A Stereoselective Tyrosinase Model Compound Derived from an m-Xylyl- l -histidine Ligand

Article abstract of DOI:10.1021/acs.inorgchem.9b00473

The aim of mimicking enzyme activity represents an important motivation for the development of new catalysts. A challenging objective is the development of chiral complexes for bioinspired enantioselective oxidation reactions. Herein, we report a new chiral dinuclear copper(II) complex based on a m-xylyl-bis(histidine) ligand (mXHI) as a biomimetic catalyst for tyrosinase and catechol oxidase. The new ligand improves a previous system also containing two tridentate N3 units derived from l-histidine that were connected by a short, rigid ethanediamine bridge. In mXHI the bridge is provided by the more extended m-xylyl moiety. The dicopper(II) complex [Cu₂(mXHI)]⁴⁺ was studied as a catalyst for stereoselective oxidations of enantiomeric couples of chiral catechols of biological interest (L/D-dopa, L/D-dopa methyl ester, and (R/S)-norepinephrine), showing excellent discrimination capability, particularly for the methyl esters of dopa enantiomers. The catechol oxidation was studied in acetate buffer as slightly acidic medium, and a role of acetate as bridging ligand between the two coppers, preorganizing the dinuclear center in a more catalytic efficient structure, could be established. The oxidation of β-naphthol and 3,5-ditertbutylphenol was studied as a model monophenolase reaction. The oxidation proceeds stoichiometrically, and the partial incorporation of ¹⁸O into β-naphthol when the reaction was performed using ¹⁸O₂ suggests the existence of two competitive reaction pathways, a genuine monooxygenase mechanism and a radical pathway. However, the more challenging reaction on derivatives of l-/d-tyrosine did not lead to the desired monooxygenase product but only to products of radical oxidation. Complex [Cu₂(mXHI)]⁴⁺ was also used for the catalytic sulfoxidation of thioanisole in the presence of hydroxylamine as cosubstrate, in a preliminary attempt to model the reaction of external monooxygenases. The reaction proceeds with 25 turnovers, but the enantiomeric excess of sulfoxide was modest.

Full text of DOI:10.1021/acs.inorgchem.9b00473

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
A Stereoselective Tyrosinase Model Compound Derived from an
m‑Xylyl‑L‑histidine Ligand
Eliana Lo Presti,† Maria L. Perrone,† Laura Santagostini,‡ Luigi Casella,† and Enrico Monzani*,†
†
̀
̀
Dipartimento di Chimica, Universita di Pavia, Via Taramelli 12, 27100 Pavia, Italy
‡
Dipartimento di Chimica, Universita di Milano, Via Golgi 19, 20133 Milano, Italy
S
* Supporting Information
ABSTRACT: The aim of mimicking enzyme activity
represents an important motivation for the development of
new catalysts. A challenging objective is the development of
chiral complexes for bioinspired enantioselective oxidation
reactions. Herein, we report a new chiral dinuclear copper(II)
complex based on a m-xylyl-bis(histidine) ligand (mXHI) as a
biomimetic catalyst for tyrosinase and catechol oxidase. The
new ligand improves a previous system also containing two
tridentate N3 units derived from L-histidine that were
connected by a short, rigid ethanediamine bridge. In mXHI
the bridge is provided by the more extended m-xylyl moiety. The dicopper(II) complex [Cu2(mXHI)]4+ was studied as a
catalyst for stereoselective oxidations of enantiomeric couples of chiral catechols of biological interest (L/D-dopa, L/D-dopa
methyl ester, and (R/S)-norepinephrine), showing excellent discrimination capability, particularly for the methyl esters of dopa
enantiomers. The catechol oxidation was studied in acetate buffer as slightly acidic medium, and a role of acetate as bridging
ligand between the two coppers, preorganizing the dinuclear center in a more catalytic efficient structure, could be established.
The oxidation of β-naphthol and 3,5-ditertbutylphenol was studied as a model monophenolase reaction. The oxidation proceeds
stoichiometrically, and the partial incorporation of 18O into β-naphthol when the reaction was performed using 18O2 suggests
the existence of two competitive reaction pathways, a genuine monooxygenase mechanism and a radical pathway. However, the
more challenging reaction on derivatives of L-/D-tyrosine did not lead to the desired monooxygenase product but only to
products of radical oxidation. Complex [Cu2(mXHI)]4+ was also used for the catalytic sulfoxidation of thioanisole in the
presence of hydroxylamine as cosubstrate, in a preliminary attempt to model the reaction of external monooxygenases. The
reaction proceeds with 25 turnovers, but the enantiomeric excess of sulfoxide was modest.
