Galactose oxidase models: Insights from 19F NMR spectroscopy

Article abstract of DOI:10.1039/b813036a

¹⁹F labelled tripodal ligands that possess a N₃O donor set (one phenol, one tertiary amine and either two pyridines or one pyridine and one quinoline) have been synthesized. The fluorine is incorporated either at the phenol O-donor (

Full text of DOI:10.1039/b813036a

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www.rsc.org/dalton | Dalton Transactions
Galactose oxidase models: insights from 19F NMR spectroscopy†
Fabien Michel,a Sylvain Hamman,a Christian Philouze,a Carlos Perez Del Valle,b Eric Saint-Amana and
Fabrice Thomas*a
Received 29th July 2008, Accepted 2nd October 2008
First published as an Advance Article on the web 28th November 2008
DOI: 10.1039/b813036a
19F labelled tripodal ligands that possess a N3O donor set (one phenol, one tertiary amine and either
two pyridines or one pyridine and one quinoline) have been synthesized. The fluorine is incorporated
either at the phenol O-donor (HLF and HLCF3) or at the quinoline N-donor (HLqOMe and HLqNO2). The
copper(II)–phenol complexes (2H)2+, (1H)2+, (3H)2+ and (4H)2+ as well as the corresponding
copper(II)–phenolate complexes have been characterized. X-Ray diffraction reveals an increase in the
+
˚
oxygen–copper bond distance of more than 0.4 A upon protonation of the phenolate moiety of (4) .
Protonation is accompanied by an axial to equatorial isomerization of the quinoline group. DFT
calculations show that stretching of the Cu–O(phenol) bond, p-stacking interactions and rotation of the
pyridine are key steps in this isomerization process. Protonation, and thus changes in the oxygen–copper
bond distance induce either a decrease ((1H)2+, (2H)2+) or an increase ((3H)2+ and (4H)2+) in the
copper–fluorine distance that could be monitored by 19F NMR. In the former case, a broadening of the
19F NMR signal is observed, whereas a sharpening is observed in the latter case. Temperature
dependent 19F NMR measurements on equimolar mixtures of the phenol and phenolate complexes of
(3)+ and (4)+ reveal rate constants for proton transfer and/or isomerization of 3000 100 s-1 and 2900
100 s-1, respectively, at the coalescence temperature. This temperature was found to be strongly affected
by the phenol para-substituent as it is 226 K and ca. 330 K for (3)+ and (4)+, respectively. A phenoxyl
radical species ((3∑)2+) could be generated and characterized for the first time by 19F NMR spectroscopy.
complex of HLOMe (Fig. 1) has been shown to be a structural
model that reproduces quite well the spectroscopic properties and
reactivity of the enzyme.6
Introduction
Galactose oxidase (GO) is a copper(II) enzyme that catalyses the
oxidation of primary alcohols into the corresponding aldehydes,
with concomitant reduction of dioxygen into hydrogen peroxide.1
The crystal structure of GO was reported in 1991, showing an
active site constituted by a copper ion in a square pyramidal
geometry.2 It is coordinated in the basal plane to two histidine
nitrogen atoms (His496), one tyrosinate oxygen (Tyr272 that is cross-
linked with Cys228), and one exogenous ligand (acetate molecule
from the buffer). The apical position is occupied by one tyrosine
oxygen (Tyr495). The apparent paradox—a two-electron oxidation
performed by a single copper ion—is explained by the involvement
of another redox center, namely a tyrosyl radical from the protein,
during the catalytic cycle. Extensive characterizations have shown
∑
that it is located at Tyr272 and that it is essential for catalysis.1
Several model compounds for the GO active site have been
developed during the last decade, contributing to an enhanced
understanding of the metal–radical interaction.3–10 In this context,
it has been shown that tripodal ligands were good structural, and
sometimes functional, models of GO.3–10 In particular, the copper
Fig. 1 Formulae of the tripodal ligands.
Techniques that are commonly used in order to characterize
such complexes are UV-vis and EPR spectroscopies, as well as
electrochemical measurements. The development of new tools
to study GO models is of major interest. For example, classical
techniques do not enable the discrimination in the phenol(ate)
position (axial vs. equatorial), and dynamic information is gen-
erally missing. We have recently shown that 19F NMR could be
a very powerful tool when it is combined with judicious ligand
labelling.7 This technique offers unique advantages as a lack of
aDe´partement de Chimie Mole´culaire, Chimie Inorganique Redox
Biomime´tique (CIRE), UMR CNRS 5250, Universite´ J. Fourier, B. P. 53,
38041 Grenoble cedex 9, France. E-mail: Fabrice.Thomas@ujf-grenoble.fr;
Fax: +33 476 514 846; Tel: +33 476 514 373
bDe´partement de Chimie Mole´culaire, Chimie The´orique, UMR CNRS 5250,
Universite´ J. Fourier, B. P. 53, 38041 Grenoble cedex 9, France
† Electronic supplementary information (ESI) available: Additional EPR,
CV and titration curves. CCDC reference numbers 661385 and 661386.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/b813036a
832 | Dalton Trans., 2009, 832–842
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Table 1 Crystal data for (1H)2+ and (3H)2+
interfering background signals, high sensitivity and distribution
of resonances over a wide spectral width. HLqNO2 (Fig. 1) is
derived from HLOMe: The fluorine atom has been incorporated
in the ligand by replacing the pyridine by a fluoroquinoline group,
while the methoxy substituent was replaced by a nitro one (in
order to monitor proton transfers and not radical formation).7
In its copper(II) complexes, we have shown that both the chemical
shift and the line width of the 19F resonance are extremely sensitive
to protonation and phenol positioning.
Is it possible to extrapolate these results to other systems? We
here address this question through the study of a series of 19F-
labelled ligands derived from HLOMe. More specifically, we explore
(i) different kinds of ligand labelling and, as this work deals with
the modelling of the GO active site, (ii) the possibility to directly
detect phenoxyl radicals by 19F NMR.
(1H) (ClO4)2
(3H)2 (ClO4)4·CH3CN
Formula
C26H29Cl2CuF3N4O9 C62H69Cl4Cu2F2N9O20
Mr/g mol-1
Crystal system
Space group
732.98
Monoclinic
P21/c
8.672(3)
40.05(2)
8.855(5)
90
101.2(1)
90
3017(3)
4
1567.18
Triclinic
¯
P1
˚
a/A
12.006(3)
17.558(4)
18.513(2)
67.13(1)
73.91(1)
82.21(2)
3453(1)
2
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
3
˚
V/A
Z
T/K
150
1.614
9.78
Graphite
150
1.507
8.55
Dc/g cm-3
m/cm-1
The ligand HLqOMe (Fig. 1) differs from HLqNO2 by its phenol
para-substituent, a strongly electron-donating methoxy group.
This substituent is known to stabilize phenoxyl radicals very
efficiently.3–6 HLqOMe is thus expected to be the precursor of stable
Monochromator
Graphite
MoKa (0.71073 A)
26 103
˚
˚
l/nm
Reflections collected
MoKa (0.71073 A)
29 657
Independent reflections
Observed reflections
Rint
R1
wR2
7549
4419 [I > 2s(I)]
0.1255
0.0436
0.0396
9241
6328 [I > 2s(I)]
0.1088
0.0719
0.0972
phenoxyl radicals, as does HLOMe
.
HLCF3 and HLF belong to another family of F-labelled tripodal
ligands in which the fluorine atom is introduced on the phenol
moiety and not on a N-donor (Fig. 1). The ligands differ to each
other by the phenolate para-substituent, a trifluoromethyl group
in HLCF3 or a fluorine atom in HLF.
Synthesis of these ligands and their copper(II) complexes is
described, as well as their characterization by electrochemical and
spectroscopic methods (including 19F NMR).
