Galactose oxidase models: Tuning the properties of Cu II-phenoxyl radicals

Article abstract of DOI:10.1002/chem.200304880

Four tripodal ligands with an N₃O coordination sphere were synthesized: (2-hydroxy-3-tert-butyl-5-nitrobenzyl)bis(2-pyridylmethyl)amine (LNO₂H), (2-hydroxy-3-tert-butyl-5-fluorobenzyl)bis(2-pyridylmethyl)amine (LFH), (2-hydroxy-3,5-d

Full text of DOI:10.1002/chem.200304880

FULL PAPER
Galactose Oxidase Models: Tuning the Properties of CuII Phenoxyl Radicals
Aure¬lie Philibert,[a] Fabrice Thomas,*[a] Christian Philouze,[a] Sylvain Hamman,[a]
Eric Saint-Aman,[b] and Jean-Louis Pierre[a]
Abstract: Four tripodal ligands with an
N3O coordination sphere were synthe-
sized: (2-hydroxy-3-tert-butyl-5-nitro-
benzyl)bis(2-pyridylmethyl)amine
dized to phenoxyl radicals. These
complexes are EPR-active (S ˆ 1),
highly stable (kdecay ˆ 0.008minÀ1 for
Their copper(ii) complexes were
isolated as dimers ([CuI2I(L'OMe2)2],
[CuI2I(L'OMeNO2)2]) that are converted
to monomers on addition of pyridine.
The complexes were investigated by
X-ray crystallography and UV/Vis and
EPR spectroscopy. Their one-electron
electrochemical oxidation leads to cop-
per(ii) phenoxyl systems that are less
stable than those of the N3O complexes.
The N2O2 complexes are more reactive
than the N3O analogues: they aerobi-
cally oxidize benzyl alcohol to benzal-
dehyde at a higher rate, as well as
ethanol to acetaldehyde (40 80 turn-
overs).
.
[CuII(LOMe )(CH3CN)]2‡) and stoi-
(LNO2H),
(2-hydroxy-3-tert-butyl-5-
chiometrically oxidise benzyl alcohol.
Two additional tripodal ligands provid-
ing an N2O2 coordination sphere were
also studied: (2-pyridylmethyl)(2-hy-
droxy-3-tert-butyl-5-methoxybenzyl)-
(2-hydroxy-3-tert-butyl-5-nitrobenzyl)-
amine (L'OMeNO2H2) and (2-pyridyl-
methyl)bis(2-hydroxy-3-tert-butyl-5-
fluorobenzyl)bis(2-pyridylmethyl)amine
(LFH), (2-hydroxy-3,5-di-tert-butylben-
zyl)bis(2-pyridylmethyl)amine (LtBuH)
and (2-hydroxy-3-tert-butyl-5-methoxy-
benzyl)bis(2-pyridylmethyl)amine (LO-
MeH). Their square-pyramidal cop-
per(ii) complexes, in which the phenol
subunit occupies an axial position, were
prepared and characterized by X-ray
crystallography and UV/Vis and EPR
spectroscopy. The phenolate moieties of
the copper(ii) complexes of LtBuH and
LOMeH were electrochemically oxi-
methoxy)benzylamine
(L'OMe2H2).
Keywords: enzyme models ¥ copper
oxidation tripodal ligands
radicals
¥
¥
¥
copper ion with a tyrosinate or protonated tyrosinate ligand
weakly bound in the axial position, two histidine imidazole
units, a second tyrosinate and either H2O or acetate (replacing
the substrate) in the equatorial sites (Scheme 1).[3] The
equatorial tyrosinate group is linked to a nearby cysteine
Introduction
Various strategies are used by metalloenzymes to store
oxidizing equivalents required for multi-electron transfers.
A fascinating class of metalloenzymes use an organic redox
center (free radical) on their own polypeptide chain to
provide or abstract electrons and complete metal-driven
electron transfer in the catalytic process.[1] Galactose oxidase
(GOase), an enzyme typical of this class, is an extracellular
type II copper protein (68 kDa) of fungal origin that catalyzes
the oxidation of a broad range of primary alcohols to
aldehydes with concomitant reduction of molecular oxygen.[2]
The crystal structure of GOase contains a square-pyramidal
À
residue by an ortho C S bond. The enzyme exists in three
well-defined and stable oxidation states [Eq. (1)]: the EPR-
silent active oxidized form, in which a copper(ii) ion is
antiferromagnetically coupled to an equatorial tyrosyl radical
responsible for hydrogen atom abstraction from the substrate;
the intermediate copper(ii) form; and the reduced form, which
contains a copper(i) center.
.
II
‡
II
I
À
À
À
Cu
O
Tyr > Cu OTyr> Cu OTyr
(1)
[a] Dr. F. Thomas, A. Philibert, Dr. C. Philouze,
Dr. S. Hamman, Prof. Dr. J.-L. Pierre
The axial tyrosine residue is involved in a protonation
deprotonation process during the catalytic cycle (Scheme 1).
Recent theoretical studies[4] showed that, prior to proton
transfer from substrate to enzyme, the radical is located at the
axial tyrosinate site. The radical is shifted to the equatorial
tyrosinate group simultaneously with proton transfer.
¬
Laboratoire de Chimie Biomimetique, LEDSS, UMR CNRS 5616
¬
Universite J. Fourier, BP 53, 38041, Grenoble Cedex 9 (France)
Fax : (‡33)4-76-51-48-36
E-mail: Fabrice.Thomas@ujf-grenoble.fr
[b] Prof. Dr. E. Saint-Aman
¬
LEOPR, UMR CNRS 5630, Universite J. Fourier
Several models of the active site of GOase have been
prepared in the last few years to give an insight into the
structure and properties of its reactive center.[5] Tripodal
BP 53, 38041 Grenoble Cedex 9 (France)
Supporting information for this article is available on the WWW under
http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2003, 9, 3803 3812
DOI: 10.1002/chem.200304880
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3803

F. Thomas et al.
FULL PAPER
functional model involving two different phenolic arms in a
tripodal N2O2 ligand and obtained a radical center located on
the equatorial phenolic arm.[9]
O
(Tyr)
(His) N
O
H
H
(Tyr)
(His) N
II
O
II
O
H
Cu
H
Cu
O
O
H
H
(His) N
S
(His) N
S
Results and Discussion
(Tyr)
(Tyr)
Preparation of the ligands: LtBuH has been previously
described.[6f] We improved the synthesis by using a Mannich
reaction: in a one-pot synthesis LtBuH was obtained by
mixing bis(2-pyridylmethyl)amine and 2,4-di-tert-butyl-4-phe-
nol in the presence of one equivalent of formaldehyde. The
Mannich reaction was also used to prepare LOMeH and LFH.
Alkylation of bis(2-pyridylmethyl)amine with 2-bromometh-
yl-6-tert-butyl-4-nitrophenyl acetate was the preferred meth-
od for obtaining LNO2H. Synthesis of the N2O2 ligands
L'OMeNO2H2 and L'OMe2H2 has been previously de-
scribed.[10]
H2O2
H
O
H
O
O
H
H
H
(Tyr)
(Tyr)
I
II
Cu
O
O
O
(His) N
(His) N
(His) N
(His) N
Cu
H
S
H
O
O
O
H
O2
S
(Tyr)
(Tyr)
Copper complexes: N2O2 ligands: On mixing Cu(ClO4)2 ¥
6H2O and the N2O2 ligands in acetonitrile in the presence
of NEt3, a dimeric species was obtained (Scheme 3). Single
crystals of [CuI2I(L'OMe2)2] suitable for X-ray diffraction
Scheme 1. The galactose oxidase active site and adaptation of the catalytic
pathway proposed by F. Himo et al.[4a]
ligands have been commonly used to provide a coordination
sphere similar to that of the enzyme. Some involve an N3O
coordination sphere[6] (usually two pyridine nitrogen atoms,
one tertiary amine nitrogen atom and one phenolic oxygen
atom), and others an N2O2 coordination sphere[7] (usually one
pyridine nitrogen atom, one tertiary amine nitrogen atom and
two phenolic oxygen atoms). However, very few of these
models exhibit catalytic activity. One of the trickiest problems
encountered in the design of biomimetic models for the CuII
phenoxyl radical active form is the control of the radical
center. This control, which involves stability, magnetic proper-
ties and reactivity, depends on interdependent factors such as
protonation of the phenolic moieties, geometry (axial versus
equatorial binding to copper) and electronic properties.