the system labeled EHI.25 The resulting dinuclear copper(II)
INTRODUCTION
■
complex exhibited appreciable enantio discriminating capacity
in the aerobic oxidation of biogenic catechols. However, in the
case of phenol oxidations, the reactions did not produce the
desired monooxygenation product but oligomeric products
resulting from radical coupling reactions.25 Fine tuning of the
structural motif is thus important to direct the reactivity of the
dimetal site toward the desired monooxygenase reactivity,
because this requires a strong cooperation between the metal
centers. For this reason, we reasoned that replacing the short
and relatively rigid ethanediamide linker in EHI with the more
flexible and extensively used meta-xylyl-diamine moiety could
allow the two copper centers to approach and facilitate an
intramolecular dioxygen binding and activation. Dicopper
complexes with such meta-xylyl-diamine linkers have been
shown to form various types of copper-dioxygen complexes, μ-
η2:η2-peroxodicopper(II), bis(μ-oxo)dicopper(III), or trans-μ-
η1:η1-peroxodicopper(II).26−31 Among these, the dicopper
complexes derived from L66 ligands are unique in exhibiting
The important advancements that have been made in the past
decade in the biomimetic oxidations by tyrosinase model
complexes, with systems reproducing catalytic oxidations of
phenols1−12 and sulfides,9,13,14 show that dinuclear complexes
referring to the active site of this enzyme bear potential interest
in synthetic applications. An important field of application
would be that of asymmetric oxidations, where the number of
well-characterized chiral copper(II) complexes and, in
particular, dinuclear copper(II) complexes, is still scarce.15−17
This depends on the difficulties involved in the synthesis of
suitable chiral multifunctional molecules with nitrogen donor
ligands. To obtain dinucleating ligands with these character-
istics, we followed through the years two different approaches,
based on the functionalization of chiral L-histidine18−21 or
binaphthyl22−24 residues, with a design that in several cases
allowed obtaining ligands with trinucleating capabilities.
Recently, we tried to simplify the ligand design focusing on
the combination of two tridentate metal binding motifs
constituted by amino-bis(imidazole) residues containing the
chiral L-histidine group, yielding a pair of fused five-membered
and six-membered chelate rings, and connected by a linker in
Received: February 18, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b00473
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Chart 1. Structure of [Cu2(mXHI)]4+(left) and [Cu2(EHI)]4+(right) Complexes (Trt = triphenyl methyl)
a
Scheme 1. Synthetic Pathway to mXHI
a
(i) Carbonyl diimidazole, dimethylformamide, 60−70 °C; (ii) benzyl bromide, CH3CN, reflux (yield: 93%); (iii) benzyl alcohol, diisopropyl ethyl
amine, CH3CN, rt (yield: 68%); (iv) H2, Pd/C, EtOH, rt (quantitative yield); (v) isophthaldialdehyde, STABH, dichloromethane, rt (yield: 98%);
(vi) 1-methyl-1H-imidazole-2-carboxaldehyde, STABH, dichloroethane, rt (yield: 78%).
both reversible dioxygen binding properties and phenol
monooxygenase activity.32−37 The new ligand introduced in
this paper, mXHI, then combines the chiral tridentate binding
motif for copper of EHI, conserving the biomimetic N6 donor
set with four imidazole donors, with the meta-xylyl-diamine
linker characterizing L66-type of ligands (Chart 1). Herein, we
describe the stereochemical and binding properties and the
chiral discrimination capacity in biomimetic oxidations of the
complex [Cu2(mXHI)]4+.
1 and Figures S1−S4. The near-UV range is dominated by the
intraligand absorption of the xylyldiamine chromophore, near
RESULTS AND DISCUSSION
■
Synthesis of the Ligand mXHI. The synthetic route to
mXHI is outlined in Scheme 1. The Nτ-benzyl substitution at
histidine groups of mXHI replaces the more sterically hindered
trityl group used in EHI25 or the smaller methyl group in
PHI.19,21 The histidyl-containing arms of the ligand were built
in by reacting N-benzyl histidine methyl ester, intermediate 5,
with isophthaldialdehyde.38 The xylyldiamine intermediate 6
was directly used in a reductive amination reaction with 1-
methyl-1H-imidazole-2-carboxaldehyde and sodium triacetoxy
borohydride (STABH) as reducing agent to give the mXHI
ligand 7. The dicopper(II) complex [Cu2(mXHI)]4+ was
obtained in almost quantitative yield by reacting the free ligand
with copper(II) perchlorate salt.
Figure 1. CD spectra recorded upon titration of [Cu2(mXHI)]4+ with
azide, up to a molar ratio of azide/Cu2 = 2:1in MeOH/MeCN, 9:1
(v/v).
280 nm, although it is expected that ligand-to-metal charge
transfer (LMCT) bands of type σ(amino) → CuII should also
contribute in this range.39 A weak shoulder on the low-energy
tail, around 350 nm, that is better resolved in the CD
spectrum, is typical of LMCT transitions of type π(imidazole)
→ CuII, as discussed before.39−43 The visible spectrum
contains the broad absorption of the d−d envelope centered
Spectroscopic Properties, Conformational Character-
ization, and Ligand-Binding Characteristics of
[Cu2(mXHI)]4+. The electronic and circular dichroism (CD)
spectra of [Cu2(mXHI)]4+ and its adducts are shown in Figure
B
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at 630 nm, which corresponds to a band of negative sign
maximizing around 650 nm in the CD spectrum. This feature
indicates that the histidine chelate ring bears a preference for
the conformation of λ chirality, with the carboxymethyl ester
side chain in pseudoequatorial disposition and the imidazole
group bound in a CuII axial position (Scheme 2). This
a change in the disposition of the imidazole donor group, from
axial to equatorial coordination position (Scheme 2). The
strong increase in CD intensity of the LF bands for the
bis(azido)−dicopper(II) adduct indicates strong stabilization
of the δ conformation. The shift of the d−d envelope to higher
energy in the absorption spectrum (∼615 nm) and the
increase in intensity agree with the strengthening of the
equatorial field.
