A dinuclear biomimetic Cu complex derived from l-histidine: synthesis and stereoselective oxidations

Article abstract of DOI:10.1039/C7DT00147A

A dinuclear copper(ii) complex derived from the chiral N₆ ligand (2S,2′S)-N,N′-(ethane-1,2-diyl)bis(2-((1-methyl-1H-imidazol-4-ylmethyl)-amino)-3-(1-trityl-1H-imidazol-4-yl)propanamide) (EHI) was synthesized and studied as a catalyst in stereoselective oxidation reactions. The ligand contains two sets of tridentate binding units, each of them giving rise to a coordination set consisting of a pair of 5- and 6-membered chelate rings, connected by an ethanediamide linker. Stereoselectivity effects were studied in the oxidations of a series of chiral l/d biogenic catechols and the pair of l/d-tyrosine methyl esters, in this case as their phenolate salts. The oxidation of β-naphthol has also been studied as a model monooxygenase reaction. The catechol oxidation was investigated in a range of substrate concentrations at slightly acidic pH and exhibited a marked dependence on the concentration of the [Cu₂EHI]⁴⁺ complex. This behavior has been interpreted in terms of an equilibrium between a monomeric and a dimeric form of the catalyst. Binding studies of l- and d-tyrosine were performed as a support for the interpretation of the stereoselectivity effects observed in the reactions. In general, [Cu₂EHI]⁴⁺ exhibits a binding preference for the l- rather than the d-enantiomer of the substrates, but it appears that in the catecholase reaction the oxidation of the d-isomer occurs at a faster rate than for the l counterpart. The same type of enantio-discriminating behavior is observed in the oxidation of l-/d-tyrosine methyl esters. In this case the reaction produces a complex mixture of products; the main product consisting of a trimeric compound, likely formed by radical coupling reactions, has been isolated and characterized. The oxidation of β-naphthol yields an additional product of the expected quinone but labeling experiments with 18-O₂ show no oxygen incorporation into the product, confirming that the oxidation likely proceeds through a radical mechanism.

Full text of DOI:10.1039/C7DT00147A

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PAPER
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Efficient extraction of sulfate from water using
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A dinuclear biomimetic Cu complex derived from L-histidine:
Synthesis and stereoselective oxidations
Maria L. Perrone,a Elena Salvadeo,a Eliana Lo Presti,a Luca Pasotti,a,b Enrico Monzani,a Laura
Santagostini,c Luigi Casella*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
aDipartimento di Chimica, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy
bNoxamet Srl, Dipartimento di Chimica, Università di Pavia, 27100 Pavia, Italy
cDipartimento di Chimica, Università di Milano, 20133 Milano, Italy
www.rsc.org/
The dinuclear copper(II) complex derived from the chiral N6 ligand (2S,2'S)-N,N'-(ethane-1,2-diyl)bis(2-((1-methyl-1H-
imidazol-4-ylmethyl)-amino)-3-(1-trityl-1H-imidazol-4-yl)propanamide) (EHI) was synthesized and studied as catalyst in
stereoselective oxidation reactions. The ligand contains two sets of tridentate binding units, each of them giving rise to a
coordination set consisting in a pair of 5- and 6-membered chelate rings, connected by an ethanediamide linker.
Stereoselectivity effects were studied in the oxidations of a series of chiral L/D biogenic catechols and the couple of L/D-
tyrosine methyl esters, in this case as their phenolate salts. The oxidation of β-naphthol has also been studied as a model
monooxygenase reaction. The catechol oxidation was investigated in a range of substrate concentrations at slightly acidic
pH and exhibited a marked dependence on the concentration of [Cu2EHI]4+ complex. This behavior has been interpreted in
terms of an equilibrium between a monomeric and a dimeric form of the catalyst. Binding studies of L- and D-tyrosine
were performed as a support for the interpretation of the stereoselectivity effects observed in the reactions. In general,
[Cu2EHI]4+ exhibits a binding preference for the L- rather than the D-enantiomer of the substrates, but it appears that in
the catecholase reaction the oxidation of the D-isomer occurs at a faster rate than for the L counterpart. The same type of
enantio-discriminating behavior is observed in the oxidation of L-/D-tyrosine methyl esters. In this case the reaction
produces a complex mixture of products; a main product consisting of a trimeric compound, likely formed by radical
coupling reactions, has been isolated and characterized. The oxidation of β-naphthol yields an addition product of the
expected quinone but labeling experiments with 18-O2 show no oxygen incorporation into the product, confirming that
the oxidation likely proceeds through a radical mechanism.
oxygen transfer reactions to the substrates:1-3,8,9 the μ-η2:η2-
peroxodicopper(II), bis(μ-oxo)dicopper(III) or trans-μ-η1:η1-
peroxodicopper(II) species. The currently available evidence on
INTRODUCTION
The dicopper enzyme tyrosinase continues to be an important
source of inspiration for biomimetic chemists interested in
developing effective catalysts for oxygenation reactions.1-4
Indeed, recent reports described the first examples of catalytic
phenol hydroxylation5,6 and sulfoxidation7 by dinuclear copper
tyrosinase indicates that the enzyme performs the phenol
hydroxylation through
μ-η2:η2-peroxodicopper(II)
a
intermediate.8 An important development for synthetic
applications of biomimetic tyrosinase models would be the
possibility to perform catalytic stereoselective oxidations. The
potential of copper salts, or simple complexes in mediating
stereoselective reactions, in the presence of chiral additives, is
well known and steadily expanding in synthetic organic
chemistry.9 However, the nature of the active species and its
nuclearity in these reactions is usually unknown, and therefore
developing well characterized, biomimetic systems capable of
promoting asymmetric reactions will open the field to the
possibility to design chiral complexes for specific applications.
We have been interested for some time in the synthesis of
polydentate nitrogen ligands to obtain chiral dinuclear and
complexes in the presence of dioxygen.
A wealth of
spectroscopic and structural studies on dicopper biomimetic
model compounds of tyrosinase have shown that three types
of copper-dioxygen intermediates are competent to perform
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The dicopper(II) complex [Cu2L]4+ was obtained by reacting the
free ligand with copper(II) salt in almost quantitative yields.
trinuclear complexes.10-12 These ligands typically contained a
set of eight donor nitrogens and were designed to host three
copper(II) centers, although for comparison purposes also the
corresponding dinuclear analogues were in parallel developed.
The reason for introducing a third metal center, in addition to
the pair of copper centers acting as dioxygen activation site,
was to exploit the binding properties of this center for
anchoring the substrate close to the catalytic unit. Indeed, the
trinuclear complexes generally exhibited better chiral
discrimination properties with respect to the corresponding
dinuclear counterparts. However, the synthetic routes to such
chiral N8 ligands are extremely lengthy and time spending, due
to intrinsic complexity of the polyfunctional molecules and to
the fact that the synthesis must comply with the need to
protect the chiral centers in every step of the procedure. We
want therefore to develop a parallel, simpler approach to
dinucleating N6 ligands that would give the chance to proceed
more systematically with stepwise improvements and
adjustments on the basis of the performance of the resulting
dicopper complexes in terms of catalytic efficiency and
recognition capacity. Herein, we report the synthesis and
reactivity characteristics of [Cu2EHI]4+, the first member of a
new family of chiral biomimetic tyrosinase model complexes,
where the ligand EHI provides a coordination environment to
each copper ion constituted by tridentate amino-
bis(imidazole) residues derived from L-histidine (Scheme 1).