Here we compare the solution chemistry, the stability of the
phenoxyl radicals and the catalytic properties of copper(ii)
complexes prepared from N2O2 and N3O ligands (Scheme 2).
The four N3O ligands have a phenolic arm that is substituted
in the para position by electron-donating or electron-with-
drawing groups. In a complex of a related non-tert-butylated
ligand the nitrophenolate group occupies an axial position,[8]
and this suggests that the phenolate group of the correspond-
ing tert-butylated ligand will also occupy an axial position in
its copper complex. In a preliminary report, we described a
1 L'H2 + 1 Cu(ClO4)2
CH3CN + N(Et)3
(i)
<[(CuII)2(L')2]>
CH2Cl2
CH3CN
[CuII(L'OMeNO2)(CH3CN)]
or [(CuII)2(L'OMe2)2]
[(CuII)2(L')2]
Pyr
Pyr
[CuII(L')(Pyr)]
Scheme 3. Solution chemistry of the N2O2 ligands, where L' represents
L'OMe2 or L'OMeNO2 (i: [L] > 0.05m).
studies were obtained by recrystallization from acetonitrile
after 12 h at 277 K (Table 1). The crystal structure of
[CuI2I(L'OMe2)2] is depicted in Figure 1, and selected bond
angles and lengths are reported in Table 2. The two copper
atoms have similar pentacoordinate environments that share
two oxygen atoms from two ligands. The geometry around the
copper(ii) center is intermediate between trigonal-bipyrami-
dal and square-pyramidal, as reflected by a t value[11] of 0.35.
Each copper atom is coordinated to two nitrogen and three
oxygen atoms: the pyridine nitrogen atom (N2), the tertiary
amino nitrogen atom (N1), one phenolate oxygen atom (O2)
OMe
À
N
and two m-phenoxo oxygen atoms (O1, O1'). The Cu O2
OH
HO
OH
Z2
À
bond length (1.895(3) ä) is shorter than Cu O1' (1.984(3) ä
N
À
or Cu O1 2.045(2) ä); the O1-Cu-O1' angle (73.88) is
N
N
N
significantly different from the Cu-O1-Cu angle (90.88).
Consequently, the CuOCuO unit is not flattened, but shows
a butterfly structure with a dihedral angle of 126.58 and a short
copper copper distance of 2.869 ä.
In [CuI2I(L'OMeNO2)2], which was previously reported but
not described,[9] the same geometry was observed (t ˆ 0.34
Z1
Z2= OMe (L'OMe2H2),
NO2 (L'OMeNO2H2)
Z1= OMe (LOMeH), tBu (LtBuH), F
(LFH), NO2 (LNO2H)
Scheme 2. Chemical structures of the ligands.
3804
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3803 3812

Galactose Oxidase Models
3803 3812
Table 1. Crystallographic data for [CuII(LtBuH)(CH3CN)](ClO4)2, [CuII(LOMeH)(CH3CN)](ClO4)2, [CuII(LFH)(CH3CN)](ClO4)2, [CuII(LNO2H)-
(CH3CN)](ClO4)2, [CuII(LNO2)(Cl)] and [CuI2I(L'OMe2)2].
[CuI2I(L'OMe2)2]
[CuII(LtBuH)(CH3CN)] (ClO4)2
[CuII(LNO2)(Cl)]
formula
M
symmetry
morphology
crystal dimensions [mm]
a [ä]
b [ä]
c [ä]
C60H76Cu2N4O8
1108.37
monoclinic
brown prism
0.41 Â 0.37 Â 0.25
28.49(3)
9.690(4)
27.72(3)
90
C29H38Cl2CuN4O9
721.09
C23H25ClCuN4O3
504.47
triclinic
dark green platelet
0.39 Â 0.35 Â 0.17
8.2328(6)
11.9330(9)
12.3562(6)
70.5(3)
monoclinic
blue platelet
0.48 Â 0.45 Â 0.25
13.2504(6)
11.745(1)
22.804(3)
90
a [8]
b [8]
131.1(1)
90
5770(10)
293.0
C2/c
4
97.7(4)
90
3517(2)
293.0
P21/a
80.7(3)
73.9(3)
1097(2)
g [8]
V [ä3]
T [K]
space group
Z
150.0
≈
P1
4
2
monochromator
l(MoKa) [ä]
graphite
0.71073
0.793
graphite
0.71073
0.826
graphite
0.71073
1.152
g [mmÀ1
]
no. of reflections (measured)
no. of reflections (unique)
12727
6433
46219
8150
12569
4481
no. of reflections
Rint
R
3090 (I > 2s)
0.148
0.073
3398 (I > 1.5s)
0.211
0.063
3262 (I > 3s)
0.039
0.036
R(w)
0.060
0.068
0.042
[CuII(LFH)(CH3CN)](ClO4)2
[CuII(LOMeH)(CH3CN)](ClO4)2
[CuII(LNO2H)(CH3CN)](ClO4)2
formula
M
C25H29Cl2CuFN4O9
682.98
C26H32Cl2CuN4O10
726.30
C28H33Cl2CuN5O11
764.56
symmetry
triclinic
triclinic
triclinic
morphology
crystal dimensions [mm]
a [ä]
b [ä]
c [ä]
a [8]
blue prism
0.48 Â 0.28 Â 0.15
8.2493(3)
9.5464(4)
19.0287(9)
93.8(2)
blue block
0.28 Â 0.18 Â 0.16
8.2869(2)
9.4031(4)
20.3281(8)
80.9(2)
blue-green prism
0.76 Â 0.61 Â 0.58
9.9996(5)
11.0806(9)
15.6455(9)
99.4(4)
b [8]
99.1(2)
78.8(1)
98.0(2)
g [8]
100.9(2)
1446(1)
80.3(1)
98.7(3)
1666(3)
V [ä3]
T [K]
1518.8(9)
293.0
293.0
150.0
≈
≈
≈
space group
Z
P1
P1
P1
2
2
2
monochromator
l(MoKa) [ä]
graphite
0.71073
1.005
graphite
0.71073
0.955
graphite
0.71073
0.883
m [mmÀ1
]
no. of reflections (measured)
no. of reflections (unique)
21596
8029
18879
8693
13014
7110
no. of reflections
Rint
R
4387 (I > 3s)
0.081
0.059
5170 (I > 2s)
0.074
0.057
5904 (I > 2s)
0.040
0.052
R(w)
0.093
0.076
0.086
and 0.39 for the two copper(ii) centers) with a bridging
methoxyphenolate and a nonbridging nitrophenolate group:
the electron-donating methoxyl group compensates more
efficiently than the nitro group for the electron deficiency on
the bridging oxygen atom. The substitution thus orientates the
position of the phenolate groups in these dimers according to
their electronic properties.
[CuI2I(L'OMeNO2)2] and [Cu2II(L'OMe2)2] are EPR-silent in
the noncoordinating solvent CH2Cl2 due to strong coupling
between the two CuII centers mediated by the phenolato
bridge.[7d) ] Dissolving [Cu2II(L'OMeNO2)2] in acetonitrile
results in the appearance of an EPR signal typical for a
mononuclear copper complex. However, no EPR signal was
observed after dissolution of [CuI2I(L'OMe2)2] in acetonitrile.
The dimeric complex [Cu2II(L'OMe2)2] can be converted to the
monomeric species by addition of a coordinating base such as
pyridine (Pyr). Titration of the dimers with pyridine was
monitored by UV/Vis spectroscopy: the intensity of the ligand
to metal charge-transfer (LMCT) band in the dimers was
higher than in the monomers, while a shift in lmax was
observed (Table 3). From the equilibrium of Equation (2)
KPyr
*
L'Cu2 ‡ 2Pyr )
2
2L'CuPyr
(2)
Chem. Eur. J. 2003, 9, 3803 3812
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3805

F. Thomas et al.
FULL PAPER
Table 2. Selected bond lengths [ä] and angles [8] of the complexes
[CuII(LtBuH)(CH3CN)](ClO4)2,
[CuII(LOMeH)(CH3CN)](ClO4)2,
[CuII(LFH)(CH3CN)](ClO4)2, [CuII(LNO2H)(CH3CN)](ClO4)2, [CuII-
(LNO2)(Cl)] and [CuI2I(L'OMe2)2].