We used the UV−vis data of azide spectral titration to
estimate the binding constants for the mono- and bis-azido
adducts of [Cu2(mXHI)]4+ in the same solvent mixture used
before for [Cu2(L66)]4+, [Cu2(L55)]4+, [Cu2(PHI)]4+, and
[Cu2(EHI)]4+, to make a reliable comparison of the binding
constants. The first azido binding step to one of the two CuII
centers cannot be isolated, as it is also evident from the CD
titration in Figure 2, showing that the exciton couplet is
Scheme 2. Conformational Preference of the L-Histidine
Chelate Ring in [Cu2(mXHI)]4+ (left), and the Effect of
Ring Conformational Inversion Caused by Azide Binding
a
(right)
a
The imidazole donor group shifts from the axial to an equatorial
coordination position in this process.
conformational preference is the same as for CuII−PHI
complexes19 but opposite to that of [Cu2(EHI)]4+,25 where
the side chain of the histidine chelate ring is actually
constituted by the linker ethanediamide chain.
The study of the binding of azide to dicopper(II) complexes
provides important information about the characteristics of the
adducts with small molecules. In the related dicopper(II)
complexes with ligands L66 and L55, azide binds as a bridging
ligand (in μ-1,3 and μ-1,1 modes, respectively),43 whereas in
the complexes derived from PHI19,20 or EHI,25 azide binds as
terminal ligand. The addition of azide to solutions of
[Cu2(mXHI)]4+ produces the growth of the usual π(azido)
→ CuII LMCT band in the electronic spectrum near 400 nm
(Figure S1). The shape of the LMCT band is perfectly
symmetric, suggesting that the ligand binds in the terminal
mode to both copper(II) centers. However, the corresponding
CD spectrum is bisignate, with a positive maximum at higher
energy (∼380 nm) and a negative maximum at lower energy
(∼430 nm) (Figure 1), featuring an evident exciton couplet44
for the LMCT transitions of the two azido-CuII chromophores.
The pattern of CD bands is the same as that observed for
[Cu2(PHI)]4+,20 and we refer to the analysis described there to
deduce the Δchirality of the skew arrangement of the Cu−
N(azido) linkages in the bis(azido)-dicopper(II) adduct
[Cu2(mXHI)(N3)2]2+ (Scheme 3).
Figure 2. CD spectra of [Cu2(mXHI)]4+ (4.13 mM) in 10:1 MeOH/
acetate buffer 0.3 mM (solid line) and in 10:1 MeOH/acetate buffer
15 mM (dashed line).
observed at azide/Cu2 molar ratios below 1:1. The binding
constants were obtained through the fitting of absorbance
changes at 396 nm with HypSpec.45 The species distribution
corresponding to the following equilibria are also given in
Figure S1.
[Cu2(mXHI)]4+ + N3− F [Cu2(mXHI)(N3)]3+
log K1 = 6.48 0.01
(1)
[Cu2(mXHI)(N3)]3+ + N3− F [Cu2(mXHI)(N3)2]2+
K2 = 4.74 0.01
(2)
The ligand field (LF) region of the CD spectrum provides
further stereochemical information about the histidine chelate
ring when azide binds to CuII. In fact, the positive optical
activity within the d−d bands for the azido adducts implies a
conformational inversion from λ to δ for the chelate ring, with
[Cu2(mXHI)]4+ + 2N3− F [Cu2(mXHI)(N3)2]2+
log β = 11.22 0.01
(3)
The log K values determined here for both azido binding steps
appear to be both larger (by 1 to 2 orders of magnitude) than
those previously found for the parent dicopper(II) complexes
of EHI,25 PHI,20 and even L55 and L66.43 The higher K values
may be due to a closer approach of the copper(II) centers in
mXHI (when compared to the spacers in EHI and PHI)
because of m-xylyl spacer and to a lower steric hindrance
around the metal centers (L55 and L66 have benzimidazole
ligands; in EHI the imidazole is protected by bulky trityl
groups).
Scheme 3. Chirality of the Skew Arrangements of the Two
Cu-Azido Bonds in [Cu2(mXHI)(N3)2]2+
C
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Table 1. Kinetic Parameters for the Catalytic Oxidation of Catechol Substrates by [Cu2(mXHI)]4+ in Methanol/50 mM
Aqueous Acetate Buffer, pH 5.1, 10:1 (v/v) at 25 °C
a
a
KM (mM)
kcat (s−1
)
k
cat/KM (M−1 s−1
)
Rk cat/KM (%)
Rk cat (%)
L-dopa
(3.13 0.83) × 10−1
(3.09 0.78) × 10−1
(4.43 0.82) × 10−1
(6.34 0.90) × 10−3
(7.16 1.93) × 10−2
(7.42 0.16) × 10−2
(1.01 0.87) × 10−3
(1.22 0.12) × 10−3
(7.73 0.63) × 10−2
(1.33 0.02) × 10−2
(8.69 0.55) × 10−4
(7.84 0.37) × 10−4
3.3
3.9
175
2098
12.1
10.6
−8
−85
7
−9
71
5
D-dopa
L-dopaOMe
D-dopaOMe
R-(−)-norepinephrine
S-(+)-norepinephrine
a
R > 0 indicates preference for L (or R for norepinephrine) enantiomer, and R < 0 indicates preference for D (or S for norepinephrine) enantiomer.