O
O
O
O
H
N
N
NH2
N
H
H
H2N
N
Fmoc
N
H
H
Fmoc
N
H
Piperidine
CH2Cl2
N
N
N
N
N
Trt
N
N
N
Trt
Trt
Trt
3
2
CHO
N
CH2Cl2
N
DIPEA
PyBOP
CH2Cl2
NH2
H2N
CH2Cl2
O
NaB(OAc)3H
O
H
H
N
H
Fmoc
COOH
N
N
N
H
N
N
N
N
N
H
N
N
N
N
N
N
Trt
Trt
Trt
1
L
Scheme 2. Synthesis of chiral diamino-tetraimidazole ligand EHI (L) from Nτ-protected
L-histidine. Fmoc is the fluorenylmethyloxycarbonyl protecting group.
The design of the EHI ligand features two tridentate amino-
bis(imidazole) chelating arms connected through a linker
chain, similarly to the previously studied ligands of the PHI
family.11bc,12b However, here the linker chain is built on the α-
carbon atoms of L-histidine residues, whereas in the PHI
ligands the linker was bound as a substituent at the nitrogen
atom of the amino group. This change is important because it
combines a large flexibility in ligand design with a great
simplification in the synthesis of the chiral ligand.
O
O
N
Spectroscopic and conformational characterization of [Cu2EHI]4+.
The electronic spectrum of [Cu2EHI]4+ (Figure 1S) in the UV
region is dominated by the absorption bands of the N-trityl
N
H
H
NH
NH
Cu2+
N
Cu2+
N
N
N
N
N
substituent of the histidine residues.
A weak shoulder
N
N
Trt
Trt
detectable on the low-energy tail of the intense UV band, near
370 nm, can be aꢀributed to π(imidazole)→Cu(II) LMCT
transitions, as discussed previously for Cu-PHI complexes.11b
The visible portion of the electronic spectrum features a broad
absorption band comprising the copper(II) LF transitions. The
maximum occurs near 630 nm, but the band is asymmetric and
extends to the near-IR. As for [Cu2PHI]4+ complexes,11bc,12b the
CD spectrum of [Cu2EHI]4+ provides important details. The
visible region is dominated by a CD band of positive sign
centered at 670 nm, which is flanked by a much weaker
negative band at higher energy, near 550 nm (Figure 1).
Scheme 1. Proposed structure of the dinuclear complex [Cu2EHI]4+
; Trt is the
triphenylmethyl protecting group.
RESULTS AND DISCUSSION
Synthesis. The synthetic route to the diamino-tetraimidazole
ligand EHI (L) is outlined in Scheme 2. It involves the
preparation of Fmoc-protected bis(L-histidine) intermediate
by condensation of the protected amino acid with
ethylenediamine, followed by deprotection and condensation
of the diamine with two molecules of N-methyl imidazole-
2
1
3
carboxaldehyde, and reduction of the resulting intermediate.
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a
c
b
Figure 1. CD spectra in the near-UV and visible range of complex [Cu2EHI]4+ (a, black trace), and after the addition of 1 (b, red trace) and 2 (c, blue trace) equiv. of azide in
MeOH/MeCN 9:1 (v/v) solution.
This pattern is opposite to that exhibited by Cu(II)-PHI Azide binding experiments to [Cu2EHI]4+ were performed to
complexes and implies a different conformational preference gain information about the mode of binding of small ligands to
by the chelate ring of the L-histidine residues. This is of δ the copper(II) centers of the complex. The experiments were
chirality for [Cu2EHI]4+, as shown in Scheme 3, implying that all carried out in the same solvent mixture of MeOH/MeCN 9:1
three nitrogen donor atoms of the ligand occupy equatorial (v/v) previously used for the parent [Cu2PHI]4+ complex to
positions of Cu(II), but the preferred conformation was λ in allow a direct comparison between the optical features and
[Cu2PHI]4+. The difference is dictated by the orientation of the binding constants of the small ligand in the two complexes.
linker chain (R in Scheme 3) attached to the histidine chelating The addition of azide to a solution of [Cu2EHI]4+ is accompanied
arm, which is axial in the former case and equatorial in the by the development of the characteristic UV band, here near
latter.11bc,12b In [Cu2EHI]4+ the R substituent on the α-carbon is 385 nm, due to π(azido)→Cu(II) LMCT. The band is symmetric
more far removed from the Cu center with respect to the N- and grows steadily in intensity, with only a small shift in the
linked position it occupies in [Cu2PHI]4+ complexes, which wavelength maximum, with stepwise addition of azide up to a
-
forces the substituent to adopt an equatorial disposition and molar ratio of 2 N3 /[Cu2EHI]4+ (ε382 = 4600 M-1 cm-1). A weak
the chelate ring into the opposite (λ) conformational band of positive sign in the same position develops in the CD
arrangement (Scheme 3). This allows the L-histidine chelate spectrum (Figure 1) and, together, these spectral features
ring of [Cu2EHI]4+ to adopt a preferred13 δ conformation. These indicate that an azido ligand binds to each copper center of
stereochemical considerations are important because they [Cu2EHI]4+ in the terminal mode and that the two Cu-azido
may be involved in the output of the asymmetric reactions chromophores are well separated in space, with negligible
catalyzed by the complexes.
interaction between the LMCT transition moments. The
situation is thus different from the case of [Cu2PHI]4+, where
the spatial proximity between the two Cu-azido chromophores
allowed their π(azido)→Cu(II) LMCT transiꢁon moments to
interact, yielding a peculiar and intense exciton couplet in the
CD spectrum.11c In addition, while for complexes of [Cu2PHI]4+
type azide binding produced an inversion of the conformation
of the L-histidine chelate ring, from λ to δ,11c,12b in the case of
[Cu2EHI]4+ the ligand strengthens the optical activity of the LF
bands without changing their sign (Figure 1), indicating that
the δ conformation is maintained.
Me
N
Me
N
X
N
N
X
N
X
N
X
R
Cu
Cu
R
N
N
H
N
AcO
N
Trt
Me
δ
λ
Scheme 3. Representation of the δ conformation of the L-histidine chelate ring
adopted by the copper centers of [Cu2EHI]4+, and the λ conformation adopted by the
corresponding chelate rings of [Cu2PHI]4+ complexes; R stands for the linker chain
connecting the two halves of the dinuclear complex and X are solvent molecules or
donor ligands, like azide.
By optical titration it was possible to separate the two steps of
azide binding to [Cu2EHI]4+ (Figure 2S). Analysis of the spectral
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data using a non-linear least-squares procedure, enabled to temperature range between -15 and +45 °C, as the mixture is
estimate two binding constants, corresponding to the stable in these conditions.
following equilibria:
‾
[Cu2EHI]4+ + N3
[Cu2EHI(N3)]3+
[Cu2EHI(N3)2]2+
Table 1. Paramagnetic shift of the reference NMR signal (in
‾
[Cu2EHI(N3)]3+ + N3
Hz), magnetic susceptibility, and magnetic moment (per
The equilibrium constants were determined as log Kb1 = 4.61 ± copper) determined at different temperatures for [Cu2(EHI)]4+.
0.01 and log Kb2 = 3.59 ± 0.01. These values are in the same
range as those for azide binding to [Cu2PHI]4+ complex,11c and
Temperature (K)
Δν (Hz)
18.04
15.98
14.15
11.94
10.90
χM (m3/mol)
µ (μB)
1.64
1.59
1.54
1.44
1.42
indicate moderate affinity of copper(II) centers with similar
ligand environment for the exogenous ligand.
258
273
288
301
318
1.64
1.45
1.28
1.08
0.99
×
×
×
×
×
10-8
10-8
10-8
10-8
10-8
Magnetic coupling in [Cu2(EHI)]4+. Magnetic susceptibility
measurements can give information about the presence of
coupling of magnetic moments in dinuclear complexes. NMR
spectra can give indication about the existence of magnetic
The data in Table 1 show that the magnetic moment (per Cu)
appreciably decreases with increasing temperature, which
suggests the presence of a weak ferromagnetic interaction.