[CuII(LtBuH)(CH3CN)]
À
À
À
Cu O1 2.456(3)
Cu N1 2.037(4)
Cu N2 1.987(4)
À
À
Cu N3 1.977(4)
Cu N4 1.974(6)
O1-Cu-N1 90.5(1)
O1-Cu-N4 90.5(2)
N1-Cu-N4 178.5(2)
N3-Cu-N4 97.4(2)
O1-Cu-N2 97.2(1)
N1-Cu-N2 83.4(2)
N2-Cu-N3 164.4(2)
O1-Cu-N3 91.6(1)
N1-Cu-N3 83.6(2)
N2-Cu-N4 95.4(2)
[CuII(LFH)(CH3CN)]
À
À
À
Cu O1 2.404(3)
Cu N1 2.024(4)
Cu N2 1.976(4)
À
À
Cu N3 1.975(3)
Cu N4 2.008(4)
O1-Cu-N1 89.8(1)
O1-Cu-N4 92.6(1)
N1-Cu-N4 177.4(2)
N3-Cu-N4 96.9(2)
O1-Cu-N2 98.0(1)
N1-Cu-N2 83.7(2)
N2-Cu-N3 163.6(1)
O1-Cu-N3 92.6(1)
N1-Cu-N3 84.0(1)
N2-Cu-N4 95.0(2)
[CuII(LOMeH)(CH3CN)]
À
À
À
Cu O1 2.411(2)
Cu N1 2.028(3)
Cu N2 1.983(3)
À
À
Cu N3 1.974(3)
Cu N4 2.002(3)
O1-Cu-N1 89.9(1)
O1-Cu-N4 93.3(1)
N1-Cu-N4 176.4(1)
N3-Cu-N4 97.3(1)
O1-Cu-N2 98.2(1)
N1-Cu-N2 83.3(1)
N2-Cu-N3 164.4(1)
O1-Cu-N3 91.1(1)
N1-Cu-N3 84.3(1)
N2-Cu-N4 94.6(1)
Figure 1. ORTEP plot showing 25% displacement ellipsoids and partial
atomic labeling for [CuI2I(L'OMe2)2]. Hydrogen atoms have been omitted
for clarity; selected distances and angles are reported in Table 2.
[CuII(LNO2H)(CH3CN)]
À
À
À
Cu O1 2.430(2)
Cu N1 2.014(2)
Cu N2 1.989(2)
À
À
Cu N3 1.988(2)
Cu N4 1.962(2)
where L' ˆ ligand and Kpyr ˆ [CuII(L')(Pyr)]2/([Cu2II(L')2]-
[Pyr]2), we obtained Kpyr[Cu2II(L'OMeNO2)2] ˆ 103.7 mÀ1 and
Kpyr[CuI2I(L'OMe2)2] ˆ 102.3 mÀ1.
O1-Cu-N1 87.14(7)
O1-Cu-N4 95.03(8)
N1-Cu-N4 177.46(9)
N3-Cu-N4 96.4(1)
O1-Cu-N2 88.23(8)
N1-Cu-N2 83.84(8)
N2-Cu-N3 165.91(9)
O1-Cu-N3 92.87(8)
N1-Cu-N3 82.18(9)
N2-Cu-N4 97.52(9)
This confirmed the easier cleavage of the dimeric structure
[CuI2I(L'OMeNO2)2]. These results cannot be explained on the
basis of the X-ray analysis, which shows a butterfly structure
for both dimers and similar interatomic distances. However,
nitro as opposed to methoxyl substitution makes the coordi-
nated CuII atom more electrophilic and thus favors nucleo-
philic attack by pyridine. The solution chemistry of the N2O2
ligands in the presence of copper(ii) is summarized in
Scheme 3.
[CuII(LNO2)(Cl)]
À
À
À
Cu Cl 2.2651(7)
Cu O1 2.190(2)
Cu N1 2.057(2)
À
À
Cu N2 1.994(2)
Cu N3 1.996(2)
Cl-Cu-O1 97.38(5)
Cl-Cu-N3 95.81(6)
O1-Cu-N3 95.66(8)
N2-Cu-N3 157.18(9)
Cl-Cu-N1 170.33(6)
O1-Cu-N1 92.29(7)
N1-Cu-N2 82.35(8)
Cl-Cu-N2 95.53(6)
O1-Cu-N2 102.44(8)
N1-Cu-N3 83.16(8)
[CuI2I(L'OMe2)2]
À
À
À
Cu O1' 1.984(3)
Cu O1 2.045(2)
Cu O2 1.895(3)
À
À
À
Cu N1 2.126(3)
Cu N2 2.284(3)
Cu Cu 2.869(3)
O1-Cu-O1' 73.8(1)
O1'-Cu-N2 92.0(1)
O1-Cu-N2 132.4(1)
N1-Cu-N2 79.8(1)
O1'-Cu-O2 165.1(1)
O1-Cu-O2 93.1(1)
O2-Cu-N1 93.2(1)
Cu-O1-Cu 90.8(1)
O1'-Cu-N1 93.7(1)
O1-Cu-N1 144.5(1)
O2-Cu-N2 102.2(1)
N3O ligands: The solution chemistry of the copper(ii)
complexes of the N3O ligands differs notably from that of
their N2O2 analogues (Scheme 4). Monomeric complexes
were obtained on mixing stoichiometric amounts of the N3O
ligands and Cu(ClO4)2 ¥ 6H2O in acetonitrile in the presence
of NEt3. Under these conditions the complexes were isolated
in their protonated form, in which the phenolic subunit
remains protonated and only d d transitions are observed in
the visible spectrum. The ligand LNO2H was an exception:
[CuII(LNO2H)(CH3CN)]2‡ was obtained in the absence of
base.
Blue single crystals of [CuII(LOMeH)(CH3CN)]2‡,
[CuII(LtBuH)(CH3CN)]2‡ and [CuII(LFH)(CH3CN)]2‡ were
obtained on slow diffusion of di-n-butyl ether into solutions of
the complexes in acetonitrile. For [CuII(LNO2H)(CH3CN)]2‡
toluene was used instead of di-n-butyl ether. The ORTEP
plots of their structures are depicted in Figure 2; selected
bond lengths and angles are listed in Table 2.
Table 3. Electronic properties of the copper(ii) complexes in acetonitrile solution
(UV/Vis spectra recorded at 298 K).
Complex
lmax [nm] (e [mÀ1 cmÀ1])
[CuII(LOMeH)(CH3CN)]2‡ 400 (320),[a] 590 (230)[a]
[CuII(LOMe)(CH3CN)]
565 (1040)
546 (950)
600 (180)[a]
553 (935), 950 (180)[a]
531 (750), 850 (180)[a]
550 (240)
305 (3400), 535 (920), 930 (130)[a]
309 (3800), 514 (780), 870 (140)[a]
388 (14830), 606 (90)
390 (11200), 517 (636)
396 (17280), 500 (401),[b] 644 (139), 868 (130)
‡
[CuII(LOMe)(Pyr)]
‡
[CuII(LtBuH)(CH3CN)]2‡
[CuII(LtBu)(CH3CN)]
‡
[CuII(LtBu)(Pyr)]
‡
[CuII(LFH)(CH3CN)]2‡
[CuII(LF)(CH3CN)]
‡
[CuII(LF)(Pyr)]
‡
[CuII(LNO2H)(CH3CN)]2‡
[CuII(LNO2)(CH3CN)]
‡
[CuII(LNO2)(Pyr)]
‡
[CuII(L'OMeNO2H)(OAc)] 298 (10300), 393 (20000), 500 (760),[b] 600 (510)[a]
The geometry around the metal centers is square-pyramidal
with a phenolic unit that weakly coordinates the copper atom
in an axial position.
In [CuII(LOMeH)(CH3CN)]2‡ the copper atom is coordi-
nated in the square plane by one tertiary nitrogen atom N1,
[CuI2I(L'OMeNO2)2]
[CuII(L'OMeNO2)(Pyr)]
[CuI2I(L'OMe)2]
296 (11400), 391 (28100), 500 (5100)[b]
305 (6300), 401 (15000), 500 (1210),[b] 700 (300)[a]
302 (16700), 445 (4330), 650 (1600)[a]
310 (20000), 488 (1400)
[CuII(L'OMe2)(Pyr)]
[a] Broad absorption. [b] Shoulder.