Another important feature of dinuclear copper(II) com-
S6), which enables fitting with simple Michaelis−Menten
equation. According to a genuine biomimetic catecholase
reaction, the catalytic cycle should proceed in two steps, with
two catechol molecules oxidized per cycle and dioxygen
reduction to water.46,47 The two steps are difficult to separate,
because this requires that their rate constants should differ by
∼1 order of magnitude. Therefore, as in other cases,14,16,25 we
assume a simplified mechanism implying fast initial dicopper-
(II) reduction by catechol, followed by a series of reactions
involving binding of dioxygen and a second molecule of
catechol to the dicopper(I) species, substrate oxidation, and
quinone release. The quinone then undergoes fast rearrange-
ment to aminochrome.48 In any case, to confirm the genuine
catecholase mechanism, we checked the eventual formation of
hydrogen peroxide during the reaction with the I3− assay,46 and
in fact the amount detected was only ∼5% of produced
quinone. As the complex does not display catalase activity, we
can assume that hydrogen peroxide is a byproduct of the
catalytic reaction; this side reaction, probably the decom-
position of the putative Cu2O2 species, competes with low
efficiency with the oxidation of the second molecule of
catechol.
plexes, with possible relevance to their catalytic behavior, is the
increased acidity of coordinated water resulting when they can
form hydroxo-bridged species. Titration of [Cu2(mXHI)]4+
with methanolic sodium hydroxide in MeOH/MeCN, 9:1 (v/
v), shows the development of a medium-intensity hydroxo →
CuII LMCT band at 350 (ε 1200 M−1 cm−1), which maximizes
at a OH−/Cu2 molar ratio of 2 and corresponds to the
II
formation of [Cu2(mXHI)(OH)2]2+ (Figures S2 and S3). The
near-UV band of the complex undergoes a red-shift to ∼300
nm. The titration data at 350 nm do not show an intermediate
monohydroxo [Cu2(mXHI)(OH)]3+ species, and this seems
confirmed by the CD changes accompanying the titration
(Figure S4). Here the prominent CD band corresponds to the
band near 300 nm, whereas the 350 nm LMCT appears as a
shoulder. These features suggest that also the 300 nm band is
contributed by a hydroxo → CuII LMCT component. In the
visible region, a prominent CD band of positive sign near 550
nm dominates the spectrum, indicating that also formation of
complex [Cu2(mXHI)(OH)2]2+ occurs with conformational
inversion to a δ chelate ring (see Scheme 2).
To obtain further information about the bis-μ-hydroxo-
dicopper(II) species, the formation of this complex upon
addition of sodium hydroxide to [Cu2(mXHI)]4+ was followed
by NMR (Figure S5). Upon addition of hydroxide to a
[Cu2(mXHI)]4+, many signals markedly sharpen, and the two
copper(II) ions appear to couple. It is also evident that there is
no accumulation of a monohydroxo [Cu2(mXHI)(OH)]3+
species, as the NMR spectrum of [Cu2(mXHI)]4+/OH− 1:1
corresponds to a mixture of [Cu2(mXHI)]4+ and
[Cu2(mXHI)(OH)2]2+. We can therefore assume that
hydroxide anions are bridging the copper(II) centers in
[Cu2(mXHI)(OH)2]2+ at room temperature (rt), with a rather
strong antiferromagnetic coupling of the two copper(II)
centers. The spectroscopic and magnetic properties of
[Cu2(mXHI)(OH)2]2+ thus resemble those of the parent
[Cu2(L55)(OH)2]2+ complex.43
The kinetic parameters kcat and KM determined for the
oxidation of the chiral catechols are collected in Table 1. The
discrimination capacity of the copper catalyst against the
enantiomeric substrates was evaluated using the stereo-
14,15,25
selectivity indexes Rk cat/KM and Rk cat
,
expressed as
percentages, given by the following equations (with
norepinephrine, L refers to the R enantiomer, and D refers
to the S enantiomer).
(kcat/Km)L − (kcat/Km)D
(kcat/Km)L + (kcat/Km)D
Rkcat/Km% =
× 100
(kcat)L − (kcat)D
(kcat)L + (kcat)D
Rkcat% =
× 100
Catalytic Oxidation of Chiral Catechols. To facilitate
the comparison of the catecholase activity of [Cu2(mXHI)]4+
with that of other chiral dicopper(II), in particular,
[Cu2(EHI)]4+,25 the studies were performed on the same set
of enantiomeric catechols of biological interest, that is, L/D-
dopa, L/D-dopa methyl ester, and R/S-norepinephrine. The
kinetics of oxidation were investigated by monitoring the
increase of the absorption band due to the formation of
aminochrome, the only intermediate with sufficient lifetime of
catechol oxidation.20 The reactivity studies were performed in
10:1 (v/v) mixture of methanol/aqueous acetate buffer (50
mM) at pH 5.1, because in these conditions catechol
autoxidation is negligible. The rate plots at increasing substrate
concentration typically exhibited hyperbolic behavior (Figure
Rk cat/KM and Rk cat give indications on the discriminating effect
observed in the catalytic oxidations at low and saturating
substrate concentrations, respectively.