The μ value is slightly reduced from the spin-only value due to
population of the singlet excited state of the dimer.
Ferromagnetic coupling is much less frequent in dinuclear
copper(II) complexes than the antiferromagnetic behavior
usually observed.14 We actually suspect that in the present
case the weak interaction, probably mediated by a solvent
molecule, occurs intermolecularly, because the rigidity of the
bis-amide linker that connects the two tridentate chelating
arms and the steric hindrance of the trityl substituent groups
are expected to hinder the close approach of the two
copper(II) centers within the same molecule. Support to this
interpretation comes from the catalytic experiments described
below. On the other hand, we did not observe appreciable
temperature or concentration dependence in the electronic
spectra of the complex.
coupling and indeed the H-NMR spectra of [Cu2(EHI)]4+ show
1
temperature dependence (Figure 3S). However, as signal
assignment is difficult without
a thorough analysis we
preferred to obtain information on the nature of the coupling
through the Evans method. Measurements of magnetic
susceptibility on [Cu2(EHI)]4+ were then performed in
deuterated methanol/acetate buffer (50 mM, pH = 5.1) 10:1
v/v, to replicate the conditions to be used in the study of
catalytic oxidations. Acetone was chosen as internal standard
and added in 10 % (v/v), a concentration much larger than the
paramagnetic substance. Figure 2 shows that temperature
variation has a strong effect in both position and signal shape,
but the most interesting parameter is the shift between the
acetone signal of the solution in the absence and presence of
[Cu2(EHI)]4+. In the latter solution, the acetone signal is
affected by interaction with the paramagnetic compound. This
causes a low-field shift of the signal with respect to the
reference signal. The analysis was performed in the
(a)
(b)
Figure 2. (a) 1H-NMR spectra collected at increasing temperature (from -15 to +45 °C) of [Cu2(EHI)]4+ in deuterated methanol/acetate buffer (pH 5.1, 50 mM) 10:1 (v/v), containing
10 % (v/v) acetone as reference compound; (b) Magnification of the range centered on acetone signal near 2.2 ppm. 1: -15 °C; 2: 0 °C; 3: 15 °C; 4: 28 °C; 5: 45 °C.
Kinetic experiments of catechol oxidation. The oxidation of a set corresponding aminochromes. The latter are relatively stable
of chiral biogenic catechols, L-/D-Dopa, the corresponding intermediates produced by cyclization of the initially formed
methyl esters L-/D-DopaOMe, and R-/S-norepinephrine quinone, which undergo further oxidation and oligomerization
(Scheme 4), was studied through the increase of the reactions (Scheme 5).15 Therefore, aminochromes are suitable
absorption band near 475 nm due to the formation of the compounds for the determination of initial rates of the
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reactions. As solutions of catecholic substrates revealed
relatively high air instability when the pH was greater than 7,
For R-/S-norepinephrine the plots display a sigmoidal behavior
(Figure 4S), which suggests the presence of a mechanism of
the rate dependence of catechol oxidation catalyzed by substrate activation, through cooperative binding. The
[Cu2EHI]4+ was determined at pH 5.1, even though in this pH
following equation (for derivation see Supplementary
Information) was therefore used to extrapolate the kinetic
parameters from the experimental data (S=substrate):
range the catalytic efficiency of the complex is significantly
reduced. However, the catecholic substrates do not undergo
appreciable autoxidation by air oxygen up to pH 6, so that in
these experimental conditions there is no need to subtract the
contribution of non-catalytic reaction.
This behavior can be explained by considering that the binding
of the first molecule of substrate results in an altered affinity
or increase in the rate of product formation for a second
molecule of substrate. It is difficult to hypothesize the
mechanism of this effect, however we note that for an
efficient catechol oxidation, the substrate should be bound as
a bridging ligand to the dicopper(II) center.10 In the latter
equation, K’ is a constant comprising the interaction factors
leading to the complex bound to two substrate molecules and
has no longer the meaning, nor the units, of KM. A comparison
of the two constants is inappropriate, and therefore the
enantiodifferentiating potential of the complex against the
two norepinephrine enantiomers was evaluated as a ratio
between the oxidation rate of the two isomers (vR/vS) at low
(0.015 mM) and high (0.8 mM) substrate concentrations,
respectively. The comparison of the kinetic parameters,
summarized in Table 2, reveals that the oxidation of L-/D-Dopa
occurs with the highest rate among the series of substrates,
with kcat values almost one order of magnitude larger than the
other catechols. This result is unusual among the reactions
catalyzed by chiral dinuclear complexes studied before,12,16 for
which the reactivity is larger for DopaOMe. On the other hand,
chiral recognition of L-/D-Dopa is almost negligible. For Dopa
substrates, it is likely that an unproductive binding occurs to a
single copper(II) center of [Cu2EHI]4+, through the amino and
carboxylate groups. The reaction then probably occurs with
negligible recognition on a second substrate molecule binding
through the catecholato residue.
NH2
COOH
NH2
COOMe
NH2
HO
OH
OH
OH
OH
(A)
OH
(B)
OH
(C)
Scheme 4. Structure of the chiral catechol substrates: (A) Dopa, (B) DopaOMe, (C)
norepinephrine.
R
R
R
R
HO
HO
O
O
O
O
HO
HO
[O]
[O]
NH2
NH2
N
H
N
H
aminochrome
(475 nm)
quinone
(400 nm)
catechol
leukoaminochrome
Scheme 5. Oxidation pathway of catecholamine derivatives; the aminochrome is the
only observable intermediate in normal conditions.
The rate dependence on substrate concentration was thus
studied by keeping the catalyst concentration constant at 1.0
μM and varying that of substrate between 0.05 mM and 1.0
mM. For the oxidation of L-/D-Dopa and L-/D-DopaOMe
(Figure 4S), the plots of the initial rates exhibited a hyperbolic
behavior and the kinetic parameters (kcat, KM) could be
determined using simple Michaelis–Menten treatment. This
enables to consider a simplified reaction scheme implying fast
initial copper(II) reduction by one molecule of catechol,
followed by
a
complex series of events involving pre-
second catechol molecule and
equilibrium binding of
a
dioxygen to the dicopper(I) species and, finally, substrate
oxidation and irreversible release of quinone product.
The plots clearly show a negligible enantiodifferentiation for L-
/D-Dopa and only a modest preference for the L-enantiomer of
DopaOMe (Figure 4S). The enantioselectivity indexes Rk
cat/KM
and Rkcat, expressed as percentages, are used as useful
parameters to evaluate the discriminating effect observed in
the catalytic oxidations at low and saturating substrate
concentration, respectively:17
(kcat/Km)L - (kcat/Km)D
Rkcat/Km % =
x 100
(kcat/Km)L + (kcat/Km)D
(kcat)L - (kcat D
)
x 100
R
kcat % =
(kcat)L + (kcat D
)
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Table 2. Kinetic parameters for the enantioselective oxidation of L-/D-Dopa, L-/D-DopaOMe and R/S-norepinephrine at pH 5.1 and 20 °C, using 1.0 and 5.0 μM concentrations of
[Cu2EHI]4+
.