3806
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3803 3812

Galactose Oxidase Models
3803 3812
two pyridine nitrogen atoms N2 and N3 and one acetonitrile
nitrogen atom N4, with copper nitrogen bond lengths of
2.028(3) (N1), 1.983(3) (N2), 1.974(3) (N3) and 2.002(3) ä
1 LH + 1 Cu(ClO4)2
CH3CN + NEt3
(i)
<[CuII(LH)(CH3CN)]>
À
(N4). The Cu O1 distance of 2.411(2) ä is significantly longer
than those reported for axially coordinated phenolates.[8] It is
within the range of that (2.567 ä) reported for the protonated
CH3CN
CH3CN + NEt3
[CuII(LH)(CH3CN)]
[CuII(L)(CH3CN)]
‡
complex [CuII(LtBuH)(Cl)] bearing a chloride ion as an
exogenous ligand in place of acetonitrile.[6f] The protonated
phenolic unit thus binds weakly to the copper atom, which lies
0.120(3) ä above the basal plane and towards the axial O1
atom. The coordination modes of [CuII(LtBuH)(CH3CN)]2‡,
[CuII(LFH)(CH3CN)]2‡ and [CuII(LNO2H)(CH3CN)]2‡ are
Pyr
Pyr
[CuII(L)(Pyr)]
with L = LOMe, LtBu, LF
II
2‡
À
similar to that of [Cu (LOMeH)(CH3CN)] , with Cu O1
1 LNO2H + 1 Cu(ClO4)2
distances of 2.456(3), 2.404(3) and 2.430(2) ä, respectively.
CH3CN
CH3CN + NEt3
(i)
(i)
À
À
The Cu N2 and Cu N3 distances range between 1.975(3) and
À
1.989(2) ä, the Cu N1 distances between 2.014(2) and
<[CuII(LNO2)(CH3CN)]>
CH3CN
<[CuII(LNO2H)(CH3CN)]>
À
2.037(4) ä and the Cu N4 distances between 1.962(2) and
2.008(4) ä.
CH3CN
+ NEt3
CH3CN
The copper(ii) complex of LNO2H could also be prepared in
[CuII(LNO2H)(CH3CN)]
[CuII(LNO2)(CH3CN)]
‡
its deprotonated form [CuII(LNO2)(CH3CN)] by mixing
stoichiometric amounts of Cu(ClO4)2 ¥ 6H2O and HLNO2 in
acetonitrile in the presence of one equivalent of NEt3. Slow
diffusion of diethyl ether into this solution afforded dark
Pyr
Pyr
[CuII(LNO2)(Pyr)]
‡
green crystals of [CuII(LNO2)(CH3CN)] , which, however,
Scheme 4. Solution chemistry of the N3O ligands (i: [L] > 0.05m).
were not suitable for X-ray analysis. To obtain single crystals
suitable for a X-ray analysis, CuCl must be used instead of
Cu(ClO4)2 ¥ 6H2O to prepare the complex.[12] Based on the
structural similarity between [CuII(LtBuH)(CH3CN)]2‡ and
‡
[CuII(LtBuH)(Cl)] ,[6f] no significant geometrical changes are
expected when acetonitrile is replaced by a chloro ligand. The
geometry around the copper atom in [CuII(LNO2)(Cl)] can be
À
described as square-pyramidal. The Cu O1 distance in
[CuII(LNO2)(Cl)] differs from those of [CuII(LNO2H)-
(CH3CN)]2‡ and [CuII(LOMeH)(CH3CN)]2‡. It is significant-
ly shorter (2.190(2) ä) and within the range of those reported
for axially coordinated phenolates.[8] The copper atom is
displaced 0.235(2) ä above the basal plane towards the axial
À
oxygen atom, as expected for a stronger Cu O1 bond
(0.036(2) ä in [CuII(LNO2H)(CH3CN)]2‡). The bond lengths
between the copper atom and the donor nitrogen atoms are
À
À
À
Cu N1 2.057(2), Cu N2 1.994(2) and Cu N3 1.996(2) ä, and
À
the Cu Cl bond length is 2.2651(7) ä. An axial position of the
nitrophenolate moiety was already observed in a related
complex[8] and explained[5a] in terms of the short methylene
spacer between the pyridine and tertiary amino groups: the
two five-membered chelate rings are preferentially located in
the basal plane. The deprotonated complexes of the other
N3O ligands bearing electron-donating substituents could not
be isolated in the solid state, even in the presence of NEt3, due
to their higher pKa.
After dissolution of the protonated complexes in acetoni-
trile (Scheme 4), only d d transitions were observed in the
visible region of the electronic spectrum (Table 3); this
suggests that the phenolic subunit remains protonated in
solution. The EPR spectrum reveals a mononuclear CuII
complex. On addition of one equivalent of NEt3 the solution
turns violet; the UV/Vis spectrum shows emergence of an
LMCT transition (the electron-withdrawing NO2 group
induces a hypsochromic shift, while the electron-donating
Figure 2. ORTEP plots showing 25% (a, b, c) or 50% (d, e) displacement
ellipsoids and partial atomic labeling for [CuII(LOMeH)(CH3CN)]2‡ (a),
CuII(LtBuH)(CH3CN)]2‡ (b), [CuII(LFH)(CH3CN)]2‡ (c), [CuII(LNO2H)-
(CH3CN)]2‡ (d) and [CuII(LNO2)(Cl)] (e). Hydrogen atoms have been
omitted for clarity; selected distances and angles are reported in Table 3.
Chem. Eur. J. 2003, 9, 3803 3812
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3807

F. Thomas et al.
FULL PAPER
groups tBu and OMe induce a bathochromic shift); this
indicates that the weakly coordinating phenolic arm is
deprotonated on addition of NEt3 and coordinates to the
copper atom. Use of excess pyridine (50 equiv) in place of
NEt3 also results in the appearance of phenolate to copper
charge-transfer band at a somewhat shorter wavelength,
which indicates that pyridine acts both as a base and as an
exogenous ligand. The solution chemistry of the copper
complexes of N3O ligands is summarized in Scheme 4.
oxidized complex (charge: ‡2) and the perchlorate anions of
the supporting electrolyte (TBAP). Under these conditions,
electrolysis could not be performed.[13]
‡
The CV curves of [CuII(LOMe)(Pyr)] , [CuII(LOMe)-
‡
‡
‡
(CH3CN)] , [CuII(LtBu)(Pyr)] , [CuII(LtBu)(CH3CN)] ,
[CuII(L'OMe2)(Pyr)] and [CuII(L'OMeNO2)(Pyr)] reveal re-
versible one-electron (as estimated by coulometric titration)
redox waves at ‡0.02, ‡0.01, ‡0.17, ‡0.15, À0.16, and
À0.08 V, respectively. No significant change in E1/2 was
observed for the N3O complexes when the exogenous
pyridine ligand was replaced by acetonitrile, as also observed
by Taki et al.[7a] As expected, oxidation occurs on the
methoxyphenolate moieties of [CuII(L'OMeNO2)(Pyr)], with
a E1/2 value close to that obtained for [CuII(L'OMe2)(Pyr)].
The EPR spectra of the electrogenerated species
Electrochemistry of the protonated ligands and complexes:
The electrochemical behaviour of the protonated ligands and
complexes was studied in acetonitrile by cyclic voltammetry
(CV). Only the results obtained with N3O ligands are
presented. The electrochemistry of the N2O2 ligands was
previously described.[10]
.
.
[CuII(LOMe )(Pyr)]2‡, [CuII(LOMe )(CH3CN)]2‡, [CuII-
(LtBu )(Pyr)]2‡, [CuII(LtBu )(CH3CN)]2‡, [CuII(L'OMe2 )-
.
.
.
The CV curve of the free ligands displays irreversible
anodic signals, as expected for phenol-centered electron
transfer. Oxidation of the nitrophenol to its phenoxyl radical
.
‡
‡
(Pyr)] and [CuII(L'OMeNO2 )(Pyr)] show a DMS ˆÆ 2
signal centred at 150 mT, associated with DMS ˆÆ 1 transi-
tions typical of an S ˆ 1 system (Figure 3). The UV/Vis spectra
‡
occurs at the highest potential (‡0.65 V versus Fc/Fc ); the
fluorine-substituted phenol has an oxidation potential
(‡0.46 V) close to that of an unfluorinated parent com-
pound,[10] and the tBu- and OMe-substituted phenols have the
lowest oxidation potentials (‡0.37 and ‡0.29 V, respective-
ly), in agreement with the electron-donating properties of
their substituents: A plot of Eap as a function of the Hammet
‡
constants s shows a linear dependence (Supporting Infor-
mation).
The CV curves of all the protonated complexes show
irreversible redox signals around ‡0.8 V. These values are
close to that previously reported by Shimazaki et al.[6f] for
[CuII(LtBuH)(Cl)]2‡. Oxidation occurs at higher potentials
relative to the free ligands due to the positive charge on the
copper atom, which decreases the electron density on the
phenolic unit and thus makes it less easily oxidizable.