It can be noted that the R indexes are striking for the L-/D-
dopaOMe enantiomeric couple, whereas they are modest in
the other cases. In fact, the complete inversion of R indexes for
L-/D-dopaOMe from low to high substrate concentration has
no precedent in the large series of studies performed before by
our group.15,25 The interpretation of this unusual behavior is
facilitated by the large difference between the KM and kcat
values for the two enantiomeric substrates. D-DopaOMe
exhibits an affinity for the active species of [Cu2(mXHI)]4+ ∼2
orders of magnitude larger than that of L-dopaOMe, whereas
the latter substrate reacts more than six times faster than its D
D
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counterpart. Therefore, KM rules the reaction rate at low
substrate concentration, while kcat is the dominant factor at
high substrate concentration, that is, under saturation
conditions.
When we tried to perform the phenol oxidation using the
tetrabutyl ammonium (TBA) salts of N-acetyl tyrosine ethyl
esters, with the protections needed to prevent copper binding
through the amino group at the α-carbon, and also hinder
intramolecular Michael addition in the case of eventual
formation of quinone, the reaction was performed with 0.01
mM solution of [Cu2(mXHI)]2+ in dry acetone and an excess
of 20 equiv of TBA salt of the tyrosine derivatives. The
temperature was kept at −80 °C, to prevent oxidative
polymerization of the products, with a slight modification of
a procedure reported in the literature.2 After 16 h reaction time
and acidic quenching, the products isolated by column
chromatography were analyzed by mass spectrometry (MS)
and 1H NMR. As for the reaction performed with
[Cu2(EHI)]2+, the major products are trimeric compounds
resulting from C−C and C−O coupling reactions.25 This does
not exclude that some minor product from tyrosinase-like
reaction is formed, but we were unable to identify it in the
complex reaction mixture.
A peculiar feature of the reaction is the increase of reaction
rate upon increasing the concentration of acetate buffer, which
was studied in detail for L-dopaOMe (Figure S7). Acetate can
bridge between the copper centers facilitating the binding of
the catechol as bridging ligand between the two coppers in the
transition state, thereby favoring simultaneous two-electron
transfer. The possibility for acetate to act as bridging ligand
was confirmed by NMR, through magnetic susceptibility
measurements of [Cu2(mXHI)]4+ with Evans’ method. The
magnetic moment per copper at 15 °C was found to decrease
from 1.45 μB with 0.3 mM acetate to 1.17 μB at 15 mM acetate
(an almost constant decrease, between 20% and 30%, in the
observed magnetic moment was observed in the temperature
range from −15 to +40 °C), indicating that this anion can
moderately couple the two copper(II) centers. The more
compact structure of the acetate-bridged complex stabilizes the
λ conformation of the amino acid ring and axial positioning of
the histidine imidazole, as shown by the CD spectra (Figure
2). The d−d envelope shifts at higher energies (570 nm),
indicating strengthening of square-planar field, and also the
LMCT band near 300 nm undergoes a blue shift, with better
resolution of a shoulder near 360 nm (Figure S8).
Titration of [Cu2mXHI]4+ with L-/D-Tyrosinato. As an
attempt to assess why we could not observe hydroxylation of
the tyrosinato derivatives, we performed binding experiments
of the enantiomeric phenolates to the dicopper(II) complex, as
we did before for the parent [Cu2(EHI)]4+.25 The binding of
the potassium salts of N-acetyl-L- and D-tyrosine ethyl ester to
[Cu2(mXHI)]4+ was thus studied in methanol solution. In
both experiments, titration of the ligand gave rise to UV bands
near 300 and 350 nm, which are typical for bis-μ-hydroxo
bridged dicopper(II) complex (Figure S13), instead of the
expected O(phenolato)→Cu(II) LMCT band near 420 nm
that was observed before in the case of [Cu2(EHI)]4+.25,50
Thus, the effect of the basic phenolate solution is the
deprotonation of coordinated water rather than coordination
of the phenolate ligand. Additional coordination of the
phenolate to the [Cu2(mXHI)(OH)2]2+ is apparently impos-
sible, and as the structural features of this complex resemble
those of the putative peroxodicopper(II) complex
([Cu2(mXHI)(O2)]2+), resulting from oxygenation of
[Cu2(mXHI)]2+, this can explain the lack of hydroxylation of
the tyrosine derivatives.