KM (mM)
kcat (s-1)
K’ (mM2)
Rkcat/KM
%
Rkcat %
[Cu2EHI]4+ / 1.0 μM
L-Dopa
D-Dopa
(2.10±0.58)×10-1
(2.22±0.56)×10-1
(9.93±0.96)×10-3
(1.08±0.09)×10-2
-
-
-1
-4
L-DopaOMe
(2.52±0.29)×10-1
(1.99±0.08)×10-3
(1.50±0.11)×10-3
(2.84±0.01)×10-3
(3.32±0.09)×10-3
-
+15
1.2a,c
+14
D-DopaOMe
(2.55±0.52)×10-1
-
R-norepinephrine
-
-
(4.15±0.01)×10-2
(4.15±0.59)×10-2
1.2b,c
S-norepinephrine
[Cu2EHI]4+ / 5.0 μM
L-Dopa
(6.89±2.63)×10-1
(5.18±0.82)×10-1
(3.89±0.01)×10-2
-
(2.57±0.56)×10-1
(3.34±0.75)×10-1
(1.68±0.36)×10-3
(1.27±0.09)×10-3
(5.36±0.01)×10-4
(6.05±0.01)×10-4
(4.07±0.31)×10-4
(4.98±0.39)×10-4
-
∼
0
+14
0.9b,c
-10
D-Dopa
-
L-DopaOMe
D-DopaOMe
-
12.8a,c
(6.63±0.01)×10-2
-
R-norepinephrine
S-norepinephrine
+3
-
a Ratio of vR/vS at low [S], corresponding to the ratio between the oxidation rate for the
R and S enantiomers extrapolated at the
b
substrate concentration of 0.015 mM. Ratio of vR/vS at high [S], corresponding to the ratio of the two oxidation rates,
c
extrapolated at the substrate concentration of 0.8 mM. Values of vR/vS ratio above 1.0 suggest a preference for the
R
enantiomer of norepinephrine, whereas for values of this ratio lower than 1.0 the discrimination favors the opposite enantiomer.
Surprisingly, when the catalytic oxidations were studied at a the more reactive monomeric form of the dinuclear complex is
larger concentration (5.0 μM) of [Cu2EHI]4+, the reaction rates present (Figure 3).
significantly dropped, with kcat almost one order of magnitude
smaller (Table 2). In addition, in these conditions, the
sigmoidal behavior was observed only for the oxidation of D-
DopaOMe (Figure 5S), which suggests a peculiar interaction of
this particular substrate with the chiral complex. The marked
decrease in the oxidation rates suggests the existence of
different forms of the complex when the concentration is
changed. We assume that, for concentrations of 5 μM, or
higher, the active form of the complex in the catalytic reaction
could be better represented by a dimeric species, that could
be promoted, for instance, by an intermolecular bridge formed
by a water molecule or hydroxo group (Scheme 6). The two-
electron transfer by the substrate to the dicopper(II) center
occurs intermolecularly, after the substrate displaced the
bridging ligand to activate the catalytic site. This step is not
contemplated at lower complex concentrations (1 μM), when Scheme 6. Hypothesis on the formation of a dimeric species of the complex and its
involvement in catalysis (L: solvent molecule; S: catecholic substrate.
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factor of almost 13 (Table 2). This corresponds to >90% ee and
appears to be the highest ee so far reported for this type of
reaction by any biomimetic copper complex. Surprisingly,
though, the enantioselectivity progressively decreases at
higher substrate concentrations, which suggests that the
binding of L-DopaOMe to dimeric [Cu2EHI]4+, although
sterically favored, gives rise to an adduct characterized by a
slower electron transfer rate with respect to the opposite D-
enantiomer. This can only depend on the less favorable spatial
arrangement of the orbitals of the ternary CuII-catecholate-CuII
complex involved in the electron transfer process.
Binding of L-/D-tyrosine methyl ester to Cu2[EHI]4+. To gain
information about the difference in coordination affinity of
chiral catecholic and phenolic substrates, we investigated the
binding behavior of the L-/D-enantiomers of tyrosine methyl
ester to [Cu2(EHI)]4+, using their phenolate salts to improve
their coordination through the phenolate oxygen donor. To
prevent solubility problems, the spectrophotometric titration
was carried out in methanol solution and using the
diazabicycloundecene (DBU) salt of the tyrosine derivatives, as
described in the Experimental Section. The optical titration
experiments are shown in Figure 5.
Figure 3. Effect of catalyst concentration on the initial rates of air oxidation of the
enantiomers of norepinephrine by [Cu2EHI]4+ studied at pH 5.1 (black symbols:
[Cu2(EHI)]4+ = 1 µM; empty symbols: [Cu2(EHI)]4+ = 5 µM).
To probe the hypothesis of an involvement of a dimeric form
of the complex, the oxidation of L-DopaOMe was studied as a
function of [Cu2(EHI]4+ concentration. These experiments were
carried out using a substrate concentration of 0.5 mM and
varying the catalyst concentration between 5× ×
10-7 and 1 10-5
M. As shown by the plots shown in Figure 4, as the [Cu2(EHI)]4+
concentration increases, the rate constant decreases
exponentially, indicating a quadratic dependence on catalyst
concentration (see Supporting Information for derivation of
the kinetic equation).
Upon addition of tyrosine methyl ester salts, the growth of an
absorption band near 430 nm is observed, which is
attributable to a LMCT band from the phenolate to the
copper(II) ions.18 The coordination of the tyrosinato ligand to
the dicopper complex also causes a blue shift of the d-d band
to about 600 nm. By processing the spectrophotometric data
with a non linear least-square procedure, the ligand binding
process is explained as the sequence of two steps, according to
which both a 1:1 and 2:1 tyrosinato adducts of [Cu2(EHI)]4+ are
formed (Figure 6). Even though the model is simplified, as
more species may be present in solution, the calculated
spectra for the 1:1 and 2:1 adducts are very informative,
because in the first step it is clear that binding of the
tyrosinate ion to [Cu2(EHI)]4+ does not involve ligation through
the phenolate group, which does only occur in the second
step. We then hypothesize that the first tyrosinate ligand
(TyrOMe−) binds to one of the copper(II) centers through the
amino group, and only the second one to the other copper(II)
center through the expected O(phenolate) donor:
[Cu2(EHI)]4+ + TyrOMe−
[Cu2(EHI)-N(TyrOMe)]3+
N(TyrOMe)-O(TyrOMe)]2+
[Cu2(EHI)-N(TyrOMe)]3+
TyrOMe−
[Cu2(EHI)-
+
Even though we could not calculate reliable binding constants
for the various types of adducts, it is clear from the plots in
Figure 6, that formation of the 1:2 [Cu2(EHI)(tyrosinato)2]2+
complex occurs at lower ligand concentration for L-tyrosine
methyl ester than for D-tyrosine methyl ester and hence the
affinity of [Cu2(EHI)]4+ for the L-tyrosinato ligand is higher than
for D-tyrosinato isomer, in agreement with the behavior of the
corresponding chiral catechols.
Figure 4. Dependence of the oxidation rate of L-DopaOMe on the concentration of
[Cu2(EHI)]4+
divided for εaminochrome. Bottom plot: Rate data expressed as ((A/s)/3600)/[Cu2(EHI)]4+
. Upper plot: Rate data are expressed as Absorbance/time (A/s) units
.
Even if less catalytically efficient, the dimeric form exhibits a
remarkable enantioselectivity in the oxidation of DopaOMe
substrates, with a strong preference for the L-enantiomer, by a
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a)
b)
0.4
0.8
0.3
0.2
0.1
0.0
0.6
0.4
0.2
0.0
400
600
800
1000
400
600
800
1000
Wavelength (nm)
Wavelength (nm)
Figure 5. UV-Vis spectra recorded upon titration of [Cu2(EHI)]4+ (5×10-4 M) in methanol solution with the DBU salt of L-tyrosine methyl ester (a) and D-tyrosine methyl ester (b),
from 0:1 to 10:1 [tyrosinate]:[Cu2] molar ratios. The spectra were recorded at 25±0.1 °C.