Figure 3. X-band EPR spectra of the electrochemically generated com-
.
.
‡
plexes [CuII(LOMe )(Pyr)]2‡ (a) and [CuII(L'OMeNO2 )(Pyr)] (b); a
contaminant with S ˆ 1/2 (accounting for less than 5 (a) or 10% (b) of the
total copper(ii) concentration) corresponding to a monomeric CuII complex
.
.
‡
is also present; [CuII(LOMe )(Pyr)]2‡ ˆ [CuII(L'OMeNO2 )(Pyr)] ˆ 2 mm,
Tˆ 4 K, 9.44711 GHz, 0.25 mW; modulation 0.5 mT, 100 KHz.
Radical generation in the deprotonated complexes: The
‡
electrochemical behaviour of [CuII(LNO2)(CH3CN)] and
‡
[CuII(LNO2)(Pyr)] is characterized by a signal at E1/2
ˆ
of these oxidized species show sharp and intense absorptions
at around 420 nm (Figure 4, Table 4), as previously observed
for phenoxyl radicals,[14] and attributed to p p* transitions.
The LMCT transitions are no longer present in the 500
600 nm region. Taken together, these results unambiguously
show evidence for the formation of a phenoxyl radical that is
ferromagnetically coupled to the copper(ii) center.
‡0.70 V. It is not fully reversible (iPa/iPc > 1) even at low
temperature (233 K), which suggests a relatively low stability
of the oxidized species. Coulometric titration revealed a one-
electron process. Dramatic changes in the electronic spectrum
were observed during electrolysis: the 390 nm absorption (e ˆ
11200mÀ1 cmÀ1), attributed to the p p* transition of the
nitrophenolate, is strongly modified (lmax ˆ 387 nm, e ˆ
6400mÀ1 cmÀ1; shoulder at 440 nm, e ˆ 3900mÀ1 cmÀ1), while
the LMCT transition at 517 nm disappears. These results
strongly suggest the formation of a transient nitrophenoxyl
species. Assuming that no isomerization occurs during
electrolysis, this is a good example of a phenoxyl radical
axially coordinated to a metal atom. Its low stability, however,
precluded further characterization.
.
.
Both [CuII(LOMe )(CH3CN)]2‡ and [CuII(LOMe )(Pyr)]2‡
were found to be highly stable at 298 K (kdecay ˆ 0.008 and
0.0045minÀ1 respectively). To our knowledge, only
one CuII phenoxyl radical possessing such a stability has
been recently described in literature.[6d) ] The other radical
complexes are relatively stable at 298 K: kdecay ˆ 0.155
.
.
([CuII(LOMeNO2 )(Pyr)] ), 0.155 ([CuII(LtBu )(CH3CN)]2‡)
and 0.087minÀ1 ([CuII(LtBu )(Pyr)]2‡). The exogenous pyr-
‡
.
‡
The CV curves of [CuII(LF)(CH3CN)] and [CuII(LF)-
idine ligand greatly stabilizes the radical. In the case of the
N3O complexes, the lower the redox potential, the longer the
half-life of the radical (Table 4), while an inverse correlation is
observed with N2O2 complexes. Moreover, the radical com-
plexes of the N3O ligands are more stable than those of the
‡
(Pyr)] at room temperature reveal a redox couple that is not
fully reversible (iPa/iPc > 1) at ‡0.20 V. When the temperature
is lowered to 233 K, a total loss of reversibility is observed,
and only the anodic peak is seen on the CV curve; this is likely
due to the formation of passivating ion pairs between the
.
‡
N2O2 ligands. In [CuII(L'OMeNO2 )(Pyr)] , the presence of a
3808
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3803 3812

Galactose Oxidase Models
3803 3812
.
(CH3CN)]2‡ or [CuII(LtBu )-
(CH3CN)]2‡ increased the ob-
served rate constant kobs for the
comsumption of the radical, while
quantitative formation of benzal-
dehyde (relative to the catalyst)
was evidenced by GC. Monoex-
ponential decays were fitted to
obtain the pseudo-first-order rate
constants kobs. Plots of the ob-
served rate constant kobs À kdecay
versus benzyl alcohol concentra-
tion show a linear dependence
(Figure 5) for both complexes.
The rate constants kox for the
oxidation of benzyl alcohol ob-
tained from the slopes are
5.8mÀ1 minÀ1 and 13.6mÀ1 minÀ1
Figure 4. UV/Vis spectra of the copper complexes (dotted lines) and the corresponding electrogenerated
.
.
CuII phenoxyl radical complexes (solid lines): [CuII(LOMe )(Pyr)]2‡ 0.11  10À3 m (a), [CuII(LtBu )(Pyr)]2‡
.
for [CuII(LOMe )(CH3CN)]2‡ and
.
.
‡
0.11  10À3 m (b), [CuII(L'OMeNO2 )(Pyr)] 0.05  10À3 m (c), [CuII(L'OMe2 )(Pyr)]2‡ 0.11  10À3 m (d); spectra
recorded in acetonitrile (‡0.01m TBAP) at 233 K in a 1 cm path length quartz cell; * denotes an
underestimated transient absorption due to instability of the complex, even at 233 K.
.
[CuII(LtBu )(CH3CN)]2‡, respec-
tively. One equivalent of benzyl
alcohol is oxidized by one equiv-
alent of complex, that is, one mole
of the catalyst formally stores two oxidative equivalents: the
phenoxyl radical and the CuII center. A kinetic deuterium
isotope effect kHox/koDx of 8 was obtained when PhCH2OH was
replaced by PhCD2OH, that is, the rate-limiting step is H
abstraction from the substrate, as for GOase and related
nitrophenolate goup coordinated to the copper atom stabil-
izes the radical generated on the methoxyphenolate moiety.
This can be regarded as a captodative effect.
Degradation of the CuII phenoxyl radical complex: After
one week of aerobic incubation (253 K) of [CuII(LO-
Me)(CH3CN)] with one equivalent of the single-electron
chemical oxidant TPASbCl6, an unexpected degradation
product was isolated (formation of an transient
.
model compounds.[6b,9] Although [CuII(LOMe )(CH3CN)]2‡
.
‡
[CuII(LOMe )(CH3CN)] species was evidenced by EPR
and UV/Vis spectroscopy): the copper complex retained its
square-pyramidal structure and only modification of the
phenolic subunit was observed. The classical oxidative
cleavage of the tertiary amine does not occur.[7e) ] The X-ray
structure of [Cu2II(LO)2(Cl)2] ¥ CH2Cl2 ¥ Sb3Cl11, thus obtained
as light green single crystals, shows an ortho-tert-butylated
quinone moiety in place of the 2-hydroxy-3-tert-butyl-5-
methoxybenzyl group. The full description of the crystal
structure is given in the Supporting Information.
Figure 5. Plot of kobs À kdecay versus the concentration of benzyl alcohol:
kdecay is the rate constant for the radical signal decay in acetonitrile
(degradation of the catalyst); the pseudo-first-order constant kox corre-
sponds to the radical signal decay in presence of an excess of benzyl
Reactivity of the radical species towards alcohol oxidation:
.
.
‡
‡
alcohol; catalysts: [CuII(LtBu )(CH3CN)] (a), [CuII(LOMe )(CH3CN)]
Under anaerobic conditions, addition of an excess of benzyl
.
alcohol to a solution of electrogenerated [CuII(LOMe )-
(b).
Table 4. Spectroscopic and electrochemical properties of copper(ii) phenoxyl complexes in acetonitrile solution.[a]
Complex
[CuII(HL'OMeNO2 )(OAC)]
lmax [nm](e [mÀ1 cmÀ1])
EPR
E1/2 [V][b]
kdecay [minÀ1
]
t1/2 [min]
.
410 (19000)[c]
390 (15300)
425(1000)[d]
426 (5100), 552 (560)
406 (8200)
416 (1900), 650 (500)
417 (3500), 660 (800)
444 (5200), 800 (3200)
silent
S ˆ 1
S ˆ 1
S ˆ 1
S ˆ 1
S ˆ 1
S ˆ 1
silent
0.08
À 0.08
À 0.16
0.02
0.01
0.15
1.05
0.155
0.71
0.008
0.0045
0.155
0.087
0.66
5
1
87
154
5
‡
.
[CuII(L'OMeNO2 )(Pyr)]
‡
.
‡
[CuII(L'OMe2 )(Pyr)]
.
[CuII(LOMe )(CH3CN)]2‡
.
[CuII(LOMe )(Pyr)]2‡
.
