protons are significantly
sharper, suggesting they are less affected by the equilibrium, thus
supporting a rapid exchange process at Zn(II). In contrast, at
1
pH 8.2 the signals in the H NMR spectrum of the aromatic
region of 10 were sharp and well-defined and the spectrum
was significantly different from that of 8 at the same pH. For
instance, the aromatic protons from the imidazole and pyridine
rings are shifted substantially downfield, consistent with electron-
withdrawal by Zn(II). The phenolic protons are shifted upfield
compared to the phenolic protons of 8, consistent with the greater
electron-density in the ring upon deprotonation of the phenol. It is
important to note that the pH of the solution (8.2) approximately
coincides with the experimentally observed pKa of the phenol
in 10. At pH 9.6 (Fig. S1‡), Zn(II) remains bound as one well-
defined species as evidenced by sharp aromatic peaks observed in
Fig. 6 Cyclic voltammogram of 8 (dashed line) and 10 (solid line) at
pH 9.6 (1 mM analyte, 0.3 M KNO3, scan rate of 10 mV s−1).
1
the H NMR spectrum. The phenolic proton signals are shifted
IV Zn(II)–TEPA solution chemistry
further upfield at pH 9.6 compared to at pH 8.2, as expected
with an increased proportion of phenolate present at equilibrium.
The alkyl region of the 1H NMR spectrum (ethylene linkers) also
changes as a function of pH (Fig. S2‡), with a significant difference
in both the shape and the chemical shift of the signals on raising
the pH from 6.8 to 8.2. At the lower pH, the ethylene proton
signals are very broad compared to their shapes at the higher pH,
where the signals are also better resolved (compared to 8).
The experimental results in conjunction with the DFT calculations
suggest that the tertiary amine nitrogen is not, or only very
weakly, coordinated to Zn(II). Furthermore, these studies suggest
that the overall stability of the Zn–BPAIP complex increases
upon deprotonation of the phenol, as illustrated by a significant
sharpening of the NMR signals and a decrease in the imidazole–
Zn bond length (DFT). If this interpretation of the data is correct,
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several predictions can be made and experimentally tested. For
instance, a coordinating ligand similar in structure but lacking
the imidazole–phenol group, such as TEPA 9, should not display
the same pH dependence of the NMR signals. Furthermore, the
DFT results predict that the Zn–Namine distance would still be long
[Zn(TEPA)](ClO4)2 reported by Xu et al.,52 no oxidation peak at-
tributable to the tertiary amine was found in the voltammogram of
13 (Fig. 8), suggesting a significant Zn–Namine bonding interaction
in solution. Indeed, our DFT calculations reveal that protonation
of the fifth (hydroxide) ligand in [Zn(TEPA)(OH)]+ results in
dissociation of the water and yields a tetrahedral structure for
+
˚
(2.571 A) for [Zn(TEPA)Br] (11, X = Br), and hence its tertiary
amine would still be prone to electrochemical oxidation. On the
[Zn(TEPA)]2+ with a Zn–Namine distance of 2.083 A.
˚
other hand, the structure of [Zn(TEPA)](ClO4)2 has been reported
52
˚
with a Zn–Namine distance of 2.028 A and, for such a complex,
oxidation of the amine should not be observed.
To test these predictions, [Zn(TEPA)Br]Br (12) was synthesised.
1
In d7-DMF, the H NMR spectrum of 12 (not shown) displayed
one set of well-defined, sharp pyridine proton peaks, suggesting
a three-fold symmetry axis. Similarly to 10, the high resolution
mass spectrum (HRMS) of 12 isolated from MeOH provided
evidence of the bromide coordinated monocation, [Zn(TEPA)Br]+,
(m/z 475.0468). Cyclic voltammetry of 12 in DMF (Fig. 7)
demonstrated one anodic peak at the same potential (Epa
=
+893 mV) as 9 (Epa = +891 mV) consistent with little Zn–
Namine bonding interaction. An additional oxidation peak at a
much higher potential was observed with 9, most likely due to
pyridine oxidation. This peak was not observed for 12 as the
pyridine groups are presumed bound, as inferred from its 1H NMR
spectrum in d7-DMF.
Fig. 8 Cyclic voltammogram of 9 (solid line) and 13 (dashed line) in
acetone (1 mM analyte, 0.2 M [TBA][PF6], scan rate of 100 mV s−1).