Results and discussion
Preparation of the ligands
HLCF3 was obtained in a one-pot synthesis by mixing bis(2-
pyridylmethyl)amine and 2-tert-butyl-4-trifluoromethanephenol
in the presence of one equivalent of formaldehyde. HLqOMe was
prepared in a similar way to HLqNO2 by using 3-tert-butyl-2-
hydroxy-5-methoxybenzaldehyde. HLF and HLqNO2 have been
previously described.6–7
Crystal structures of the copper complexes
Monomeric complexes were obtained upon mixing stoichiometric
amounts of the N3O ligands and Cu(ClO4)2·6H2O in acetoni-
trile. Under these conditions, the complexes were isolated in
their protonated form in which the phenolic subunit remains
protonated. Single crystals of [CuII(HLCF3)(CH3CN)]2+ (1H)2+,
[CuII(HLF)(CH3CN)]2+ (2H)2+,6 [CuII(HLqOMe)(CH3CN)]2+ (3H)2+
and [CuII(HLqNO2)(CH3CN)]2+ (4H)2+7 were obtained upon slow
diffusion of di-iso-propyl ether into acetonitrile solutions of the
complexes (Table 1). When one equivalent of Cu(ClO4)2·6H2O
and HLqNO2 were mixed in acetonitrile in the presence of NEt3, the
phenolate copper complex (4)+ could be crystallized.7 The ORTEP
plots of these complexes are depicted in Fig. 2 and selected bond
lengths and angles are listed in Table 2.
Fig. 2 ORTEP plot of: (a) (1H)2+ (ClO4-); (b) (2H)2+ (ClO4-);6 (c) (3H)2+
(ClO4-); (d) (4H)2+ (ClO4
)
and (e) (4)+.7 Ellipsoids are drawn at the
-
7
50% probability level and hydrogen atoms (except the phenolic ones) are
omitted for clarity.
In (1H)2+ and (2H)2+, the geometry around the metal center is
octahedral with one phenol oxygen and one perchlorate oxygen
atom that coordinate in axial positions. An exogenous acetonitrile,
two pyridines and the tertiary amine nitrogen atoms occupy the
equatorial positions.
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◦
˚
Table 2 Selected bond distances (A) and angles ( )
(1H)·2ClO4
Cu–O1
Cu–N2
N1–Cu–N2
N2–Cu–N3
2.353(2)
1.992(2)
83.0(1)
Cu–O2
Cu–N3
N1–Cu–N3
N2–Cu–N4
2.526(2)
1.980(2)
84.3(1)
Cu–N1
Cu–N4
N1–Cu–N4
N3–Cu–N4
2.026(2)
1.988(2)
176.3(1)
98.6(1)
165.5(1)
93.9(1)
(2H)·2ClO4
Cu–O1
Cu–N3
N1–Cu–N2
N2–Cu–N3
2.404(3)
1.975(3)
83.7(2)
Cu–N1
Cu–N4
N1–Cu–N3
N2–Cu–N4
2.024(4)
2.008(4)
84.0(1)
Cu–N2
Cu–O7
N1–Cu–N4
N3–Cu–N4
1.976(4)
2.600(4)
177.4(2)
96.9(2)
163.6(1)
95.0(2)
(3H)·2ClO4
Cu1–O1
Cu1–N2
N1–Cu1–N2
N2–Cu1–N3
Cu2–O3
Cu2–N6
N5–Cu2–N6
N6–Cu2–N7
2.415(5)
1.981(4)
83.8(2)
165.9(2)
2.355(4)
2.002(4)
83.7(2)
Cu1–O8
Cu1–N3
N1–Cu1–N3
N2–Cu1–N4
Cu2–O17
Cu2–N7
N5–Cu2–N7
N6–Cu2–N8
2.631(5)
2.016(4)
82.8(2)
Cu1–N1
Cu1–N4
N1–Cu1–N4
N3–Cu1–N4
Cu2–N5
Cu2–N8
N5–Cu2–N8
N7–Cu2–N8
2.014(4)
1.980(5)
166.2(2)
100.0(2)
2.005(5)
1.978(5)
164.7(2)
102.5(2)
92.0(2)
2.631(5)
2.010(4)
82.2(2)
165.7(2)
90.6(2)
(4H)·2ClO4
Cu1–O1A
Cu1–N2A
N2A–Cu1–N1A
N2A–Cu1–N3A
Cu2–O1B
Cu2–N2B
N2B–Cu2–N1B
N2B–Cu2–N3B
2.363(3)
2.002(4)
83.86(16)
165.20(14)
2.637(4)
1.976(4)
84.67(15)
166.55(14)
Cu1–O11A
Cu1–N3A
N1A–Cu1–N3A
N5A–Cu1–N2A
Cu2–O21B
Cu2–N3B
N3B–Cu2–N1B
N5B–Cu2–N2B
2.615(4)
2.038(4)
82.24(16)
91.85(16)
2.650(4)
1.985(4)
81.90(14)
93.79(17)
Cu1–N1A
Cu1–N5A
N5A–Cu1–N1A
N5A–Cu1–N3A
Cu2–N1B
Cu2–N5B
N5B–Cu2–N1B
N5B–Cu2–N3B
2.008(4)
1.996(4)
169.83(17)
101.03(16)
1.994(3)
1.972(4)
166.45(17)
99.46(16)
(4)·ClO4
Cu–O1
Cu–N3
O1–Cu–N2
N1–Cu–N2
N2–Cu–N3
1.905(1)
2.229(1)
161.74(5)
83.53(6)
Cu–N1
Cu–N4
O1–Cu–N3
N1–Cu–N3
N2–Cu–N4
2.053(1)
2.015(2)
93.18(5)
82.62(5)
94.82(6)
Cu–N2
1.965(1)
94.27(5)
83.78(6)
168.67(5)
108.60(5)
O1–Cu–N1
O1–Cu–N4
N1–Cu–N4
N3–Cu–N4
104.46(5)
For (1H)2+, the Cu–Ntertiary amine bond distance Cu–N1 is
atoms from an exogenous acetonitrile, the pyridine, the quinoline
and the tertiary amine.
˚
2.026(2) A, while the Cu–Npyridine bond distances Cu–N2 and Cu–
˚
N3 are 1.992(2) and 1.980(2) A, respectively. They are within
In the two independent complexes present in the crystal cell of
the range of those reported for (2H)2+ (2.008(4), 1.976(4) A and
1.975(3) A for Cu–N1, Cu–N2 and Cu–N3, respectively) and
some other related complexes.3–10
(3H)2+ the Cu–Npyridine bond distances Cu1–N2 and Cu2–N6 are
˚
6
˚
˚
1.981(4) and 2.002(4) A, the Cu–Nquinoline bond distances Cu1–N3
˚
and Cu2–N7 are 2.016(4) and 2.010(4) A, while the Cu–Ntertiary
˚
The Cu–Ophenol bond distances are 2.353(2) and 2.404(3) A for
amine bond distances Cu1–N1 and Cu2–N5 are 2.014(4) and
(1H)2+ and (2H)2+, respectively. These values are much higher
than those reported for axial Cu–Ophenolate bonds (typically around
2.2 A),7–10 thus showing that the phenolic unit remains only weakly
˚
2.005(5) A. The corresponding bond distances in the previously
˚
described complex (4H)2+ were found to be roughly similar, with
˚
˚
Cu1–N2A = 2.002(4) A and Cu2–N2B = 1.976(4) A for the Cu–
˚
˚
coordinated to the metal center. In both complexes, the Cu–
Npyridine bonds, Cu1–N3A = 2.038(4) A and Cu2–N3B = 1.985(4) A
˚
˚
for the Cu–Nquinoline bonds, and Cu1–N1A 2.008(4) A and Cu2–
Operchlorate bond distance is much longer (2.526(2) and 2.600(4) A for
(1H)2+ and (2H)2+, respectively), in agreement with an even weaker
N1B 1.993(3) A for the Cu–Ntertiary amine bonds.7 The increase in
˚
Cu–Operchlorate bond. The phenol unit is found to be p-stacked over
bond length observed for the Cu–Nquinoline bond compared to the
Cu–Npyridine one is ascribed to steric hindrances around the nitrogen
atom of the quinoline that consequently weakens the Cu–N bond.
Similar increases in bond lengths have been reported in copper(II)
complexes of tripodal ligands when pyridines are a-methylated.9
one pyridine ring with a distance between the two centroids of
2+
4.04 and 4.09 A for (1H) and (2H)2+, respectively.