[CuII(LtBu )(CH3CN)]2‡
.
[CuII(LtBu )(Pyr)]2‡
0.17
À 0.23
8
active GOase
‡
[a] UV/Vis spectra recorded at 233 K, EPR spectra at 100 K. kdecay and t1/2 at 298 K. [b] Versus Fc/Fc , 298 K in acetonitrile, 0.1m TBAP. [c] In CH3CN/CH2Cl2
(50/50). [d] Underestimated due to the low stability of this complex.
Chem. Eur. J. 2003, 9, 3803 3812
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3809

F. Thomas et al.
FULL PAPER
.
and [CuII(LtBu )(CH3CN)]2‡ oxidize benzyl alcohol more
proposition that tyrosyl radicals axially coordinated to metal
atoms could be of major biological interest in enzymatic
catalysis.[16]
.
slowly than [CuII(L'OMeNO2 )], the ratio kox/kdecay is higher.
It is also 100 times higher than the value reported for a
previously described complex.[6b]
[CuII(LNO2 )(CH3CN)]2‡ and [CuII(LF )(CH3CN)]2‡ were
not stable enough to study their reactivity at room temper-
ature in the presence of a substrate. Moreover, excess pyridine
competitively inhibits substrate binding.
.
.
Experimental Section
General: All chemicals were of reagent grade and used without purifica-
Ethanol was oxidized aerobically to acetaldehyde by the
CuII phenoxyl radical complex of [CuI2I(L'OMe2)2]: 40 Æ 5
turnovers were achieved in 30 min at 298 K.[15] On the
tion. NMR spectra were recorded on a Bruker AM 300 (1H at 300 MHz, 13
C
at 75 MHz). Chemical shifts are given relative to tetramethylsilane (TMS).
Mass spectra were recorded on a Thermofunnigen (EI/DCI) or a Nermag R
101 C (FAB ‡ ) apparatus. Microanalyses were performed by the Service
Central d'Analyse du CNRS (Lyon, France).
.
.
other hand, [CuII(LOMe )(CH3CN)]2‡ and [CuII(LtBu )-
(CH3CN)]2‡ oxidized ethanol to acetaldehyde only stoichio-
metrically, even in the presence of O2 and a base. However,
electrocatalysis in the presence of a catalytic amount of NEt3
allows 5 Æ 1 turnovers to be achieved for the oxidation of
ethanol. In constrast to the reduced complexes of N2O2
ligands, the CuII phenoxyl complexes of N3O ligands after
oxidation of the substrate are not regenerated with O2. Proton
transfer from the substrate to O2 cannot be achieved
efficiently in the absence of a coordinating phenolate. This
unambiguously shows that the axial phenol/phenolate in the
copper complexes of the N2O2 ligands is involved in a
protonation deprotonation process during the catalytic cy-
cle, as in GOase.
UV/Vis spectroscopy: UV/Vis spectra were recorded on a Perkin Elmer
Lambda 2 spectrophotometer equiped with a temperature controller unit
set at 298 K. The quartz cell path length was 1.000 cm. Low-temperature
Vis spectra were recorded on an UVIKON spectrophotometer equipped
with a quartz Dewar (H.S. Martin Inc.) containing a quartz cell of 1.000 cm
path length.
Electrochemistry: Electrochemical measurements were carried out using a
PAR model 273 potentiostat equipped with a Kipp-Zonen x y recorder.
Experiments were performed in a standard three-electrode cell under
argon atmosphere. An Ag/AgNO3 (0.01m) reference electrode was used.
‡
All the potentials given in the text are referred to the regular Fc/Fc redox
couple used as an internal reference (‡87 mV versus Ag/AgNO3). A
vitrous carbon disc electrode (5 mm diameter) was used as working
electrode and was polished with 1 mm diamond paste. The electrochemical
behaviour of the ligands and complexes was studied by cyclic voltammetry
(CV) in acetonitrile solutions containing 0.1m tetrabutylammonium
perchlorate (TBAP) as supporting electrolyte. Electrolysis was performed
at 233 K using the same apparatus and a carbon-felt working electrode.
Conclusion
EPR spectroscopy: X-band EPR spectra were recorded on a Bruker ESP
300E spectrometer equipped with a Bruker nitrogen flow cryostat, or a
Bruker EMX spectrometer equipped with an ESR 900 helium flow cryostat
(Oxford Instruments). Samples of the protonated copper complexes of the
N3O ligands were prepared in acetonitrile with a few drops of toluene and
CH2Cl2 to improve the resolution of the spectrum. Spectra were treated
with the WINEPR software and simulated using the Bruker SIMFONIA
software.
The solution chemistry of the copper complexes of N2O2
ligands (2 phenol, 1 pyridine group) differs significantly from
that of N3O ligands (1 phenol, 2 pyridine groups): the former
afforded stable dimeric structures that can be converted to
monomers by addition of a coordinating base, while the latter
were isolated only as monomers. The properties of the
electrogenerated CuII phenoxyl radical species can be finely
tuned by changing 1) the number of phenolic arms, 2) the
electronic properties of the phenol p-substituent and 3) the
exogenous ligand. The monomeric copper complexes of the
N2O2 ligands have lower redox potentials that favor formation
of a radical species. The electrochemically generated N2O2
CuII phenoxyl species are highly reactive and much less
stable than their N3O analogues. All the CuII phenoxyl
complexes stoichiometrically oxidize benzyl alcohol to ben-
zaldehyde in the absence of O2 with a rate limiting step that is,
as for GOase, H abstraction from the substrate. The reaction
proceeds much more slowly when N3O ligands are used. The
aerobic oxidation of ethanol to acetaldehyde by the CuII
phenoxyl copper complexes of N3O ligands proceeds stoichio-
metrically, while 40 80 turnovers are achieved with the N2O2
complexes: the acido-basic phenol is needed (and cannot be
replaced by acido-basic pyridine) for regeneration of the
catalyst.
Crystal structure analysis: For all structures, collected reflections were
corrected for Lorentzian and polarization effects but not for absorption.
The structures were solved by direct methods and refined with TEXSAN
software. 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. CCDC-198361
([CuI2I(L'OMe2)2]),
CCDC-198776
CCDC-199022
([CuII(LNO2)(Cl)]),
([CuII(LtBuH)(CH3CN)](ClO4)2),
CCDC-198520
([CuII(LFH)-
(CH3CN)](ClO4)2), CCDC-198701 ([CuII(LOMeH)(CH3CN)](ClO4)2),
CCDC-198860 ([CuII(LNO2H)(CH3CN)](ClO4)2), and CCDC-199566
([CuI2I(LO)2(Cl)2]) contain the supplementary crystallographic data for
this paper; these data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html.
Oxidation of ethanol: The nonradical N2O2 complex was dissolved in
ethanol (0.1m, TBAP) to give a 1 mm solution, and one-electron electro-
chemical oxidation was performed at low temperature (233 K). The base
was then added, and the temperature raised to 298 K. The solution was
stirred for 30 min at 298 K under ambient O2 pressure. Prior to analysis, the
aliquots were passed through a column containing the ion-exchange resin
Dowex 50X2-200. Acetaldehyde was titrated spectrophotometrically by
using the 3-methyl-2-benzothiazolone hydrazone test (E. Sawicki, T.R.
Hauser, T.W. Stanley, W. Elbert, Anal. Chem. 1961, 33, 93).
Formation of a transient phenoxyl radical axially coordi-
nated to the copper atom has been evidenced; it constitutes a
model of the GOase active site in its CuII radical state prior to
proton transfer from the coordinated substrate to the axial
tyrosyl group according to the theoretical scheme proposed by
F. Himo et al.[4a] These results also support the recent
The electrocatalysis experiment was performed with the N3O catalysts:
Electrolysis of the nonradical complex (1 mm in ethanol) was conducted
directly in the presence of base at 298 K until
a residual current
corresponding to 1% of the initial current was obtained. Coulometric
titration revealed five exchanged electrons at this time. The aliquots were
analyzed in a similar way as described above.
3810
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3803 3812

Galactose Oxidase Models
3803 3812
‡
Oxidation of benzyl alcohol: A 2 mm solution of [CuII(LtBu)(CH3CN] or
isobutane): m/z: 392 (LOMeH); elemental analysis (%) calcd for
C24H29N3O2: C 73.63, H 7.47, N 10.73; found: C 73.65, H 7.64, N 10.74.