Catalytic Sulfoxidation of Thioanisole. As tyrosinase is
able to promote asymmetric oxidation of thioanisole in
presence of dopa as sacrificial reducing agent,51 we replicated
this activity using [Cu2(mXHI)]4+ as catalyst. Hydroxylamine
was chosen here as reducing agent, as in previous biomimetic
studies, as N2 is the only byproduct deriving from its
oxidation.13 Preliminary results of thioanisole oxidation were
collected by analyzing both yield and enantiomeric excess of
the sulfoxide at increasing reducing agent concentration
(Figure 3). The data show an exponential decrease in reaction
yield with excess of hydroxylamine concentration, while there
is no apparent relationship between concentration of hydroxyl-
amine and enantiomeric excess. In all cases, the enantiose-
lectivity of sulfoxide formation was rather low (ranging from 8
to 16% ee); however, we note that similar experiments
performed using [Cu2(EHI)]4+ led to racemic sulfoxide (data
not shown). Also worthy of note is the fact that the reaction is
catalytic, and the complex performs 25 cycles in 16 h.
Oxidation of Phenolate Derivatives by [Cu2(mXHI)]2+/
O2. Because of the presence of a five-membered ring and a six-
membered chelate ring, the reduced [Cu2(mXHI)]2+ complex
is expected to display an intermediate behavior between
[Cu2(L55)]2+, characterized for its high reactivity to O2,49 and
[Cu2(L66)]2+, capable of forming a relatively stable Cu2O2
complex at low temperature.33 As all attempts to isolate a
copper−dioxygen adduct of [Cu2(mXHI)]2+ were unsuccessful
(in acetone at −80 °C, in propionitrile at −80 °C, in
dichloromethane at −90 °C, and in 2-methyl-tetrahydrofuran
at −110 °C), we believe that the structure of this complex, like
that of [Cu2(L55)]2+,49 essentially contains a linear bis-
(imidazole) ligation, with very weak or absent interaction
between the xylylamine N donors and the metal ions, though,
like [Cu2(L55)]2+,32 [Cu2(mXHI)]2+ is capable to perform
phenol hydroxylation in the presence of molecular oxygen. In
this experiment, we reacted the 1,5-diazabicyclo(5.4.0)undec-
5-ene (DBU) salt of 3,5-ditertbutylphenol and [Cu2(mXHI)]2+
in 10:1 ratio in acetone at −40 °C overnight, followed by
exposure to O2, and found that 3,5-ditertbutylquinone was
formed quantitatively in a stoichiometric reaction (Figure S9).
With β-naphthol, as DBU salt, as substrate, hydroxylation of
the phenol ring was also observed. The reaction between
[Cu2(mXHI)]2+ and DBU naphtholate, followed by exposure
to dioxygen, was performed in acetone at room temperature, in
the same conditions as for [Cu2(EHI)]2+.25 As before, the
reaction yields the conjugate addition product between
naphthoquinone and excess naphtholate (Figures S10 and
S11). However, unlike [Cu2(EHI)]2+, the experiment
performed using 18O2 showed significant incorporation (almost
50%) of labeled oxygen into the product (see Figure S12).
This result could be indicative of the presence of two parallel
mechanisms, one occurring through a radical reaction pathway
and the other through inner-sphere concerted oxygen transfer
from Cu2O2 complex to coordinated naphtholate, that is, a
tyrosinase-like reaction.
CONCLUSIONS
■
Among tyrosinase biomimetic complexes with chiral ligands
reported so far by our group, namely, [Cu2(PHI)]4+,20
[Cu2(EHI)]4+,25 and [Cu2(L55Bu4)]4+,14 and others,15 com-
E
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cooperate;10,52−55 this facilitates the formation of the Cu-SQ
species. The copper complex of mXHI is binuclear and shows
higher reactivity when the two ions are forced to cooperate (as
shown by the effect of acetate anion); therefore, we believe
that a Cu-SQ species, if formed, would be only a minor
intermediate.
Complex [Cu2(mXHI)]4+ is catalytically active in the
sulfoxidation of thioanisole, and although the enantioselectivity
of the reaction is modest, it is observed here for the first time
by a biomimetic dicopper catalyst.
Further work is in progress to develop new chiral tyrosinase
biomimetic model compounds.15
EXPERIMENTAL SECTION
■
Materials and Methods. All reagents were of reagent grade and
purchased from either Sigma-Aldrich Chemical Co. or VWR
International and used without further purification. Solvents were
dried over standard drying agents. UV−Vis spectra were recorded on
Agilent HP 8453 diode-array spectrophotometers. Elemental analyses
were obtained at the Microanalysis service of the Department of
Chemistry of University of Milano. CD spectra were obtained with a
Jasco J500 spectropolarimeter; the spectra were recorded at 0.2 nm
resolution in the range between 300 and 700 at 50 nm min−1 scan
rate, with three scans acquired for each spectrum. The NMR spectra
were recorded with a Bruker AVANCE 400 spectrometer operating at
Figure 3. Yield of sulfoxidation calculated on the basis of the
concentration of hydroxylamine. The reactions were performed in 9:1
MeOH/acetate buffer (50 mM, pH 5.1), for 16 h at room
temperature. The concentration of [Cu2(mXHI)]4+ was 10 μM, and
that of thioanisole was 10 mM.
plex [Cu2(mXHI)]4+ exhibits the best discrimination capacity,
as assayed in the catecholase reaction, with R index up to 85
for the best substrates, that is, L- and D-dopaOMe. The reason
why chiral recognition is displayed with these substrates can be
ascribed to the bulkiness of the methyl ester groups, which may
force an interaction with the benzyl substituents present on the
L-histidyl ligands. With the more polar substrates, like L-/D-
dopa, the smaller size of the polar groups attached to the Cα
atom make such steric interaction negligible. For the same
reason we observe little enantioselectivity in the sulfoxidation
reaction. It should also be taken into account that for L-/D-
dopa and R-/S-norepinephrine the polar groups on the side
chain compete with the catechol residue in the binding to the
dicopper center. This explains the smaller rate constants
observed for the catecholase reaction with respect to D-/L-
dopaOMe (Table 1).