˚
In both (3H)2+ and (4H)2+, the crystal cell contains two
independent complexes. The geometry around the metal center is
close to that observed in (1H)2+ and (2H)2+, i.e. octahedral with the
axial positions occupied by one phenol oxygen and one perchlorate
oxygen atom. The equatorial positions are occupied by nitrogen
The Cu–Ophenol bond distances in (3H)2+ are Cu1–O1 = 2.415(5) A
˚
˚
and Cu2–O3 = 2.355(4) A, whereas the Cu–Operchlorate bond lengths
˚
˚
Cu1–O8 and Cu2–O17 are longer (2.631(5) A and 2.631(5) A,
834 | Dalton Trans., 2009, 832–842
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respectively). This trend is similar to that observed in (1H)2+,
(2H)2+ and (4H)2+. As in these latter complexes, the phenol is
Table 3 Electronic properties of the copper(II) complexes
Complex
UV-vis [lmax/nm (e/M-1 cm-1)]a
˚
p-stacked over the pyridine ring at a distance of 3.85 and 3.99 A
(between the two centroids).
(1H)2+
(1)+
605 (85)
In (4)+, the geometry was found to be quite different:7 the
copper(II) ion resides within a square pyramidal geometry. The
quinoline nitrogen binds in the axial position, while the phenolate
moiety, an exogenous acetonitrile, the pyridine and the tertiary
amine nitrogen atoms occupy the equatorial positions. The Cu–
Ophenolate bond is stronger than in (4H)2+ as reflected by a shorter
500 (880), 930 (130)
(2H)2+
(2)+
550 (240)b
535 (920), 930 (130)b
617 (115)
(3H)2+
(3)+
560 (1330), 660 (700) sh
(4H)2+
(4)+
630 (120)c
510 (910), 654 (260)c
˚
a CH3CN solutions, T = 298 K, sh = shoulder. b From ref. 6. c From
Cu–O bond distance (1.905(1) A). In addition, it is the phenolate
ref. 7.
and the quinoline rings that are involved in intramolecular p-
stacking interactions (and not the phenol with one pyridine unit).
In the CuII-phenol complexes (1H)2+, (2H)2+, (3H)2+ and (4H)2+,
the Cu ◊ ◊ ◊ OH(Ar) bond is weak and consequently the phenol
systematically occupies the more labile, i.e. axial, position. Weak
interactions between the copper ion and one perchlorate molecule
(as a sixth ligand) could also be detected at the solid state. In the
CuII-phenolate complex (4)+, the Cu ◊ ◊ ◊ O-(Ar) bond is stronger,
as reflected by the shorter Cu–O bond distance, and no more
interaction with the perchlorate anion exists. Some X-ray crystal
structures of CuII-phenolate complexes built from N3O ligands de-
rived from the bis(pyridylmethyl)amine or bis(pyridylethyl)amine
have been reported.7–9 In the bis(pyridylmethyl)amine derivatives,
the phenolate occupies the axial position, except when one (or
both) pyridine(s) is(are) a-methylated;9 in this particular case, the
X-ray crystal structure analysis reveals an equatorial positioning
of the phenolate. Steric hindrances around the quinoline nitrogen
of (4)+ (as in a-methylpyridines), as well as the p-stacking
interactions between the phenolate and quinoline rings (see below)
may contribute to the isomerization.
Fig. 3 UV-vis spectra of 0.2 mM CH3CN solutions of (1)+, (2)+, (3)+ and
(4)+. l = 1.0 cm, T = 298 K.
Spectroscopic and electrochemical properties of the
copper complexes
The UV-vis spectra of the phenol complexes (1H)2+, (2H)2+,
(3H)2+ and (4H)2+ in CH3CN are characterized by copper(II)
d–d transitions at around 600 nm, in agreement with a square
planar, or elongated octahedral geometry, around the metal. The
addition of NEt3 to these complexes results in the appearance of a
phenolate-to-copper CT transition as expected for deprotonation
of the phenolic moiety.6 The so-obtained deprotonated species
(1)+, (2)+, (3)+ and (4)+ exhibit a CT transition at 500, 535, 560
and 510 nm, respectively (Fig. 3, Table 3). In each sub-series
(ligands bearing either two pyridines or one pyridine and one
quinoline), the lmax is shifted according to the electronic properties
of the phenolate substituent, with the highest lmax observed for
the electron-donating methoxyl group and the lowest for the
electron-withdrawing nitro and trifluoromethyl groups.6 In their
EPR spectra all the complexes exhibit a (S = 1/2) signal with
copper(II) hyperfine structure, indicating that the mononuclear
structures are preserved in CH3CN (Fig. 4).
Fig. 4 X-Band EPR spectra of a 1 mM CH3CN (+0.1 M TBAP)
solution of (a) (3)+ and (b) the electrogenerated (3∑)2+. Microwave frequency
9.419 GHz, power 20 mW, modulation frequency 100 kHz, amplitude
0.197 mT, T = 100 K. The same scale is used along the y axis for both
complexes. No signal was detected below 250 mT in the case of (3)+.
into methoxyphenoxyl radicals,6,12 further confirming that the
redox process corresponds to the oxidation of the phenolate into
a phenoxyl radical. The signal was found to be reversible or
quasi-reversible for (1)+, (2)+ and (3)+, but not for (4)+. This is
not surprising as the stability of phenoxyl radicals substituted by
electron-withdrawing groups is expected to be low.6
The phenolate complexes (1)+, (2)+, (3)+ and (4)+ exhibit in
their CV curves a one-electron oxidation wave centered at E1/2
=
0.56, 0.20, 0.09 and 0.83 V vs. Fc/Fc+, respectively. These values
+
correlate with the s Hammet of the phenolate para-substituent,
suggesting that the ligand is involved in the oxidation process. In
addition, the value of 0.09 V for (3)+ is close to those reported
for the oxidation of copper(II) coordinated methoxyphenolates
The electrochemical behaviour of the complexes has been
studied by cyclic voltammetry in CH3CN at 298 K (in the presence
of 0.1 M TBAP as supporting electrolyte).
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The phenol complexes (1H)2+, (2H)2+, (3H)2+ and (4H)2+ exhibit,
in the anodic region of potentials, an irreversible signal at values
higher than 0.8 V vs. Fc/Fc+ attributed to the primary oxidation of
the phenol moiety into a phenoxyl radical cation that decomposes
irreversibly.11
Electrolysis of the phenolate complexes was performed at 233 K
in CH3CN under an inert atmosphere. Under these conditions
only the one-electron oxidized species (3∑)2+ could be generated
(the other oxidized species were found to be too unstable). Its UV-
vis spectrum is characterized by an intense transition at 425 nm
(2940 M-1 cm-1) and a lower intensity one at 545 nm (690 M-1 cm-1)
(Fig. 5). These bands are similar to those reported for the CuII-
∑
phenoxyl radical complex [CuII(LOMe )(CH3CN)]2+ (lmax = 426
and 550 nm)6 supporting the fact that (3∑)2+ has a CuII-phenoxyl
radical character. They are attributed to the p–p* transitions of the
phenoxyl radical.13 In addition, the 100 K EPR spectrum of (3∑)2+
exhibits a signal at 150 mT corresponding to a forbidden DMS =
2 transition as well as broader signals in the higher field region
corresponding to DMS = 1 transitions (Fig. 4). This shows that
the copper(II) spin and the ligand radical spin are magnetically
interacting in (3∑)2+, and thus that the phenoxyl radical remains
coordinated to the metal center. (3∑)2+ was found to be relatively
stable at 298 K as it decomposes in a first order process with kdec
=
0.013 min-1 (t1/2 = 53 min). Its stability is somewhat lower than
∑
that of [Cu(LOMe )(CH3CN)]2+ (kdec = 0.008 min-1, t1/2 = 87 min at
298 K)6 showing that the N-donor ability of the ligand influences
the stability of the CuII-phenoxyl radical species.9
Fig. 6 Titration of 60 mM solution of (a) (1H)+ and (b) (2H)+ with NEt3.
NMR spectra recorded in (CD3CN–CH3CN) (1 : 4) at 298 K. The numbers
correspond to the molar equivalents of copper added and the spectra are
normalized.
Fig. 5 UV-vis spectrum of a 0.2 mM CH3CN solution (+0.1 M TBAP)
of the electrogenerated (3∑)2+. l = 1.0 cm, T = 233 K. The insert represents
DA at 425 nm during decomposition of a 0.12 mM CH3CN solution of the
electrogenerated (3∑)2+ at 298 K.
Table 4 19F chemical shifts of the ligands and complexes
Species
d/ppma
HLCF3
(1H)2+
(1)+
100.6
99.6 (16)
125 (3000)
34.0
41.9 (23)
> 60 (n.d.)