[CuII(LOMe)(CH3CN] was electrochemically oxidized at 233 K (CH3CN,
‡
TBAP 0.1m). A 25 mLaliquot was quickly heated to 298 K and diluted in
475 mLof a solution of CH 3CN containing benzyl alcohol (298 K). The
disappearance of the 410 nm absorption was monitored by UV/Vis
spectroscopy. The yield of benzaldehyde was determined by GC with
mesitylene as an internal standard (experimental error 10%).
[CuI2I(L'OMe2)2]: L'OMe2H2 (200 mg, 0.407 mmol) was treated with two
equivalents of NEt3 in acetonitrile (10 mL) prior to addition of Cu(ClO4)2 ¥
6H2O (152 mg, 0.407 mmol). After 12 h at 277 K, the dark brown single
crystals of [CuI2I(L'OMe2)2] that had formed were collected by filtration.
Elemental analysis (%) calcd for C60H76N4Cu2O8 ¥ 1H2O: C 63.98, H 6.98, N
4.97, Cu 11.28; found: C 63.94, H 6.92, N 4.95, Cu 11.20; FAB ‡ MS: m/z:
1107 ([CuI2I(L'OMe2)2]); UV/Vis (CH3CN): lmax [nm] (e [LmolÀ1 cmÀ1]):
302 (16.7 Â 103), 445 (4.3 Â 103), 650 (1.6 Â 103).
(2-Hydroxy-3-tert-butyl-5-nitrobenzyl)bis(2-pyridylmethyl)amine
(LNO2H): 2-bromomethyl-6-tert-butyl-4-nitrophenyl acetate (3.36 g,
10 mmol), prepared according to a described method,[10] bis(2-pyridyl-
methyl)amine (1.99 g, 10 mmol), NaI (1.5 g, 10 mmol) and Na2CO3 (3.18 g,
30 mmol) in THF (90 mL) were stirred for 3 d at room temperature. The
solution was filtered and the solvent evaporated. The crude acetate salt was
hydrolyzed with sodium hydroxide (25 mL, 40% in water) and ethanol
(25 mL). Ethanol was removed and HCl was added to neutralization. The
solution was poured into water, and the mixture extracted with CH2Cl2.
Organic layers were dried (Na2SO4) and evaporated. Column chromatog-
raphy on silica gel (hexane/ethyl acetate 1/1) yielded LNO2H (4g, 60%) as
a white solid; 1H NMR (300 MHz, CDCl3, 298 K, TMS): d ˆ 1.49 (s, 9H;
tBu), 3.38 (s, 2H; H7), 3.91 (s, 4H; H8 and H9), 7.23 (ddd, 3J(H2,H1) ˆ
4.98 Hz, 3J(H2,H3) ˆ 7.41 Hz, 4J(H2,H4) ˆ 1.08 Hz, 1H; H2), 7.28 (d,
3J(H4,H3) ˆ 7.56 Hz, 1H; H4), 7.64 (ddd, 3J(H3,H2) ˆ 3J(H3,H4) ˆ
[CuII(LNO2)(CH3CN)](ClO4):
L NOH (100 mg, 0.246 mmol) and Cu-
2
(ClO4)2 ¥ 6H2O (92 mg, 0.246 mmol) were dissolved in CH3CN/CH2Cl2 (3/1,
18 mL) in the presence of one equivalent of NEt3. The solution was stirred
for 3 h. Dark green hygroscopic microcrystals of [CuII(LNO2)(CH3CN)]-
(ClO4) were obtained by slow diffusion of diethyl ether into the CH3CN/
CH2Cl2 solution. Elemental analysis (%) calcd for C25H28N5CuClO7 ¥
2H2O ¥ 0.25CH2Cl2: C 45.48, H 4.91, N 10.50, Cu 9.53; found: C 45.96, H
4.76, N 10.61, Cu 9.54; EPR (9.41 GHz, 100 K, CH3CN): gxx ˆ 2.050, gyy
ˆ
2.074, gzz ˆ 2.247, Axx ˆ 0.5 mT, Ayy ˆ 1.5 mT, Azz ˆ 17.2 mT; FAB ‡ MS:
m/z: 469 ([CuII(LNO2)]); UV/Vis (CH3CN): lmax [nm] (e [LmolÀ1 cmÀ1]):
390 (11.2 Â 103), 517 (6.4 Â 103).
4
4
7.56 Hz, J(H3,H1) ˆ 1.80 Hz, 1H; H3), 7.89 (d, J(H5,H6) ˆ 2.82 Hz, 1H;
H5 or H6), 8.14 (d, 4J(H5,H6) ˆ 2.82 Hz, 1H; H5 or H6), 8.57 ppm (d,
3J(H1,H2) ˆ 4.98 Hz, 1H; H1); 13C NMR (75 MHz, CDCl3, 298 K, TMS):
d ˆ 29.52 (q), 35.53 (s), 57.16 (t), 59.22 (t), 122.75 (d), 123.18 (d), 123.56 (s),
123.64 (d), 124.64 (d), 137.21 (d), 138.10 (s), 139.43 (s), 149.25 (d), 157.90 (s),
163.65 ppm (s); MS (DCI, NH3, isobutane): m/z: 407 (LNO2H); elemental
analysis (%) calcd for C23H26N4O3: C 67.96, H 6.45, N 13.78; found: C 68.22,
H 6.66, N 13.74.
[CuII(LNO2H)(CH3CN)](ClO4)2: L NOH (200 mg, 0.492 mmol) and Cu-
2
(ClO4)2 ¥ 6H2O (184 mg, 0.492 mmol) were dissolved in acetonitrile (4 mL),
and the solution was stirred for one day. Blue microcrystals of [CuII(L-
NO2H)(CH3CN)](ClO4)2 were obtained by slow diffusion of toluene into a
solution of the complex in CH3CN/CH2Cl2 (3/1). Elemental analysis (%)
calcd for C25H29N5CuCl2O11: C 42.29, H 4.12, N 9.86, Cu 8.95, Cl 9.99;
found: C 42.08, H 4.24, N 10.16, Cu 8.91, Cl 10.13; EPR (9.41 GHz, 100 K,
CH3CN): gxx ˆ 2.066, gyy ˆ 2.066, gzz ˆ 2.230, Axx ˆ 0.5 mT, Ayy ˆ 1.5 mT,
Azz ˆ 18.0 mT; FAB ‡ MS: m/z: 470 ([CuII(LNO2H)]); UV/Vis (CH3CN):
lmax [nm] (e ˆ [LmolÀ1 cmÀ1]): 255 (12.8  103); 388 (14.8  103); 606
(0.09 Â 103). Caution: on exposure to air, the blue crystals rapidly
decompose to a green powder.
(2-Hydroxy-3-tert-butyl-5-fluorobenzyl)bis(2-pyridylmethyl)amine (LFH):
2-tert-Butyl-4-fluorophenol (168 mg, 1 mmol), prepared according to
Vanholm et al.,[17] bis(2-pyridylmethyl)amine (238 mg, 1.2 mmol) and
formaldehyde (375 mLof a 37% aqueous solution) in EtOH/H 2O (4/6,
10 mL) were heated for 24 h at 353 K. The reaction mixture was added to
water (50 mL) and extracted with CH2Cl2 (2 Â 50 mL). The organic layer
was dried (Na2SO4) and evaporated. The ligand was purified by column
chromatography on silica gel with hexane/ethyl acetate (9/1 to 5/5) as
eluent. LFH (342 mg, 90%) was obtained as a white solid; 1H NMR
(300 MHz, CDCl3, 298 K, TMS): d ˆ 1.43 (s, 9H; tBu), 3.76 (s, 2H; H7),
3.84 (s, 4H; H8 and H9), 6.58 (dd, 4J(H5,H6) ˆ 3.10 Hz, 3J(H5,F) or
3J(H6,F) ˆ 8.97 Hz, 1H; H5 or H6), 6.90 (dd, 4J(H5,H6) ˆ 3.10 Hz,
3J(H5,F) or 3J(H6,F) ˆ 8.97 Hz, 1H; H5 or H6), 7.14 (ddd, 3J(H2,H1) ˆ
5.37, 3J(H2,H3) ˆ 8.31, 4J(H2,H4) ˆ 0.90 Hz, 1H; H2), 7.32 (d, 3J(H4,H3) ˆ
8.31 Hz, 1H; H4), 7.61 (ddd, 3J(H3,H4) ˆ 3J(H3,H2) ˆ 8.31 Hz,
4J(H3,H1) ˆ 1.77 Hz, 1H; H3), 8.52 (d, 3J(H1,H2) ˆ 5.37 Hz, 1H; H1);
13C NMR (75 MHz, CDCl3, 298 K, TMS): d ˆ 29.38 (q), 35.02 (s), 57.51 (t),
59.25 (t), 112.88 (d), 113.18 (d), 113.58 (d), 113.89 (s), 122.43 (d), 123.61 (d),
136.85 (d), 148.99 (d), 152.31 (s), 152.33 (s), 157.88 ppm (s); 19F NMR
(282.54 MHz, CDCl3, 298 K): d ˆ 35.11 ppm (dd, 3J(H5,F) ˆ 3J(H6,F) ˆ
8.8 Hz); MS (DCI, NH3, isobutane): m/z: 380 (LFH); elemental analysis
(%) calcd for C23H26FN3O: C 72.80, H 6.91, N 11.07, F 5.01; found: C 73.09,
H 6.89, N 10.94, F 4.83.