9.37 T with frequencies of 400.13 and 100.6 MHz for H and 13C
1
NMR, respectively. The data acquisition and processing were
performed with a standard Bruker software package (Topspin 1.3).
Mass spectra were recorded with a Thermo-Finnigan LCQ 371 ADV
MAX spectrometer. All reactions involving copper(I) species were
performed using standard Schlenk techniques under an inert
atmosphere of N2 unless otherwise stated.
Synthesis of (S)-Methyl 5-oxo-5,6,7,8-tetrahydroimidazo[1,5-
c]pyrimidine-7-carboxylate (2). This compound was prepared
according to a procedure described in the literature.56 1H NMR
(400 MHz, CDCl3) δ 8.25 (s, 1H), 6.93 (s, 1H), 6.73 (s, 1H), 4.41
(ddd, J = 7.9, 5.4, 2.2 Hz, 1H), 3.83 (s, 3H), 3.41 (dd, J = 15.8, 5.3
Hz, 1H), 3.19 (dd, J = 16.2, 8.4 Hz, 1H); MS (electrospray ionization
(ESI)) [M+H+] + 196 (m/z).
Synthesis of 2-Benzyl-7-(methoxycarbonyl)-5-oxo-5,6,7,8-
tetrahydroimidazo[1,5-c]pyrimidin-2-ium (3). The product was
synthesized with a modification of a literature procedure.56 To a
suspension of 2 (1.0 g, 5.33 mmol) in 15 mL of acetonitrile, 3 equiv of
benzyl bromide (1.9 mL, 16 mmol) were added. The reaction mixture
was refluxed for 2 h, and the product precipitated as a white powder.
The mixture was then cooled, and the solid was filtered off and
washed with diethyl ether (3 × 30 mL); the yield was 93%. 1H NMR
(400 MHz, MeOD) δ 9.77 (d, J = 1.6 Hz, 1H), 7.54 (d, J = 1.4 Hz,
1H), 7.52−7.42 (m, 5H), 5.49 (d, J = 1.7 Hz, 2H), 4.63 (t, J = 5.4 Hz,
1H), 3.77 (s, 3H), 3.47−3.39 (m, 2H). MS (ESI) [M+H+] + 286 (m/
z).
Although complex [Cu2(mXHI]2+/4+ proved to be capable of
performing phenol hydroxylation of simple substrates, the
more challenging reaction on derivatives of L-/D-tyrosine did
not lead to the desired monooxygenase product. We believe
that this is due to structural limitations in the mode of
assembly of the three partners (Cu2, O2, and TyrO−) in the
transition state. The dioxygen adduct of [Cu2(mXHI)]2+ does
not accumulate in solution for its apparent low stability, and
this makes formation of the ternary complex with tyrosinate
substrates virtually impossible. With phenolic substrates
lacking, the bulky substitution at the α-carbon formation of
the key ternary intermediate, and hence phenol hydroxylation,
is possible. Though, as shown by the partial 18-O
incorporation into β-naphthol, the hydroxylation occurs
probably through competing pathways involving radical and
genuine monooxygenase reactions. Our results on the C−O
formation and 18-O incorporation do not allow to exclude the
possible formation of a Cu(II)-semiquinone radical, Cu-SQ,
during the reactions, also because its optical bands are too
weak to be detected in our experiments (they would be
covered by the products bands, which have a much higher
extinction coefficient). But in the model systems described in
the literature showing the formation of the Cu(II)-semi-
quinone radical, the two copper ions are not tightly forced to
Synthesis of N-α-(Benzyloxycarbonyl)-1-benzyl-L-histidine meth-
yl ester (4). To a solution of 3 (1.0 g, 2.80 mmol) in acetonitrile (100
mL), benzyl alcohol (1.45 mL, 14 mmol) and diisopropylethylamine
(0.98 mL, 5.6 mmol) were added. The solution was stirred overnight
at rt. The solvent was removed in vacuo, obtaining a highly viscous
yellow oil, which was dissolved in the minimum amount of
dichloromethane and precipitated with diethyl ether. The crude
product was purified by silica chromatography with a mixture of
hexane−ethyl acetate 1:1 (v/v) as eluent; the product was obtained
1
upon evaporation of the solvent (yield 68%). H NMR (400 MHz,
CDCl3) δ 7.45 (s, 1H), 7.40−7.30 (m, 8H), 7.16−7.08 (m, 2H), 6.65
(s, 1H), 6.37 (d, J = 8.1 Hz, 1H), 5.12 (s, 2H), 4.63 (dt, J = 8.2, 5.0
Hz, 1H), 3.66 (s, 3H), 3.08 (ddd, J = 19.2, 14.6, 4.9 Hz, 2H). MS
(ESI) [M+H+] + 393 (m/z).