46.2
19F NMR characterization of the copper complexes
HLF
(2H)2+
(2)+
The 19F NMR spectra of (1H)2+ and (2H)2+ in CD3CN–CH3CN
(1 : 4) are shown in Fig 6 and the chemical shifts are given in
HLqOMe
(3H)2+
(3)+
Table 4 (relative to C6F6 used as an external reference, dC6
=
F
53.6 (345)
49.0 (54)
46.0b
6
-162.17 ppm vs. CFCl3). (1H)2+ and (2H)2+ show single 19F
HLqNO2
(4H)2+
(4)+
resonances at 99.6 ppm (W1/2 = 16 Hz) and 41.9 ppm (W1/2
=
53.7 (250)b
49.0 (25)b
23 Hz), respectively. For comparison, the free ligands HLCF3 and
HLF exhibit 19F resonances at 100.6 and 34.0 ppm, respectively.
The chemical shift is thus highly sensitive to the presence of
metal and thus to complexation. The addition of NEt3 to (1H)2+
and (2H)2+ induces both a downfield shift of the 19F signal and
a
d
given relative to C6F6 used as an external reference (dC
=
6 F6
-162.17 ppm vs. CFCl3); W1/2 (Hz) is indicated for the copper(II)
complexes. b From ref. 7.
836 | Dalton Trans., 2009, 832–842
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dramatic line broadening. This indicates that the CuII-phenol and
CuII-phenolate complexes are in fast equilibrium at 298 K. For
(1H)2+, the 19F resonance could be distinguished up to one molar
one molar equivalent of NEt3 is added, the signal becomes much
sharper (d = 49.0 ppm, W1/2 = 25 Hz), thus showing that the
(4H)2+ and (4)+ species are in fast equilibrium at 298 K. A more
complicated behaviour was observed upon addition of NEt3 to
(3H)2+; a progressive disappearance and broadening of the (3H)2+
signal is observed, concomitant with the appearance of a new
broad signal originating from (3)+ that increases in intensity. When
exactly one molar equivalent of NEt3 is added, the signal becomes
much sharper, as observed for (4)+. This behaviour is interpreted
by the fact that 298 K roughly corresponds to the coalescence
temperature in the (3H)2+ and (3)+ mixture.
The phenolate complexes (3)+ and (4)+ thus exhibit a 19F NMR
signal that is significantly upfield shifted and much narrower than
in the corresponding phenol species. This behaviour contrasts
sharply with that of (1)+ and (2)+. In these latter cases, the signal
observed for the phenolate complexes was found to be much
broader and downfield shifted when compared to the phenol
species.
equivalent of NEt3 added. At this amount of NEt3 added, the 19
F
resonance is observed at dC ª 125 ppm with a line width of ca.
F
6
6
W1/2 = 3000 Hz. For (2H)2+, the signal becomes barely observable
at 0.3 molar equivalents of NEt3 added, while it could not be
observed above when 0.4 molar equivalents were added.
The sharper signal observed for (1)+ compared to (2)+ could be
interpreted in terms of an increase in the fluorine–metal distance.
The extra methylene group between the Cu and F atoms in
(1)+ makes the fluorine nucleus less sensitive to the copper(II)
paramagnetism, resulting in a sharpening of the 19F resonance.
Spin rotation is also expected to contribute to the line width in a
lesser amount.
The 19F resonance was also found to be much sharper in the
phenol than in the phenolate complexes. Deprotonation induces
a contraction of the Cu–Ophenol bond distance, and subsequent
shortening of the fluorine–metal distance. In addition, a better
electronic communication (through the p-system) exists between
the copper and the fluorine atoms in the phenolate complexes.
The fluorine atom thus becomes more sensitive to the copper
paramagnetism in the phenolate complexes, inducing a dramatic
broadening of the 19F resonance.
As shown above, deprotonation induces a contraction of the
Cu–Ophenol bond. When the fluorine atom is incorporated on
the phenol group, as in (1H)2+ and (2H)2+, the Cu–F distance
varies concomitantly with the Cu–Ophenol bond and the fluorine
atom becomes more sensitive to the Cu2+ paramagnetism in the
deprotonated complex.
Similar experiments have been undertaken on the phenol
complexes (3H)2+ and (4H)2+. They exhibit single 19F resonances
at 53.6 ppm (W1/2 = 345 Hz) and 53.7 ppm (W1/2 = 250 Hz),
respectively, higher values than those of the free ligands (46.0 and
46.2 ppm for HLqOMe and HLqNO2, respectively). One can note that
the chemical shifts are poorly influenced by the nature of the phe-
nol para-substituent, which is not surprising as there is no signif-
icant communication between the phenol and the quinoline ring.
Upon addition of NEt3 to (4H)2+, a broadening as well as an
upfield shift of the 19F signal is observed (Fig. 7). When exactly
In (4H)2+, the X-ray structural analysis has shown that de-
protonation is followed by a geometrical rearrangement; the
phenolate shifts from the axial to the equatorial position, resulting
in a shortening of the Cu–O bond. Simultaneously, the 6-
fluoroquinoline moiety moves from the equatorial to the axial
position. Axial bonds are known to be larger than equatorial ones;
the enlargement of the Cu–Nquinoline, and consequently Cu–F, bond
distances in (4)+ makes the fluorine atom less sensitive to the Cu2+
paramagnetism in the presence of base. The 19F NMR resonance
is then sharper in (4)+ compared to (4H)2+.
Although the CuII-phenolate species (3)+ could not be crystal-
lized, the close similarity in the 19F NMR spectra of (3)+ and (4)+
suggests an axial positioning of the quinoline group in the latter
complex.14
Interconversion between (4)+ and (4H)2+
As shown above, protonation of (4)+ into (4H)2+ is accompanied by
a molecular rearrangement. In (4H)2+, the quinoline occupies the
equatorial position, and the phenol weakly coordinates in the axial
position, whereas in (4)+, the quinoline moiety occupies an axial
position, and the phenolate coordinates in an equatorial position.
In order to get dynamic informations on the interconversion, we
have recorded 19F NMR spectra of equimolar mixtures of the CuII-
phenol and CuII-phenolate complexes of HLqNO2 and HLqOMe in
(CD3CN–C2H5CN) (1 : 4) as a function of the temperature.
For the (4)+ + (4H)2+ mixture, a broad resonance is observed
down to 226 K, while at lower temperatures, the spectra consists of
two distinct resonances at 49.0 and 53.7 ppm (Fig. 8). These signals
are attributed to the (4)+ and (4H)2+ complexes that are in slow
equilibrium at low temperature. At the coalescence temperature
TC = 226 K, according to eqn (1), a rate constant k = 3000
100 s-1 could be calculated for the equilibrium in eqn (2).7,15
This rate constant is large, indicating that proton transfer and/or
molecular re-arrangement (isomerization) is extremely rapid.
Fig. 7 Titration of (4H)2+ (60 mM) with NEt3; 19F NMR spectra were
recorded in (CD3CN–CH3CN) (1 : 4) at 293 K. The numbers correspond
to the molar equivalents of base added and the intensities are normalized.
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Fig. 9 Variable temperature 19F NMR spectra of a (1 : 1) mixture of (3)+ +
(3H)2+ (total = 20 mM) recorded in (CD3CN–C2H5CN) (1 : 4); intensities
are normalized, temperature (in K) indicated at the right. The stars denote
unidentified diamagnetic impurities that appear upon an increase in the
temperature (the temperature was lowered to 265 K and then increased up
to 349 K during the experiment).
Fig. 8 Variable temperature 19F NMR spectra of a (1 : 1) mixture of
(4)+ + (4H)2+ (total = 2 mM) recorded in (CD3CN–C2H5CN) (1 : 4);
intensities are normalized, temperature (in K) is indicated at the right.
p ¥ Dn
k =
(1)
(2)
21
2
(4)+ + (4H)2+ ꢀ (4H)+ + (4)2+
A
B
A
B
(A and B represent two distinct complexes that interconvert.)