120
100
80
60
40
20
0
0.5
0.4
0.3
0.2
0.1
0.0
120
100
80
60
40
20
0
0.25
0.20
0.15
0.10
0.05
0.00
0
2
4
6
8
10
0
2
4
6
8
10
eqv of D-tyrosinato
eqv of L-tyrosinato
Figure 6. Absorbance profile at 428 nm during the titration of [Cu2(EHI)]4+ with methyl L-tyrosinate (left) and methyl D-tyrosinate (right), superimposed to species distribution
diagram. Diamonds: experimental absorbance values; dotted line: calculated absorbance; long dashed line: distribution of free [Cu2(EHI)]4+; short dashed line: distribution of the
1:1 [Cu2(EHI)-tyrosinato]3+ complex; dashed and dotted line: distribution of the 1:2 [Cu2(EHI)(tyrosinato)2]2+ complex.
Hydroxylation of N-acetyl-L/D-tyrosine ethyl ester. The (20 equiv.) followed by exposure to air oxygen. We also tried in
hydroxylation of tyrosine, and other phenolic substrates, is the several attempts, albeit unsuccessfully, to characterize a
most important activity of tyrosinase.19 The enzyme exhibits a copper/dioxygen adduct in various organic solvents at low
marked preference for L-tyrosine than for D-tyrosine, and also temperature (down to -80 °C in dry acetone, -90 °C in dry
for L-Dopa vs. D-Dopa,20 and the difference depends dichlorometane, -120 °C in dry 2-methyl tetrahydrofuran). The
exclusively on KM. It was therefore interesting to investigate Cu2O2 complex does not accumulate because as soon as it
the oxidation of the two enantiomers of tyrosine by the forms it reacts with dicopper(I), and for this reason exposure
dicopper-EHI complex. As the phenol hydroxylation is much to O2 of the reaction solution was always allowed after adding
slower than catechol oxidation, here the problem of the high the phenolic substrate to [Cu2(EHI)]2+. In any case, the color of
reactivity of the quinone is more dramatic than before. the solution turns brown, indicating that the reaction occurs,
Therefore, these experiments were performed using the and we made several attempts trying to isolate and
tetrabutylammonium salt of N-acetyl L-/D-tyrosine ethyl characterize the oxidation products. However, the material
esters, to prevent cyclization to aminochrome and trying to isolated after work-up is probably a mixture of oligomeric
slow down the reactivity of the quinone. As the dicopper(II) products. The high reactivity of quinones towards nucleophiles
form of the complex is not competent in the phenol is well known also for the enzymatic reaction.21 We could
hydroxylation,10,19 the reactions were carried out by preparing actually separate
a main product of the reaction by
the dicopper(I) complex, [Cu2(EHI)]2+, anaerobically, at -80 °C in chromatography and tried to characterize it by NMR and MS
dry acetone and adding an excess of the phenolate substrate (Figure 6S-8S). The MS data (405 a.m.u.) and the pattern of
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NMR signals would be in agreement with the following reaction Scheme 7. Labeling experiments with 18O2 showed
trimeric structure (or some isomeric structure):
complete absence of 18-O atom into the product, which
suggests that also the naphthol hydroxylation reaction, as
tyrosine oxidation, likely occurs through a radical mechanism.
NH2
NH2
O
OH
OH
N
H
which appears to derive from C-C and C-O radical coupling
reactions of phenolic rings, decarboxylation and cyclization of
one free amino group of the side chains, after deacetylation.
As a main scope of the present investigation is to assess the
enantiodiscriminating properties of the dicopper-EHI system,
we monitored the oxidation of the enantiomeric L-/D-tyrosine
derivatives spectrophotometrically, after exposing to air a
solution of [Cu2(EHI)]2+ and excess phenolate salts of N-
protected tyrosine ethyl esters (20 equiv.) initially prepared
anaerobically. In the initial part of the reaction, a broad
absorption band centered around 400 nm, attributable to
some quinone product(s), increases with time (Figure 7 and
9S). The time profile of this band clearly shows a steeper
increase for the L-tyrosinate substrate with respect to the D
enantiomeric counterpart, indicating a faster reaction. The
initial rates, in Δabsorbance/s units, are in fact 7.4×10-5 for
ethyl N-acetyl-L-tyrosinate and 3.4×10-5 for ethyl N-acetyl-D-
tyrosinate, that is in line with the binding preference for the L-
tyrosinato ligand with respect to D-tyrosinato by [Cu2(EHI)]4+.
Scheme 7. The steps involved in the hydroxylation of β-naphthol (as DBU salt) by
[Cu2(EHI)]2+/O2, with the subsequent addition of β-naphtholate to the naphthoquinone
product of the monooxygenase reaction.
Conclusion. The dinucleating N6 ligand EHI, obtained
through a relatively easy synthetic approach, represents the
first member of a new family of chiral hexadentate ligands
with potential applications in stereoselective catalysis. The
different type of linkage of the two tridentate coordinating
units with respect to the previous ligands of the PHI
family,11,12b appears to be advantageous in terms of
stereochemical rigidity of the chelating rings, which are not
inverted by the binding of exogenous ligands such as azide.
Apparently, the opposite conformational preference of the
chelate rings of [Cu2EHI]4+ with respect to copper-PHI type
complexes11,12b does not change the binding preference
toward L-Dopa vs. D-Dopa and L-tyrosine vs. D-tyrosine
derivatives. However, while the enantioselectivity effects in
the catalytic oxidations of catechols performed with copper-
PHI complexes were entirely due to the L-/D binding
discrimination, for [Cu2EHI]4+ marked differences occur in the
electron transfer step, indicating a differential orientation of
the aromatic ring of the bound enantiomeric substrates with
respect to the catalytic center.
0.10
a)
0.08
0.06
b)
Although we were unable to obtain
a full structural
0.04
characterization of [Cu2EHI]4+, because we could not obtain
suitable crystals, the catecholase experiments clearly indicate
that the current limit of the EHI structure is in the relatively
rigid ethanediamide moiety connecting the tridentate binding
units, which makes easier an intermolecular binding of the
catechol substrates at relatively low concentration. The
presence of equilibria between monomeric and dimeric forms
of the dinuclear complexes complicates a full understanding of
the catalytic reactions. However, it is worthy of note that the
chiral discrimination capacity of [Cu2EHI]4+ towards catecholic
substrates, in the best conditions, favorably compares with
that of the large family of dicopper complexes with related
chiral ligands studied before. High enantioselectivity has been
observed in particular for the oxidation of R-/S-norepinephrine
isomers, which usually occurs with poor discriminating
behavior, probably because of the benzylic position of the
chiral center. Work is currently in progress to replace the
ethanediamide linker of EHI with longer and more flexible
residues to make intramolecular cooperation between the two
metal centers easier. This should enable to obtain dicopper
complexes with genuine monooxygenase activity against
phenolic substrates, which for the present dicopper-EHI
complex occurs through a non-biomimetic radical mechanism.
0.02
0.00
0
1000
2000
3000
Time (s)
Figure 7. Time profile of the absorption at 400 nm during the oxidation of the
tetrabutylammonium salts of ethyl N-acetyl-L-tyrosinate (a) and ethyl N-acetyl-D-
tyrosinate (b) by [Cu2(EHI)]2+/O2 in acetone solution at 25 °C.
Hydroxylation of β-naphthol. In order to probe the capability of
the [Cu2(EHI)]2+/O2 system to perform phenol hydroxylation,
we used a hydrophobic substrate, β-naphthol, that could
enable a easier separation and characterization of the product.