[CuII(LtBuH)(CH3CN)](ClO4)2: LtBuH (200 mg, 0.478 mmol) and Cu-
(ClO4)2 ¥ 6H2O (179 mg, 0.478 mmol) were dissolved in acetonitrile (8 mL)
in the presence of one equivalent of NEt3. The solution was stirred for three
days. Blue hygroscopic microcrystals of [CuII(LtBuH)(CH3CN)](ClO4)2
were obtained by slow diffusion of diethyl ether into a solution of the
complex in acetonitrile. Elemental analysis (%) calcd for
C29H38N4Cl2CuO9 ¥ 0.5H2O: C 47.71, H 5.38, N 7.67, Cu 8.70, Cl 9.71; found:
C 47.99, H 5.31, N 7.64, Cu 8.68, Cl 10.01; EPR (9.41 GHz, 100 K, CH3CN):
gxx ˆ 2.048, gyy ˆ 2.079, gzz ˆ 2.233, Axx ˆ 1 mT, Ayy ˆ 1 mT; Azz ˆ 18.0 mT;
ESI MS: m/z: 481 ([CuII(LtBuH)]); UV/Vis (CH3CN): lmax [nm]
(e [LmolÀ1 cmÀ1]): 600 (0.18 Â 103).
[CuII(LOMeH)(CH3CN)](ClO4)2: LOMeH (200 mg, 0.512 mmol) and
Cu(ClO4)2 ¥ 6H2O (190 mg, 0.512 mmol) were dissolved in acetonitrile
(8 mL) in the presence of one equivalent of NEt3. The solution was stirred
for three days. [CuII(LOMeH)(CH3CN)](ClO4)2 was obtained as blue
crystals by slow diffusion of di-n-butyl ether into the acetonitrile solution.
Elemental analysis (%) calcd for C26H32N4CuCl2O10: C 44.93, H 4.64, N
8.06, Cu 9.14; found: C 45.05, H 4.61, N 8.14, Cu 8.98; EPR (9.41 GHz,
(2-Hydroxy-3,5-di-tert-butylbenzyl)bis(2-pyridylmethyl)amine (LtBuH):
LtBuH was synthesized according to the procedure used for LFH using
2,4-di-tert-butyl-4-phenol instead of 2-tert-butyl-4-fluorophenol. LtBuH
was previously described.[6f]
100 K, CH3CN): gxx ˆ 2.066, gyy ˆ 2.066, gzz ˆ 2.233, Axx ˆ 0.5 mT, Ayy
ˆ
1.5 mT, Azz ˆ 18 mT; FAB ‡ MS: m/z: 454 ([CuII(LOMeH)]); UV/Vis
(CH3CN): lmax [nm] (e [LmolÀ1 cmÀ1]): 400 (0.32 Â 103); 590 (0.23 Â 103).
(2-Hydroxy-3-tert-butyl-5-methoxybenzyl)bis(2-pyridylmethyl)amine
(LOMeH): LOMeH was synthesized according to the procedure used for
HLF using 2-tert-butyl-4-methoxyphenol instead of 2-tert-butyl-4-fluoro-
phenol. LOMeH (1.028 g, 55%) was obtained as a pale orange solid;
1H NMR (300 MHz, CDCl3, 298 K, TMS): d ˆ 1.44 (s, 9H; tBu), 3.73 (s,
3H; OCH3), 3.79 (s, 2H; H7), 3.86 (s, 4H; H8 and H9), 6.48 (d,
4J(H5,H6) ˆ 2.94 Hz, 1H; H5 or H6), 6.80 (d, 4J(H5,H6) ˆ 3.06 Hz, 1H;
H5 or H6), 7.16 (ddd, 3J(H2,H1) ˆ 4.99 Hz, 3J(H2,H3) ˆ 7.55 Hz,
4J(H2,H4) ˆ 1.15 Hz, 1H, H2), 7.34 (d, 3J(H4,H3) ˆ 7.94 Hz, 1H; H4),
7.61 (ddd, 3J(H3,H4) ˆ 3J(H3,H2) ˆ 7.68 Hz, 4J(H3,H1) ˆ 1.79 Hz, 1H;
H3), 8.70 ppm (d, 3J(H1,H2) ˆ 4.08 Hz, 1H; H1); 13C NMR (75 MHz,
CDCl3, 298 K, TMS): d ˆ 29.83 (q), 35.33 (s), 56.12 (q), 58.47 (t), 112.57 (d),
113.49 (d), 116.71 (s), 122.66 (d), 123.40 (s), 123.99 (d), 137.08 (d), 138.29
(s), 149.33 (d), 150.62 (s), 152.00 (s), 158.39 ppm (s); MS (DCI, NH3,
[CuII(LFH)(CH3CN)](ClO4)2: LFH (200 mg, 0.528 mmol) and Cu(ClO4)2 ¥
6H2O (197 mg, 0.528 mmol) were dissolved in CH3CN/CH2Cl2 (3/1, 8 mL)
in the presence of one equivalent of NEt3. The solution was stirred for three
days. Blue, hygroscopic microcrystals of [CuII(LFH)(CH3CN)](ClO4)2 were
obtained by slow diffusion of diethyl ether (2 mL) into the solution of the
complex in acetonitrile. Elemental analysis (%) calcd for C25H29N4CuF-
Cl2O9 ¥ 0.5H2O: C 43.39, H 4.37, N 8.10, Cu 9.18, Cl 10.25; found: C 42.78, H
4.27, N 7.71, Cu 9.07, Cl 10.69; EPR (9.41 GHz, 100 K, CH3CN): gxx ˆ 2.064,
gyy ˆ 2.064, gzz ˆ 2.233, Axx ˆ 0.5 mT, Ayy ˆ 1.5 mT, Azz ˆ 18.0 mT; ESI MS:
m/z: 442 ([CuII(LFH)]); UV/Vis (CH3CN): lmax [nm] (e [LmolÀ1 cmÀ1]):
550 (0.24 Â 103).
Caution: Perchlorates are potentially explosive and should be handled with
special care.
Chem. Eur. J. 2003, 9, 3803 3812
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3811

F. Thomas et al.
FULL PAPER
Hildenbrandt, K. Wieghardt, J. Biol. Inorg. Chem. 1997, 2, 444; c) C.
Ochs, F. E. Hahn, R. Frˆhlich, Eur. J. Inorg. Chem. 2001, 2427; d) D.
Aknowledgements
¬
Zurita, I. Gautier-Luneau, S. Menage, J. L. Pierre, E. Saint-Aman, J.
The authors thank the Institute of Metals in Biology of Grenoble that
Biol. Inorg. Chem. 1997, 2, 46; e) Y. Shimazaki, S. Huth, A. Odani, O.
Yamauchi, Angew. Chem. 2000, 112, 1732; Angew. Chem. Int. Ed.
2000, 39, 1666.
¬
provided facilities for performing 4 K EPR, Dr. Stephane Menage for
¡
technical assistance in these experiments and Mylene Vial for the
crystallization of [CuI2I(L'OMe2)2].
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and spectroscopic characterization.
‡
[13] To minimize this phenomenon, the [CuII(LF)(Pyr)]
and
‡
[CuII(LF)(CH3CN)] complexes were formed as their triflate salts.
The supporting electrolyte was replaced by N-lithiotrifluoromethane-
sulfonimide, and a platinum-grid working electrode was used; in this
case electrolysis was possible, but the oxidized species were not stable
enough to allow their characterization. Coulometric titration revealed
a one-electron process.
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dimer [CuI2I(L'OMe2)2] by the coordinating solvent ethanol (evi-
denced by EPR spectroscopy).
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Received: February 24, 2003 [F4880]
3812
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3803 3812