When the (4)+ + (4H)2+ mixture was replaced by the (3)+ + (3H)2+
mixture, a similar behaviour was observed but the coalescence
temperature was found to be much higher. A significant decom-
position is observed at high temperatures (sharp peaks denoted
by stars on Fig. 9), thus precluding any accurate determination
of TC. Nevertheless, 330 K represents a reasonable approximation
for the coalescence temperature. At this temperature, according
to eqn (1), the rate constant for the equilibrium in eqn (2) was
estimated to be 2900
100 s-1. Although it is found to be
roughly similar to that obtained for the (4)+ + (4H)2+ mixture,
the temperature at which it is calculated is much higher. This
suggests that proton transfer and/or molecular re-arrangement
is slower in the mixture (3)+ + (3H)2+, what may be ascribed
to the increased basicity of a methoxyphenol compared to a
nitrophenol.
The availability of crystal structures of both the (4)+ and
(4H)2+ species makes (4)+ the ideal candidate to study the acid-
promoted isomerization by DFT calculations (Fig. 10 and 11).16
The crystal structure of (4)+ has been optimized and a proton
has been added to the equatorial phenolate oxygen (structure A,
Fig. 11). The system spontaneously evolves towards a complex
involving an axial phenol in a multi-step process (Fig. 10). All
the steps were shown to have favourable reaction energies and
low activation barriers, in agreement with the high rate constant
obtained experimentally.
Fig. 10 Calculated B3LYP potential energy surface for the isomerization
of (4H)2+ at 0 K. The barriers are given by taking in account the zero point
energy.
an increase in the Cu–O bond length and subsequent rearragement
of the three arms around the metal center. An optimized structure
for the transition state (structure B) is shown in Fig. 11. It does not
exhibit any p-stacking interactions between any of the aromatic
rings. The t value that represents the degree of distortion of
a square pyramidal towards a trigonal bipyramidal geometry
(0 corresponds to a perfect square pyramidal geometry, 1 a perfect
trigonal bipyramidal geometry)17 could be calculated to 0.71 for
B, while it was only 0.09 for A. The geometry around the metal
center is thus found to be best defined as trigonal bipyramidal in
B, whereas it is essentially square planar in A. The Cu–Ophenol bond
˚
The first step (limiting step) occurs with a low barrier of 2.4 kcal
mol-1 and is found to be exothermic by 6.6 kcal mol-1. It involves
was found to be 2.27 A, while one of the hydrogen atoms of the
pyridine a-methylene group interacts with the phenol oxygen at a
838 | Dalton Trans., 2009, 832–842
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process exothermic by 3.2 kcal mol-1. Tyr495 was found to dissociate
upon protonation, as reflected by a Cu–OTyr bond distance of
˚
2.86 A, a value higher than that obtained in the X-ray crystal
˚
structure of GO (2.69 A). In both the enzyme and our system
the protonation process is thus found to be extremely rapid, even
when isomerization occurs. In addition, these works highlight the
important role of the link between one N-donor group and the
phenol in order to maintain the Ophenol atom in the close vicinity
of the copper ion.
Chemical generation and detection of a CuII-phenoxyl radical
species by 19F NMR
Upon addition of zero to one molar equivalent of copper(II) to
(3)+ at 238 K the violet CH3CN solution turns dark green.18 The
absorbance at 560 nm progressively decreases, while new peaks
appear at 425 and 550 nm. When exactly one molar equivalent
is added, the spectrum was found to be identical to that of the
electrogenerated (3∑)2+ species. A CuII-phenoxyl radical species is
thus formed according to eqn (3):20
(3)+ + CuII → (3∑)2+ + CuI
(3)
19F NMR (Fig. 12) reveals that, upon addition of copper(II), a
new resonance progressively increases in intensity at 53.7 ppm at
the expense of the original signal at 49.0 ppm (corresponding to
(3)+). When one molar equivalent is added, the spectrum exhibits a
single resonance at 53.7 ppm. This signal is attributed to the (3∑)2+
radical complex that is in slow equilibrium with (3)+ at 238 K. As
the solution at the end of the titration may contain paramagnetic
copper(II) impurities, further discussions concerning the difference
in linewidth between (3)+ and (3∑)2+ is not possible.
Fig. 11 B3LYP optimized structures for species A–E. The values indi-
cated represent the distances between: (A) the centroids of the quinoline
and phenol rings; (B–D) one hydrogen of the pyridine a-methylene group
and the phenol oxygen; (D–E) the centroids of the pyridine and phenol
rings.
˚
Ophenol–Hmethylene distance of 2.61 A. As a consequence, free rotation
of the pyridine ring is severely perturbed.
The second step occurs with a very small barrier (less than
0.1 kcal mol-1) if there is one. It involves stretching of the Ophenolate
–
Hmethylene bond that allows rotation of the pyridine ring. The
transition state D exhibits Ophenolate–Hmethylene and Cu–Ophenol bond
˚
distances of 3.90 and 2.27 A, respectively, while the metal ion
adopts a geometry that is intermediate between square pyramidal
and trigonal bipyramidal (t = 0.31). In the final species E (Fig. 11),
the pyridine has slightly moved and the Cu–Ophenol bond distance
˚
stretches to 2.36 A. This value is in perfect agreement with that
˚
of 2.363(3) A obtained experimentally from the X-ray structural
analysis. The geometry around the metal ion of E is best described
as square pyramidal (t = 0.15) with the phenol ring located at
the axial position. In addition, p-stacking interactions could be
detected between the pyridine and the phenol ring, in agreement
with the X-ray crystal structure of (4H)2+. Although no perchlorate
ion was included in our calculations, the ligand positioning in E is
in very good agreement with that found in the crystal structure of
(4H)2+, confirming the validity of our method.
These results show that stretching of the Cu–O bond, p-stacking
interactions and rotation of the pyridine are key steps in the
isomerization process. It is interesting to compare these results
with those obtained by Siegbahn et al. concerning the protonation
step of Tyr495 of GO after substrate binding.19 Prior to proton
transfer (coordinated tyrosinate and bound alcohol) a minimum
could be found in the potential surface energy only for models
incorporating the peptidic link between Tyr495 and His496 (not for
the models in which the amino acid residues are not linked).
In this case, the barrier for the protonation of the tyrosinate
residue was found to be small, lower than 3 kcal mol-1, and the
Fig. 12 Titration of (3)+ (80 mM) with copper(II) perchlorate. NMR
spectra recorded in (CD3CN–CH3CN) (1 : 4) at 238 K. The values
correspond to the molar equivalents of copper added and the spectra
are normalized.
A remarkable feature is that (3)+ and (3∑)2+ exhibit distinct
chemical shifts although the Cu–Nquinoline bond distances are not
expected to be significantly different. In fact, (3)+ and (3∑)2+ exhibit
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a different spin state, (S = 1/2) for the former and (S = 1) for the
latter (see above). The fluorine atom is thus dramatically influenced
by changes in the spin state of the complex, and not in the Cu–
F distance. This makes 19F NMR also an efficient probe for the
phenolate oxidation state.
UV-vis spectroscopy
UV-vis spectra were recorded on a Perkin Elmer Lambda 2
spectrophotometer equipped with a temperature controller unit
set at 298 K. The quartz cell path length is 1.0 cm. Low temperature
visible spectra were recorded on an UVIKON spectrophotometer
equipped with a quartz Dewar (H.S. Martin Inc.) containing a
1.0 cm path length quartz cell. The rate constant for the self-
decomposition of the radical species was obtained spectrophoto-
metrically: the absorbance decay at 425 nm was fitted using the
Biokine (Bio Logic Co., Claix, France) software.
Conclusions
Key reactions that take place at the protein ligand during the GO
catalytic cycle are proton transfer from the substrate to a tyrosinate
residue and tyrosyl radical reduction. We have addressed here the
basics concerning the use of 19F NMR to monitor such phenomena
in copper(II) complexes that model the enzyme active site.
The fluorine atom has been incorporated in N3O tripodal
ligands in two different ways: either at the phenol as a para
substituent (HLCF3 and HLF) or by replacing the pyridine group
by a 6-fluoroquinoline (HLqOMe and HLqNO2). The copper(II)
complexes of each ligand were characterized on their phenol and
phenolate protonation states. Deprotonation induces contraction
of the Cu–Ophenol bond and subsequent changes in copper–fluorine
distance that could be easily vizualized by 19F NMR. As the
copper–fluorine distance decreases in (1H)2+ and (2H)2+, the 19F
resonance broadens (the fluorine atom is more sensitive to the
metal paramagnetism). In contrast, it increases in (3H)2+ and
(4H)2+ making the 19F resonance sharper (the fluorine atom is
less sensitive to the metal paramagnetism).