The reaction was carried out using an excess of DBU salt of β-
naphthol and [Cu2(EHI)]2+ in degassed acetone, upon exposure
to air at room temperature. The color of the solution turned
visually from colorless to pale gold yellow and the reaction was
allowed to proceed for several hours. The product of the
reaction could be separated by conventional chromatography
from the excess unreacted phenolate, and was identified as
the regiospecific conjugate addition product of β-naphthol to
the initially formed 1,2-naphthoquinone, as outlined in the
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mmol) was added to a solution of 2 (1.57 g, 1.2 mmol) in
dichloromethane (10 ml). After stirring for 1.5 h at room
Experimental Section
General considerations. All preparations were carried out temperature, the solvent was rotary-evaporated giving a
using standard Schlenk techniques under an inert atmosphere yellow oil, which was washed with diethyl ether, affording a
of N2 unless otherwise stated. Solvents were dried over highly hygroscopic white powder (1.01 g, nearly quantitative
standard drying agents and stored over 3-Å molecular sieves. yield). 1H NMR (400 MHz, CDCl3): δ 7.93–7.82 (s, 2H), 7.43–
All starting materials were of reagent grade and purchased 7.30 (m, 20H), 7.20–7.06 (m, 12H), 6.65 (s, 2H), 3.66–3.56 (m,
from either Sigma-Aldrich Chemical Co. or VWR International 2H), 3.43–3.30 (m, 2H), 3.31–3.20 (m, 2H), 2.96 (dd, J = 14.4,
and used without further purification. Nα-Fmoc-N(Im)-trityl-L- 3.9 Hz, 2H), 2.75 (dd, J = 14.4, 8.0 Hz, 2H). MS (ESI): m/z +819
histidine (1
) was purchased from Novabiochem, and used [M+H+].
without further purification. Optical purity of the final ligand Synthesis of (2S,2'S)-N,N'-(ethane-1,2-diyl)bis(2-((1-methyl-
EHI was determined via HPLC using a Jasco HPLC instrument 1H-imidazol-4-ylmethyl)-amino)-3-(1-trityl-1H-imidazol-4-
equipped with two PU-1580 pumps and a MD-1510 diode yl)propanamide)
array detector (working range: 195–659 nm) with a chiral dissolved in dichloromethane (10 mL) under an inert
column Lux 5u Cellulose-1 (0.46 25 cm), eluent: hexane:2- atmosphere. solution of 1-methyl-2-imidazole
propanol = 85:15, flow rate: 0.8 ml min-1.
carboxaldehyde (230 mg, 2.09 mmol) in dichloromethane (1.5
(EHI). Compound 3 (612 mg, 0.75 mmol) was
×
A
Elemental analyses were obtained at the Microanalysis service mL) was slowly added. The reaction was stirred for 2 h at room
of the Milano Chemistry Department. CD spectra were temperature. The acid labile diimine could not be visualized by
obtained with a Jasco J500 spectropolarimeter; the spectra TLC, so the reaction was monitored by ESI–MS. Given the
were recorded at 0.2 nm resolution in the range between 300 predominant formation of the disubstituted product, the
and 700 nm at 50 nm min–1 with three scans acquired for each material was transferred directly into the reduction reaction.
spectrum. NMR spectra were recorded with a Bruker AVANCE Solid NaB(OAc)3H (12 equiv. in 4 portions) was added portion-
400 spectrometer operating at 9.37 T with frequencies of wise and the suspension was stirred at room temperature for
1
400.13 and 100.6 MHz for H and 13C NMR, respectively. The two more h. The solvent was removed and the yellow oil was
data acquisition and processing were performed with
standard Bruker software package (Topspin 1.3). The UV/Vis diethyl ether-2-propanol (5:3 v/v) and an increasing amount of
spectra were recorded with an Agilent 8453 aqueous ammonia (from 1% to 4%), to remove the reduced
a
purified by column chromatography on silica gel, with eluent
spectrophotometer. Mass spectra were recorded with
Thermo-Finnigan LCQ 371 ADV MAX spectrometer.
a
imidazole-carboxaldehyde (Rf=0.86). The final product was
recovered by chromatography on silica using a mixture of
Synthesis of bis[(9H-fluoren-9-yl)methyl]-[(2S,2'S)-(ethane- chloroform-methanol-aqueous ammonia (10:1:0.15 v/v) as
1,2-diyl-bis(azanediyl)]bis[1-oxo-3-(1-trityl-1H-imidazol-4-
eluent, giving EHI as a colorless oil (150 mg, 26 %). 1H NMR
yl)propane-2,1-diyl)]dicarbamate (2). Nα-Fmoc-N(Im)-trityl-L- (400 MHz, CDCl3): δ 7.42–7.22 (m, 20H), 7.15–7.02 (m, 12H),
histidine (2.00 g, 3.2 mmol) was suspended in 6.74 (s, 2H), 6.69 (s, 2H), 6.60 (s, 2H), 3.82–3.67 (m, 4H), 3.58
dichloromethane (8 mL) and (benzotriazol-1-yloxy)- (s, 6H), 3.47–3.15 (m, 6H), 2.91 (dd, J = 14.4, 5.7 Hz, 2H), 2.78
tripyrrolidinophosphoniumhexafluorophosphate (PyBOP) (1.66 (dd, J = 14.4, 7.3 Hz, 2H). 13C NMR (100 MHz): δ 174.81 (Q),
g, 3.2 mmol) was poured into the solution. Ethylenediamine 146 (Q), 138.6 (CH), 137.8 (Q), 130.11 (CH), 128,5 (CH), 126.95
(108 μL, 1.6 mmol) was added to the solution and, finally, N,N- (CH), 121.6 (CH), 119.9 (CH), 75.6 (Q), 62.8 (CH), 44.14 (CH2),
diisopropylethylamine (DIPEA, 2.2 mL, 12.8 mmol) was further 39.53 (CH2), 33.2 (CH3), 31.95 (CH2). MS (ESI): m/z +1007
added as a base. The reaction mixture was stirred for 3 h at [M+H+].
room temperature. Then, the solvent was rotary-evaporated, Synthesis of [Cu2EHI][ClO4]4. The ligand EHI (50 mg, 0.05
yielding a highly viscous colorless oil, which was purified by mmol) was dissolved in methanol (800 µL) yielding a yellow
silica chromatography using a mixture of dichloromethane- solution. Then, copper(II) perchlorate hexahydrate (37 mg,
methanol (from 98:2 to 0:100, v/v) as eluent. A white foamy 0.10 mmol) in methanol (500 µL), from a stock solution, was
solid product (2.57 g, quantitative yield) was obtained, after added dropwise. The color of the solution changed from
treatment with diethyl ether. 1H NMR (400 MHz, CDCl3, 298 K): yellow to shining green, until a green solid precipitated. The
δ 9.72, (br, s), δ 7.75 (d, J = 7.0 Hz, 2H), δ 7.73 (d, J = 7.0 Hz, solid was recovered by filtration, washed with diethyl ether
2H), 7.55 (d, J = 7.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.44 – 7.30 (3×1 mL) and dried in air (yield: 65%). Anal. Calcd for
(m, 24H), 7.25 (d, J = 7.0 Hz, 2H), 7.23 (d, J = 7.0 Hz, 2H), 7.19– C62H62Cl4Cu2N12O18·4CH3OH (1656.3): C 47.86, H 4.74, N 10.12;
7.11 (m, 12H), δ 7,01 (dd, 2H), 6.64 (s, 2H), 6.04 (d, J = 6.0 Hz, found
1H), 4.53–4.44 (m, 2H), 4.33–4.06 (m, 6H), 3,5 (dd, 2H), 3.41 ([[Cu2(EHI)](ClO4)3]3+)
(d, J = 13.4 Hz, 2H). 13C NMR (100 MHz, 298 K): δ 170 (Q); 156 Synthesis of L- and D-tyrosine methyl ester. The L
C
47.17,
H
4.34,
N
9.87. ESI-MS:+1431
-
or D-
(Q), 144 (Q), 142 (Q), 138 (CH), 135 (Q), 130 (CH), 128,5 (CH), tyrosine powder (100 mg, 0.552 mmol) was suspended in
125,6 (CH), 120, 8 (CH), 120,31 (CH), 76 (Q), 67 (CH2), 53.5 methanol and cooled in an ice/NaCl bath. Then, SOCl2 (1.49
(CH), 47.5 (CH), 38 (CH2), 31 (CH2). MS (ESI): m/z +1263 mmol) was slowly added under stirring and the solution was
[M+H+], +1285 [M+Na+], +1302 [M+K+].