Conclusions
A new chelating ligand (BPAIP, 8) incorporating a non-hindered
ortho-imidazole–phenol linkage has been synthesised and some of
the properties of its Zn(II) complex have been explored. Spectro-
scopic data (mass spectrometry, 1H NMR spectroscopy and cyclic
voltammetry) and theoretical studies suggest the complex exists as
the monocation, [Zn(BPAIP)(X)]+, exhibiting trigonal pyramidal
geometry with an axial bromide ligand defining the vertical plane
and the tertiary amine not, or only weakly, coordinated. The
coordination to Zn(II) results in a small decrease in the phenolic
pKa of about 0.6 log units and a slight increase in the potential
of the phenolate/phenoxyl radical couple of approximately +50
mV. Although the geometry of the oxidised CuB site in CcO is
different (distorted square planar), the ligand set in the enzyme
(three histidine ligands and a hydroxide or peroxide) is similar
to that in the Zn(II) model with three neutral nitrogen donors
(two pyridine and one imidazole) and an anionic donor (Br−,
OH−). Hence, we anticipate Cu(II) coordination to the cross-linked
imidazole in the enzyme to also have a modest effect in further
lowering the phenolic pKa. Our results therefore support the
previously suggested model that a decreased pKa facilitates proton
delivery to the active site via the cross-linked moiety situated at
the end of the K-channel.14
Fig. 7 Cyclic voltammogram of 9 (dashed line) and 12 (solid line) in
DMF (1 mM analyte, 0.2 M [TBA][PF6], scan rate of 10 mV s−1).
Under similar experimental conditions as for 10, 1H NMR
spectra were collected for 12 as a function of pH in aqueous
buffered solutions. In contrast to the results found with the Zn(II)–
BPAIP complex, 12 displays broad and undefined peaks even
at pH 9.6 (Fig. S3‡). Assuming the geometry of both Zn(II)
complexes (10, 12) are similar in aqueous solution, this suggests the
stability of 10 at pH 9.6 (Fig. S1D and S2B‡) is directly related to
the deprotonation of the unique imidazole–phenol. This conflicts
with the deprotonation of a Zn(II)-bound water as the reason for
the increased stability of 10 at pH 9.6, since a similar stabilization
should then have been observed for 12.
Several interesting features of the coordination chemistry of
the crosslink were uncovered. BPAIP (8) forms a more stable
Zn(II) complex as a function of increasing pH, as observed by
1H NMR spectroscopy. This is related to the deprotonation of
the phenol and its effect on the electron-donating ability of the
imidazole imino nitrogen, since the stability of the analogous
As a final test, a TEPA complex of Zn(II) was prepared in
the presence of a weak-field ligand (−OTf) (13) and subjected to
electrochemical studies. Consistent with the solid state structure of
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Dalton Trans., 2006, 3326–3337 | 3335
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View Article Online
[Zn(TEPA)Br]+ and [Zn(TEPA)]2+ (not shown) did not display
such a pH dependence. DFT calculations support the distorted
trigonal bipyramidal structure for both the bromide and hydroxide
complexes. The overall coordination geometry does not change
upon formal removal of a hydrogen atom (H+ + e−) from the
phenolic moiety of the imidazole–phenol ligand. However, a trend
in distortion away from trigonal pyramidal toward tetrahedral
geometry was found as a function of increased electron donation
from the imidazole–phenol ligand to Zn(II) along the equatorial
plane. In other words, the deprotonation of phenol results in
structural changes to the Zn(II) complex as well as an enhanced
binding strength of 8 in the gas phase.
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Acknowledgements
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The authors would like to thank Dr Robert Ellenwood and Prof.
Gregory Girolami (UIUC) for their assistance with the X-ray data
analysis and Meera Raja for her help with the synthesis of 8.
This work was supported by the National Institutes of Health
(GM44911). DAP acknowledges the support of the Jane Coffin
Childs Fund for Medical Research. This work was also partially
supported by the National Computational Science Alliance under
Grant CHE300057N and utilised the IBM P690 located on the
UIUC campus.
30 Bruker AXS Inc., 1998, Madison, Wisconsin, USA.
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53 Chemical formula, C53H74Br8N10O6; formula weight, 1586.50; Crystal
˚
˚
system, monoclinic; Unit-cell dimensions, a, 14.088(7) A; b, 7.523(4) A;
◦
◦
◦
3
˚
˚
c, 34.71(2) A; a, 90.00 ; b, 97.75(1) ; c , 90.00 ; volume, 3645(3) A );
temperature, 193(2) K; Space group P21/n; Z = 4; l = 4.446; Number
of reflections measured, 6388; R(Fo) = R[(Fo − Fc)]/R(Fo) = 0.087;
51 Principles of Bioinorganic Chemistry, ed. S. J. Lippard and J. M. Berg,
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the four HBr molecules could not be located.
This journal is
The Royal Society of Chemistry 2006
Dalton Trans., 2006, 3326–3337 | 3337
©