Protonation of (4)+ was found to be accompanied by a rapid
equatorial-to-axial isomerization of the phenol(ate) group. DFT
calculations show that this process occurs with a low barrier, and
that stretching of the Cu–O bond, p-stacking interactions and
rotation of the pyridine are key steps in the isomerization process.
In order to extend this study, 19F NMR spectroscopy has been
used to detect phenoxyl radicals. For this purpose, the phenol
para-substituent has to be strongly electron-donating. (3)+ has
been synthesized and successfully oxidized into a stable phenoxyl
radical. We present herein the first 19F NMR evidences for the
formation of a phenoxyl radical.
EPR
X-Band EPR spectra were recorded on a BRUKER ESP 300E
spectrometer equipped with a BRUKER nitrogen flow cryostat.
The spectra were processed using the WINEPR software and
simulated using the BRUKER SIMFONIA software.
Electrochemistry
The cyclic voltammograms of each compound (1 mM) in CH3CN
containing tetra-n-butyl ammonium perchlorate (TBAP) 0.1 M
as the supporting electrolyte, were recorded on a CH Instrument
660 potentiostat at 298 K, under an Ar atmosphere, using a glassy
carbon disc as the working electrode (3 mm diameter), a Pt wire as
the secondary electrode, and an Ag/AgNO3 0.01 M as the refer-
ence electrode. The potential of the regular ferrocene/ferrocenium
(Fc/Fc+) used as an internal reference is +0.087 V under our
experimental conditions. Electrolysis at a carbon felt electrode
(10 ¥ 10 ¥ 10 mm) was performed at 233 K, under an Ar
atmosphere, using a PAR 273 potentiostat.
Crystal structure analysis
-
-
For the structures of (1H)2+·2ClO4 and (3H)2+·2ClO4 , collected
reflections were corrected for Lorentz and polarization effects but
not for absorption. The structure was solved by direct methods and
refined using the TEXSAN software.22 All non-hydrogen atoms
were refined with anisotropic thermal parameters. Hydrogen
atoms were generated in idealized positions, riding on the carrier
atoms, with isotropic thermal parameters.
The 19F NMR technique is thus a very powerful tool that gives
access to unprecedented information. Depending on the finality
(probe for phenoxyl radical formation or highly sensitive probe
for phenol deprotonation), the judicious labelling is either that
realized on the N-donor or the O-donor.
One may envisage to use this elegant tool to study substrate
binding on model compounds or the enzymes directly.21 In
addition, the 19F signal of (2H)+ was found to be extremely sensitive
to NEt3. This property could be potentially useful to detect traces
of bases in organic media by 19F NMR.
DFT calculations
All calculations were performed using the Gaussian 03 program16
at the B3LYP level of theory and the LANL2DZ ECP basis set
for Ni, C and H and LANL2DZdp ECP basis set for N and O.
Synthesis
Experimental
2-Tert-butyl-6-{[(6-fluoro-quinolin-2-ylmethyl)-pyridin-2-ylmethyl-
General
amino]-methyl}-4-methoxyphenol
(HLqOMe). 3-Tert-butyl-2-
All chemicals were of reagent grade and used without purification.
NMR spectra were recorded on a Bruker AM 300 (1H at 300 Mhz,
13C at 75 Mhz and 19F at 282 MHz). Chemical shifts are given
relative to tetramethylsilane (TMS). C6F6 was used as an internal
reference for 19F NMR experiments (dC
CFCl3). Mass spectra were recorded on a Thermofinningen
hydroxy-5-methoxybenzaldehyde (208 mg, 1 mmol), 6-fluoro-
quinolin-ylmethyl)-pyridin-2-ylmethylamine (267 mg, 1 mmol)
and 0.1 mL glacial acetic acid were dissolved in methanol
(10 mL). Sodium cyanoborohydride (78 mg, 1.3 mmol) was
added by small amounts, during 8 h. After 12 h stirring, the
reaction mixture was neutralized by HCl (2 M), extracted with
CH2Cl2 (2 ¥ 50 mL), washed with saturated NaCl, dried over
= -162.17 ppm vs.
F
6
6
(EI/DCI) or a Nermag R 101 C (FAB+) apparatus.
840 | Dalton Trans., 2009, 832–842
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(Cq), 123.7 (CH), 123.8 (q, CH, 3J(C,F) = 4.2 Hz), 125.2 (q, Cq,
1J(C,F) = 271.3 Hz), 125.5 (q, CH, 3J(C,F) = 3.8 Hz), 137.1 (CH),
137.6 (Cq), 149.4 (CH), 158.1 (Cq), 159.9 (Cq). MS (DCI, NH3
isobutane), m/z, 428 (M + H)+. Anal. Calcd for C24H26F3N3O: C
67.12, H 6.10, N 9.78, F 13.37%. Found: C 65.02, H 6.02, N 9.33,
F 13.27%.
Na2SO4 and evaporated. 2-Tert-butyl-6-{[(6-fluoro-quinolin-2-
ylmethyl)-pyridin-2-ylmethyl-amino]-methyl}-4-methoxyphenol
was purified by silica gel column chromatography with CH2Cl2–
MeOH (30 : 1) as eluent. 2-Tert-butyl-6-{[(6-fluoro-quinolin-2-
ylmethyl)-pyridin-2-ylmethyl-amino]-methyl}-4-methoxyphenol
(275 mg, 60%) is obtained as a deep brown oil. 1H NMR
(300.12 MHz, CDCl3, 298 K, TMS) d/ppm: 1.50 (s, 9 H), 3.74 (s,
3 H), 3.84 (s, 2 H), 3.89 (s, 2 H), 4.03 (s, 2 H), 6.52 (d, 4J(H,H) =
3.1 Hz), 6.82 (d, 4J(H,H) = 3.1 Hz), 7.11 (dd, 3J(H,H) = 7.4 Hz,
7.34–7.41 (m, 3 H), 7.47 (ddd, 1 H, 3J(H,F) = 9.0 Hz, 3J(H,H) =
(1H)2+·2ClO4 . To a CH3CN solution (5 mL) of 2-tert-butyl-6-
-
{[bispyridin-2-ylmethyl-amino]-methyl}-4-trifluoromethylphenol
(190 mg, 0.443 mmol),
a CH3CN solution (1 mL) of
Cu(ClO4)2·6H2O (164 mg, 0.528 mmol) was added dropwise.
4
3
8.5 Hz, J(H,H) = 2.8 Hz), 7.56 (td, 1 H, J(H,H) = 7.7 Hz,
(1H)2+·2ClO4 was recrystallized by slow diffusion of Et2O into
-
3
4J(H,H) = 1.8 Hz), 8.01 (d, J(H,H) = 8.6 Hz), 8.19 (dd, 1 H,
the CH3CN solution. Blue single crystals of (1H)2+·2ClO4 were
-
4
3
3J(H,H) = 9.2 Hz, J(H,H) = 5.4 Hz), 8.52 (d, J(H,H) = 4.9
Hz). 13C NMR (75.465 MHz, CDCl3, 298 K, TMS) d/ppm: 30.1
(CH3, tBu), 35.4 (Cq), 53.8 (CH2), 56.1 (OCH3), 58.5 (CH2), 59.8
(CH2), 60.2 (CH2), 110.9 (d, CH, 2J(C,F) = 21.8 Hz), 112.9 (CH),
113.6 (CH), 120.0 (d, CH, 2J(C,F) = 25.6 Hz), 122.0 (CH), 122.6
(CH), 123.6 (Cq), 123.9 (CH), 128.2 (d, Cq, 3J(C,F) = 10.4 Hz),
131.7 (d, CH, 3J(C,F) = 9.3 Hz), 136.3 (d, CH, 4J(C,F) = 5.2 Hz),
137.0 (CH), 138.3 (Cq), 144.9 (Cq), 149.2 (CH), 150.7 (Cq), 151.0
collected by filtration (yield 65%). ESI MS, m/z: 491 (M - H -
CH3CN - 2ClO4)+. Anal. Calcd for C26H29Cl2CuFN4O9: C 42.60,
H 3.99, N 7.64, Cu 8.67%. Found: C 42.78, H 4.05, N 7.61, Cu
8.80%. UV-vis in CH3CN at 298 K (lmax/nm (e/M-1 cm-1)): 605
(85). EPR (9.41 GHz, CH3CN, 100 K): gxx = gyy = 2.054, gzz
=
2.240, Axx = Ayy = 2 mT, Azz = 18.2 mT.