Synthesis of (2S,2'S)-N,N'-(ethane-1,2-diyl)bis(2-amino-3-(1- solvent was removed by rotary evaporation to give the pure
brought to room temperature and stirred overnight. The
trityl-1H-imidazol-4-yl)-propanamide) (3). Piperidine (2 ml, 20 product.
10 | J. Name., 2012, 00, 1-3
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General procedure for the synthesis of the DBU salts of Kinetic studies of L-/D-Dopa oxidation. Two stock solutions of
phenolic substrates. The phenol was dissolved in 1 mL of 8×10-4 M of L- and D-Dopa were prepared in a 1:1 (v/v)
methanol and an equimolar amount of diazabicycloundecene mixture of methanol/aqueous buffer. To favor the dissolution
was added. The solution was stirred for 30 min at room in the stock solution mixture, we found it useful to dissolve the
temperature and the solvent removed by rotary evaporation, substrates first in buffer solution and then in the remaining
to give the pure product.
volume of methanol, with mild warming of the mixture. The
Azide Binding Studies. Titrations with azide were performed exact substrate concentration was then controlled on the basis
by addition of a concentrated methanolic solution of sodium of the absorbance values measured at 283 nm, the wavelength
azide to the solution of [Cu2EHI]4+ in MeOH/MeCN 9:1 (v/v). All of maximum absorbance for the catechols (εDopa = 2700 M-1
measurements were done at 25
incubation of the mixtures, as the anion binding was found to stock substrate solution to the desired concentration; after
be fast. Titration of [Cu2EHI]4+ (5.98 10-5 M) was performed by recording the blank spectrum, the reaction was initiated by the
stepwise addition of equal amounts of the azide solution addition of a small volume of a concentrated solution of the
(1.00
10-3 M) from 0.1 to 2.0 [azide]:[Cu2] ratios. It was catalyst dissolved in methanol (final concentration 5×10-6 M).
possible to separate the titration steps for [N3−]:[Cu2] between Absorption spectra of these solutions were recorded at a
0.1 and 1.0 ( max = 389 nm) and between 1.1 to 2.0 ( max = 382 regular time interval of 1 s for 5 min in the wavelength range
±
0.1 °C and without any cm-1). The kinetic measurements were performed diluting the
×
×
λ
λ
nm). The spectral data were analyzed as previously 200–800 nm. The reaction was followed by monitoring the
described,12b,15 to deduce equilibrium constants and increase in the absorbance at 475 nm (ε475 = 3600 M-1cm-1)22
stoichiometry of formation of the adducts. CD spectra of the as a function of time.
azide adducts were recorded after the addition of 1.0 and 2.0 Kinetic studies of L-/D-DopaOMe oxidation. These
equiv. of the ligand.
experiments were carried out following the same procedure
Determination of magnetic susceptibility and magnetic described before. Due to their better solubility in organic
moment of [Cu2(EHI)]4+ with the Evans method. This solvents, the stock solutions of L- and D-DopaOMe were
experiment was performed using
a coaxial NMR tube prepared in methanol, and their concentrations corrected on
containing solvent and reference compound in the presence the basis of the absorbance measured at 283 nm (εDopaOMe
=
(A) or absence (B) of the complex. Solution A was prepared as 2700 M-1 cm-1). The catecholase activity was followed
follows: 110 µL of 1 mM solution of [Cu2(EHI)]4+ in 10:1 (v/v) spectrophotometrically by monitoring the increase in the
deuterated methanol/acetate buffer (pH= 5.1, 50 mM) to absorbance at 468 nm (the ε value used was assumed to be
which 10 % volume of acetone (11 µL) was added. Solution B the same as that of dopachrome)22 as a function of time.
was prepared as follows: 660 µL of 10:1 (v/v) deuterated Kinetic studies of R-/S-norepinephrine oxidation. Also for the
methanol/acetate buffer (pH= 5.1, 50 mM) to which 10 % oxidation of S-/R-norepinephrine L-bitartrate we followed the
volume of acetone (66 µL) was added. Solution A was loaded same general procedure described before. The initial solutions
into the internal tube, while solution B loaded into the outer of the substrates were prepared in a 1:1 (v/v) mixture of
1
tube. H-NMR spectra were recorded at various temperatures methanol/aqueous buffer, and their concentrations corrected
(-15 °C, 0, 15 °C, 28 °C, 45 °C) and shifts between the acetone on the basis of the absorbance measured at 283 nm
(εnorepinephrine = 3070 M-1 cm-1). The catechol oxidations were
signals in solutions (A) and (B) were recorded.
Catalytic oxidations of o-catechols. The catecholase activity of followed spectrophotometrically by monitoring the increase in
complex [Cu2(EHI)]4+ was determined by studying the oxidation the absorbance at 480 nm (the ε value used was assumed to
of the chiral catechols L-Dopa, D-Dopa, L-DopaOMe, D- be the same as that of dopachrome)22 with time.
DopaOMe, R-(-)-norepinephrine, and S-(+)-norepinephrine. Binding of L- and D-tyrosine methyl ester to [Cu2EHI]4+.
×
The kinetic studies were carried out spectrophotometrically by Spectrophotometric titration of [Cu2EHI]4+ (5 10-4 M) in
the method of initial rates by monitoring the increase of each methanol solution was performed by addition of successive
characteristic aminochrome absorption band over time. The amounts of a methanolic solution of the DBU salt of each
reading wavelengths were: 475 nm for L-/D-Dopa, 468 nm for enantiomer of tyrosine methyl ester, 0.05 M, from 0:1 to 10:1
L-/D-DopaOMe, and 480 nm for R-/S-norepinephrine. The [tyrosinate]:[Cu2] molar ratios, maintaining
a constant
solvent used was a 10:1 (v/v) mixture of methanol/aqueous temperature of 25 0.1 °C. The spectral data were then
±
acetate buffer (50 mM, pH 5.1), saturated with atmospheric analyzed with HypSpec, 23 considering the spectral changes in
dioxygen. To check the contribution of the autoxidation the range of wavelengths between 370 and 764 nm.
reaction of the catechols, we used the same solution without Low temperature hydroxylation of tetrabutylammonium N-
adding the catalyst as internal reference. The experiments acetyl-L-tyrosinate ethyl ester. An oven-dried 100 mL round
were carried out over a substrate concentration range (from bottom flask equipped with a stirring bar and a rubber septum
5×10-5 to 1×10-3 M) at a constant temperature of 25±0.1 °C, to was degassed and backfilled with Ar, then charged with
determine the dependence of the rate on substrate anhydrous acetone (47 mL). Previously prepared solutions in
concentration. To maintain pseudo-first order conditions, a dry acetone of tetrabutylammonium salt of the tyrosine
5×10−5 M solution of [Cu2(EHI)]4+ was treated with a minimum derivative (1×10-5 mol in 1 mL) and EHI (5×10-7 mol in 1 mL)
of 10 equiv. of substrate, as the lowest concentration.