(3H)2+·2ClO4 . 2-Tert-butyl-6-{[(6-fluoro-quinolin-2-ylmethyl)-
pyridin-2-ylmethyl-amino]-methyl}-4-methoxyphenol (100 mg,
0.217 mmol) and Cu(ClO4)2·6H2O (80 mg, 0.217 mmol) were
-
4
(Cq), 158.4 (d, Cq, J(C,F) = 2.8 Hz), 158.7 (Cq), 160.7 (d, Cq,
1J(C,F) = 247.7 Hz). MS (DCI, NH3 isobutane), m/z, 459 (M +
H)+. Anal. Calcd for C38H30FN3O2: C 66.07, H 6.08, N 8.04%.
Found: C 66.08, H 6.10, N 8.41%.
-
dissolved in CH3CN (4 mL). (3H)2+·2ClO4 was recrystallized
by slow diffusion of Et2O into the CH3CN solution. Blue single
crystals of (3H)2+·2ClO4 were collected by filtration (100 mg,
-
2-Tert-butyl-4-trifluoromethylphenol. 4-Trifluoromethylphenol
(1 g, 6.16 mmol) was dissolved in CH3OH (1 mL) and tert-butanol
(5 mL, 50 mmol) was added at room temperature. Sulfuric acid
(92%, 5 mL) was then added dropwise at 273 K. After 12 h
stirring, the reaction mixture was extracted with CH2Cl2 (2 ¥
50 mL), washed with saturated Na2CO3, dried over Na2SO4 and
evaporated. 2-Tert-butyl-4-trifluoromethylphenol was purified by
silica gel column chromatography with CH2Cl2–pentane (1 : 1)
as eluent. 2-Tert-butyl-4-trifluoromethylphenol was obtained as a
50%). ESI MS, m/z: 521 (M - H - CH3CN - 2ClO4)+. Anal.
Calcd for C30H33Cl2CuFN4O10: C 47.22, H 4.36, N 7.34%. Found:
C 47.17, H 4.28, N 7.39%. UV-vis in CH3CN at 298 K (lmax /nm
(e/M-1 cm-1)): 617 (115). EPR (9.41 GHz, CH3CN, 100 K): gxx
gyy = 2.063, gzz = 2.253, Axx = Ayy = 0.5 mT, Azz = 17.6 mT.
=
Notes and references
1 For reviews, see: J. W. Whittaker, in Metal Ions in Biological Systems,
ed. H. Sigel and A. Sigel, Marcel Dekker, New York, 1994, vol. 30,
pp. 315; C. D. Borman, C. G. Saysell, A. Sokolowski, M. B. Twitchett,
C. Wright and A. G. Sykes, Coord. Chem. Rev., 1999, 190–192, 771;
M. J. McPherson, M. R. Parsons, R. K. Spooner, C. M. Wilmot, in
Handbook for metalloproteins, ed. A. Messerschmidt, R. Huber, T.
Poulos and K. Wieghardt, John Wiley and Sons, 2001, vol. 2, pp. 1272;
J. W. Whittaker, in Advances in Protein Chemistry, ed. F. M. Richards,
D. S. Eisenberg and J. Kuriyan, Academic Press, Elsevier, 2002, vol.
60, pp. 1; J. W. Whittaker, Chem. Rev., 2003, 103, 2347; M. S. Rogers
and D. M. Dooley, Curr. Opin. Chem. Biol., 2003, 7, 189; S. J. Firbank,
M. Rogers, R. Hurtado-Guerrero, D. M. Dooley, M. A. Halcrow, S. E.
V. Phillips, P. F. Knowles and M. J. McPherson, Biochem. Soc. Trans.,
2003, 31, 506.
2 N. Ito, S. E. V. Philips, C. Stevens, Z. B. Ogel, M. J. McPherson, J. N.
Keen, K. D. S. Yadav and P. F. Knowles, Nature, 1991, 350, 87–90.
3 For recent articles, see: R. C. Pratt and T. D. P. Stack, Inorg. Chem.,
2005, 44, 2367; P. Chaudhuri, K. Wieghardt, T. Weyhermu¨ller, T. K.
Paine, S. Mukherjee and C. Mukherjee, Biol. Chem., 2005, 386, 1023;
A. Dos Anjos, A. J. Bortoluzzi, B. Szpoganicz, M. S. B. Caro, G. R.
Friedermann, A. S. Mangrich and A. Neves, Inorg. Chim. Acta, 2005,
358, 3106; F. Michel, S. Torelli, F. Thomas, C. Duboc, C. Philouze, C.
Belle, S. Hamman, E. Saint-Aman and J.-L. Pierre, Angew. Chem., Int.
Ed., 2005, 44, 438; A. Berkessel, M. Doucet, S. Bulat and K. Glaubitz,
Biol. Chem., 2005, 386, 1035; M. A. Hossain, F. Thomas, S. Hamman,
E. Saint-Aman, D. Boturyn, P. Dumy and J.-L. Pierre, J. Pept. Sci.,
2006, 12, 612; E. Zueva, P. H. Walton and J. E. McGrady, Dalton Trans.,
2006, 159; L. Benisvy, E. Bill, A. J. Blake, D. Collison, E. S. Davies, C. D.
Garner, G. McArdle, E. J. L. McInnes, J. McMaster, S. H. K. Ross and
C. Wilson, Dalton Trans., 2006, 258; C. Mukherjee, T. Weyhermu¨ller,
K. Wieghardt and P. Chaudhuri, Dalton Trans., 2006, 2169; F. Michel,
F. Thomas, S. Hamman, C. Philouze, E. Saint-Aman and J.-L. Pierre,
Eur. J. Inorg. Chem., 2006, 3684; E. Safaei, T. Weyhermu¨ller, E. Bothe,
1
yellow oil with a yield of 80%. H NMR (300.12 MHz, CDCl3,
298 K, TMS) d/ppm: 1.41 (s, 9H), 7.33 (dd, 1H, 3J(H,H) =
8.3 Hz, 4J(H,H) = 2.3 Hz), 7.51 (dd, 1H, 4J(H,H) = 1.8 Hz). 13
C
NMR (75.465 MHz, CDCl3, 298 K, TMS) d/ppm: 29.7 (CH3,
tBu), 35.1 (Cq), 123.2 (q, Cq, 2J(C,F) = 32.2 Hz), 124.8 (q, CH,
3
3J(C,F) = 14.3 Hz), 124.9 (q, CH, J(C,F) = 12.9 Hz), 125.0 (q,
Cq, 1J(C,F) = 271.3 Hz), 137.2 (Cq), 157.2 (Cq). MS (DCI, NH3
isobutane), m/z, 218 (M + H)+. Anal. Calcd for C11H13F3O: C
60.54, H 6.00, F 26.12%. Found: C 59.79, H 6.04, F 26.04%.
2-Tert-butyl-6-{[bispyridin-2-ylmethyl-amino]-methyl}-4-trifluoro-
methylphenol
(HLCF3). 2-Tert-butyl-4-trifluoromethylphenol
(1 g, 4.6 mmol), bis-methylpyridylamine (800 mg, 4.0 mmol)
and paraformaldehyde (1 g, 30 mmol) were dissolved in ethanol
(20 mL) and heated to reflux during 18 h. After evaporation,
the oil was purified by silica gel column chromatography with
CH2Cl2–CH3OH (70 : 1) as eluent. 2[(Bis-pyridin-2-ylmethyl-
amino)-methyl]-6-tert-butyl-4-trifluoromethylphenol was ob-
tained as a colourless oil with a yield of 63%. 1H NMR
(300.12 MHz, CDCl3, 298 K, TMS) d/ppm: 1.47 (s, 9H), 3.85
(s, 2H), 3.89 (s, 4H), 7.13–7.18 (m, 3H), 7.29–7.31 (m, 2H), 7.41
(d, 1H, 4J(H,F) = 1.9 Hz), 7.62 (td, 2H, 3J(H,H) = 7.6 Hz,
3
4
4J(H,H) = 1.8 Hz), 8.57 (d, 2H, J(H,H) = 5.0 Hz, J(H,H) =
1.8 Hz, 4J(H,H) = 1.7 Hz). 13C NMR (75.465 MHz, CDCl3,
298 K, TMS) d/ppm: 29.7 (CH3, tBu), 35.4 (Cq), 57.7 (CH2),
2
59.5 (CH2Py), 120.3 (Cq, J(C,F) = 31.8 Hz), 122.7 (CH), 123.4
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K. Wieghardt and P. Chaudhuri, Eur. J. Inorg. Chem., 2007, 2334;
O. Rotthaus, O. Jarjayes, F. Thomas, C. Philouze, E. Saint-Aman and
J.-L. Pierre, Dalton Trans., 2007, 889; A. Mukherjee, F. Lloret and R.