were cannulated into the reaction flask. The mixture was
cooled at -80 °C with a cryostat and carefully degassed and
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kept under N2. Separately, [Cu(CH3CN)4]PF6 (1×10-6 mol) was 2. S. Itoh, in Copper-Oxygen Chemistry, K. D. Karlin, S. Itoh Eds,
dissolved in dry and degassed acetone (1 mL). This solution Wiley, Hoboken, N. J., 2011, pp.225-282.
was cooled and added to the reaction flask, via cannula, under 3. M. R. Halvagar, D. J. Salmon, W. B. Tolman, In
Ar. To the reactor was subsequently applied an O2-filled Comprehensive Inorganic Chemistry II, Volume 3, J. Reedijk, K.
balloon to assure an approximately constant pressure of O2. At Poeppelmeier Eds., Elsevier, 2013, pp. 455-486.
the end of the reaction, the vessel was opened to the 4. M. Rolff, J. Schottenheim, H. Decker, F. Tuczek, Chem. Soc.
atmosphere and the mixture was quenched at low Rev., 2011, 40, 4077-4098.
temperature with 2 mL of 1 M HClO4. The organic solvent was 5. M. Rolff, J. Schottenheim, G. Peters, F. Tuczek, Angew.
removed in vacuo and the aqueous phase was extracted with Chem. Int. Ed., 2010, 49, 6438-6422.
CH2Cl2 (3×10 mL). The combined organic fractions were then 6. A. Hoffmann, C. Citek, S. Binder, A. Goos, M. Rübhausen, O.
dried over Na2SO4, filtered and rotary evaporated to afford an Troeppner, I. Ivanović-Burmazović, E. C. Wasinger, T. D. P.
oil which was analyzed directly by 1H-NMR. The crude reaction Stack, S. Herres-Pawlis, Angew. Chem. Int. Ed., 2013, 52, 5398-
mixture was purified using column chromatography using silica 5401.
gel and hexane as eluent.
7. I. Gamba, S. Palavicini, E. Monzani, L. Casella, Chem. Eur. J.
Hydroxylation of N-acetyl-L/D-tyrosine ethyl ester. 2009, 15, 12932-12936.
Spectrophotometric study. A solution of EHI (5×10-5 M) and 8. A. Spada, S. Palavicini, E. Monzani, L. Bubacco, L. Casella,
the tetrabutylammonium salt of the tyrosine derivative (10-3 Dalton Trans., 2009, 6468-6471.
M) was prepared in freshly distilled and degassed acetone (2 9. A. Alexakis, N. Krause, S. Woodward, Eds., Copper-Catalyzed
mL) in a screw-cap thermostated optical cell, stoppered with a Asymmetric Synthesis, Wiley-VCH, Weinheim, 2014.
rubber septum. A solution of [Cu(CH3CN)4]PF6 in freshly 10. G. Battaini, A. Granata, E. Monzani, M. Gullotti, L. Casella,
distilled and degassed acetone (10-4 M, 1 mL) was added to the Adv. Inorg. Chem. 2006, 58, 185- 233.
previous solution, through a cannula, maintaining the mixture 11. (a) L. Santagostini, M. Gullotti, R. Pagliarin, E. Monzani, L.
under an inert atmosphere. The reaction was initiated by Casella, Chem. Commun. 2003, 2186-2187; (b) L. Santagostini,
exposure to dioxygen and followed by spectrophotometry, M. Gullotti, R. Pagliarin, E. Bianchi, L. Casella, E. Monzani,
monitoring the increase in intensity of the broad absorption Tetrahedron: Asymmetry 1999, 10, 281-295; (c) M. Gullotti, L.
band at 400 nm during 3600 s. The comparison of the Santagostini, R. Pagliarin, S. Palavicini, L. Casella, E. Monzani,
reactivity of the two tyrosine enantiomers was made through G. Zoppellaro, Eur. J. Inorg. Chem. 2008, 2081-2089.
the initial rates of development of the band at 400 nm.
12. (a) M. C. Mimmi, M. Gullotti, L. Santagostini, G. Battaini, E.
Hydroxylation of β-naphthol. A solution of EHI (5×10-5 M) and Monzani, R. Pagliarin, G. Zoppellaro, L. Casella, Dalton Trans.
the DBU salt of the naphthol (1×10-3 M) was prepared in 2004, 2192-2201; (b) F. G. Mutti, M. Gullotti, L. Casella, L.
degassed, dry acetone (20 mL), maintaining an argon Santagostini, R. Pagliarin, K. Kristoffer Andersson, M. F. Iozzi,
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4
M, 1 mL) was added to the above solution, with a gas-tight K. Kristoffer Andersson, L. Casella, Eur. J. Inorg. Chem. 2009,
syringe. The reaction was initiated by the addition of dioxygen 554-566.
and was stirred at room temperature overnight; the color of 13. (a) L. Casella, M. Gullotti, J. Inorg. Biochem. 1983, 18, 19-
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solvent was removed by rotary evaporation, then the crude L. Casella, M. Gullotti, Inorg. Chem. 1985, 24, 84-88.
product was dissolved in dichloromethane and a few drops of 14. I. Bertini, C. Luchinat, "NMR of paramagnetic substances",
distilled water were added, followed by a small amount of the Elsevier, Amsterdam, 1996, Chapter 5.
disodium salt of EDTA. The organic phase was separated and 15. (a) J. D. Simon, D. N. Peles, Acc. Chem. Res. 2010, 43, 1452-
evaporated to give the crude product. Purification by liquid 1460; (b) A. Napolitano, P. Manini, M. d'Ischia, Curr. Med.
chromatography, using silica gel (230-400 A mesh) and eluting Chem. 2011, 18, 1832-1845; M. d'Ischia, A. Napolitano, V. Ball,
with hexane/ethyl acetate 7:3 gives the final product (Rf= C.-T. Chen, M. J. Buehler, Acc. Chem. Res. 2014, 47, 3541-3550.
0.38). 1H NMR (400 MHz, acetone-d6) δ 8.82 (s, 1H), 8.13 (d, J = 16. M. L. Perrone, E. Lo Presti, S. Dell’Acqua, E. Monzani, L.
7.3 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.77 Santagostini, L. Casella, Eur. J. Inorg. Chem. 2015, 3493-3500.
(d, J = 7.6 Hz, 1H), 7.56 (dd, J = 11.6, 7.4 Hz, 2H), 7.44 –7.21 (m, 17. T. Plenge, R. Dillinger, L. Santagostini, L. Casella, F. Tuczek,
J = 7.1 Hz, 4H), 6.85 (d, J = 7.6 Hz, 1H), 6.46 (s, 1H).
Z. Anorg. Allg. Chem. 2003, 629, 7381-7389.
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Acknowledgments
This work was supported by the Italian MIUR. CIRCMSB is also Chem. 2002, 41, 479-491.
gratefully acknowledged.
19. (a) N. Wang, D. N. Hebert, Pigm. Cell Res. 2006, 19, 3-18;
(b) E. I. Solomon, D. E. Heppner, E. M. Johnston, J. W.
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3853 .
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Graphical abstract
A new dicopper(II) complex with histidine-derived N6 ligand
perfoms biomimetic stereoselective oxidation of catechols.
O
O
N
N
H
H
NH
NH
Cu2+
N
Cu2+
N
N
N
N
N
N
N
Trt
Trt
14 | J. Name., 2012, 00, 1-3
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