Mukherjee, Inorg. Chem., 2008, 47, 4471; A. John, M. M. Shaikh and
P. Ghosh, Dalton Trans., 2008, 2815.
4 For reviews see: H.-J. Kru¨ger, Angew. Chem., Int. Ed., 1999, 38, 627;
S. Itoh, M. Taki and S. Fukuzumi, Coord. Chem. Rev., 2000, 198, 3; B.
A. Jazdzewski and W. B. Tolman, Coord. Chem. Rev., 2000, 200–202,
633; P. Chaudhuri and K. Wieghardt, Prog. Inorg. Chem., 2001, 50,
151; W. Kaim, Dalton Trans., 2003, 761; P. Chaudhuri, K. Wieghardt,
T. Weyhermu¨ller, T. K. Paine, S. Mukherjee and C. Mukherjee, Biol.
Chem., 2005, 386, 1023; K. Ray, T. Petrnko, K. Wieghardt and F.
Neese, Dalton Trans., 2007, 1552; F. Thomas, Eur. J. Inorg. Chem., 2007,
2379.
5 S. Itoh, M. Taki, S. Takayama, S. Nagatomo, T. Kitagawa, N. Sakurada,
R. Arakawa and S. Fukuzumi, Angew. Chem., Int. Ed., 1999, 38, 2774;
F. Thomas, G. Gellon, I. Gautier-Luneau, E. Saint-Aman and J.-L.
Pierre, Angew. Chem., Int. Ed., 2002, 41, 3047; Y. Shimazaki, S. Huth,
S. Hirota and O. Yamauchi, Inorg. Chim. Acta, 2002, 331, 168; A. John,
M. M. Shaikh and P. Ghosh, Dalton Trans., 2008, 2815.
13 H. M. Chang and H. H. Jaffe, Chem. Phys. Lett., 1973, 23, 146; H.
M. Chang, H. H. Jaffe and C. A. Masmandis, J. Phys. Chem., 1975,
79, 1118; L. J. Johnston, N. Mathivanan, F. Negri, W. Siebrand and
F. Zerbetto, Can. J. Chem., 1993, 71, 1655; J. Takahashi and T. Shida,
Bull. Chem. Soc. Jpn., 1994, 67, 2038; J. Takahashi, T. Momose and T.
Shida, Bull. Chem. Soc. Jpn., 1994, 67, 964; R. Liu, K. Morokuma, A.
M. Mebel and M. C. Lin, J. Phys. Chem., 1996, 100, 9314; A. Hinchliffe,
R. E. Steinbank and M. A. Ali, Theor. Chim. Acta, 1996, 5, 95; J. G.
Radziszewski, M. Gil, A. Gorski, J. Spanget-Larsen, J. Waluk and B.
J. Mroz, J. Chem. Phys., 2001, 115, 9733.
14 If no rearrangement occurs, elongation of the Cu–N bond upon
˚
deprotonation should be less than 0.03 A, which is too small to explain
the observed differences in line widths.
15 H. Gu¨nther, NMR spectroscopy, John Wiley and Sons, New York,
2nd edn, 1992; Dn corresponds to the chemical shift difference (in Hz)
of the signals exchanging. The chemical shifts are independent of the
temperature.
16 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
J. R. Cheeseman, J. A. Montgomery, Jr., T. Vreven, K. N. Kudin, J. C.
Burant, J. M. Millam, S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci,
M. Cossi, G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M.
Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T.
Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J. E.
Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,
C. Pomelli, J. Ochterski, P. Y. Ayala, K. Morokuma, G. A. Voth, P.
Salvador, J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D.
Daniels, M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck, K.
Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui, A. G. Baboul, S.
Clifford, J. Cioslowski, B. B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R. L. Martin, D. J. Fox, T. Keith, M. A. Al-Laham, C. Y.
Peng, A. Nanayakkara, M. Challacombe, P. M. W. Gill, B. G. Johnson,
W. Chen, M. W. Wong, C. Gonzalez and J. A. Pople, GAUSSIAN 03
(Revision C.02), Gaussian, Inc., Wallingford, CT, 2004.
17 A. W. Addison, T. N. Rao, J. Reedijk, J. van Rijn and G. C. Verschoor,
J. Chem. Soc., Dalton Trans., 1984, 1349.
6 A. Philibert, F. Thomas, C. Philouze, S. Hamman, E. Saint-Aman and
J.-L. Pierre, Chem.–Eur. J., 2003, 9, 3803.
7 F. Michel, S. Hamman, F. Thomas, C. Philouze, I. Gautier-Luneau and
J. L. Pierre, Chem. Commun., 2006, 4122.
8 R. Uma, R. Viswanathan, M. Palaniandavar and M. Lakshmi-
narayanan, J. Chem. Soc., Dalton Trans., 1994, 1219; H. Adams, N.
A. Bailey, C. O. Rodriguez de Barbarin, D. E. Fenton and Q.-Y. He, J.
Chem. Soc., Dalton Trans., 1995, 2323; H. Adams, N. A. Bailey, I. K.
Campbell, D. E. Fenton and Q.-Y. He, J. Chem. Soc., Dalton Trans.,
1996, 2233; M. Vaidyanathan, R. Viswanathan, M. Palaniandavar, T.
Balasubramanian, P. Prabhaharan and T. P. Muthiah, Inorg. Chem.,
1998, 37, 6418.
9 Y. Shimazaki, S. Huth, S. Hirota and O. Yamauchi, Bull. Chem. Soc.
Jpn., 2000, 73, 1187; M. Taki, H. Hattori, T. Osako, S. Nagatomo, M.
Shiro, T. Kitagawa and S. Itoh, Inorg. Chim. Acta, 2004, 357, 3369.
10 S. Itoh, M. Taki, H. Kumei, S. Takayama, S. Nagatomo, T. Kitagawa,
N. Sakurada, R. Arakawa and S. Fukuzumi, Inorg. Chem., 2000, 39,
3708; M. Taki, H. Kumei, S. Nagatomo, T. Kitagawa, S. Itoh and S.
Fukuzumi, Inorg. Chim. Acta, 2000, 300–302, 622.
18 The temperature was lowered to 238 K in order to prevent decomposi-
tion of the radical species.
19 F. Himo, L. A. Eriksson, F. Maseras and P. E. M. Siegbahn, J. Am.
11 F. Thomas, O. Jarjayes, C. Duboc, C. Philouze, E. Saint-Aman and
J.-L. Pierre, Dalton Trans., 2004, 2662.
Chem. Soc., 2000, 122, 8031.
20 F. Michel, F. Thomas, S. Hamman, E. Saint-Aman, C. Bucher and J.
L. Pierre, Chem.–Eur. J., 2004, 10, 4115.
12 J. Mu¨ller, T. Weyhermu¨ller, E. Bill, P. Hildebrandt, L. Ould-Moussa,
T. Glaser and K. Wieghardt, Angew. Chem., Int. Ed., 1998, 37, 616; E.
Bill, J. Mu¨ller, T. Weyhermu¨ller and K. Wieghardt, Inorg. Chem., 1999,
38, 5795; A. Sokolowski, H. Leutbecher, T. Weyhermu¨ller, R. Schnepf,
E. Bothe, E. Bill, P. Hildebrandt and K. Wieghardt, JBIC, J. Biol. Inorg.
Chem., 1997, 2, 444.
21 P. K. Mykhailiuk, S. Afonin, G. V. Palamarchuk, O. V. Shishkin,
A. S. Ulrich and I. Komarov, Angew. Chem., Int. Ed., 2008, 47,
5765.
22 TEXSAN: Single Crystal Structure Analysis Software, Molecular
Structure Corporation, The Woodlands, TX, USA.
842 | Dalton Trans., 2009, 832–842
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The Royal Society of Chemistry 2009
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