Synthesis of 1-Benzyl-L-histidine methyl ester (5). The mixture of
compound 4 (0.65 g, 1.65 mmol) and 10% Pd/C (10% w/w with
respect to substrate) in ethanol (10 mL) was vigorously stirred at
F
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Article
room temperature (ca. 20 °C) under a hydrogen pressure of 1 atm
(kept through a rubber balloon) for 24 h. The reaction mixture was
filtered using diatomaceous earth, and the filtrate was washed several
times with ethanol and rotary evaporated to yield the product, as a
yellow oil, in quantitative yield. Caution! The product is highly
of methanol was added. The resulting blue solution was poured into 5
mL of diethyl ether to induce precipitation of the complex. The
suspension was centrifuged, and the blue solid was separated, washed
with diethyl ether (3 × 3 mL), and dried under vacuum (yield 68%).
−
MS (ESI) [M+2Cu2++2ClO4 ] + 566 m/z (detected as double
1
photosensitive and should be protected from light. H NMR (400 MHz,
charged ion). Anal. Calcd for C46H52Cl4Cu2N10O20 (1333.88): C
41.42, H 3.92, N 11.50. Found: C 41.31, H 3.52, N 11.23%. UV−Vis
λmax (nm), MeOH: 278 (ε 3160 M−1 cm−1), 630 (ε 140 M−1 cm−1).
Titration of [Cu2(mXHI)]4+ with Azide. The binding of azide to
[Cu2(mXHI)](ClO4)4 was studied spectrophotometrically by adding
successive and equal amounts (13 μL) of a concentrated methanol
solution of sodium azide (0.01 M) to the solution of the complex
(3.82 × 10−4 M) in MeOH/MeCN, 9:1 (v/v), until further addition
of azide did not produce any appreciable spectral changes. The
measurements were performed in a thermostated optical cell, with
magnetic stirring, at 20.0 0.1 °C. [Cu2(mXHI)]4+ ε278 = 3150 M−1
cm−1, ε396 = 185 M−1 cm−1, ε647 = 160 M−1 cm−1; [Cu2(mXHI)-
(azido)]3+ ε278 = 3070 M−1 cm−1, ε396 = 1330 M−1 cm−1, ε630 = 280
CDCl3) δ 7.42 (s, 1H), 7.38−7.21 (m, 3H), 7.11 (d, J = 6.7 Hz, 2H),
6.68 (s, 1H), 5.02 (s, 2H), 3.89−3.71 (m, 1H), 3.64 (s, 3H), 3.00
(dd, J = 14.4, 4.6 Hz, 1H), 2.88−2.77 (m, 1H), 2.46 (s broad, 2H),
1.18 (t, J = 7.0 Hz, 1H). MS (ESI) [M+H+] + 260 (m/z).
Synthesis of Dimethyl 2,2′-((1,3-phenylenebis(methylene))bis-
(azanediyl))bis(3-(1-benzyl-1H-imidazol-4-yl)propanoate) (6). In an
amber-colored flask, 1.2 mmol of 5 were dissolved in 5 mL of
dichloromethane, and solid isophthalic dialdehyde (80 mg, 0.6 mmol)
was added under stirring at room temperature under N2. After 24 h,
additional 1.2 mmol of 5 was poured into the solution, and the
mixture was left under stirring overnight. The reaction progress was
monitored by ESI-MS. STABH (380 mg, 1.8 mmol) was added, and
the mixture was stirred at room temperature under a N2 atmosphere
for 30 min. An excess of reducing agent (380 mg, 1.8 mmol) was then
used to completely reduce the bis-imine product. The reaction
mixture was quenched by removing the solvent and treating the
resulting borate salts with dichloromethane. The suspension was
centrifuged to allow sedimentation of the white salts, while the solvent
was rotary-evaporated to give a viscous green oil. The final product
was recovered by chromatography on silica using a mixture of
hexane−ethyl acetate−methanol−aqueous ammonia (1:9:1:0.3 v/v)
as eluent, giving 6 as a colorless oil (98% yield). 1H NMR (400 MHz,
CDCl3) δ 7.42 (s, 2H), 7.33−7.25 (m, 6H), 7.15 (t, J = 7.3 Hz, 2H),
7.09 (dd, J = 6.7, 5.0 Hz, 6H), 6.64 (s, 2H), 6.23 (d, J = 44.9 Hz,
11H), 5.01 (d, J = 5.8 Hz, 4H), 3.79−3.69 (m, 2H), 3.63−3.53 (m,
10H), 2.96−2.82 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 175.44
(q), 140.14 (q), 138.90 (q), 137.26 (q), 129.45 (CH), 128.64 (CH),
128.64 (CH), 128.51 (CH), 127.63 (CH), 127.27 (CH), 117.34
(CH), 61.21 (CH), 52.26 (CH2), 52.08 (CH2), 51.16 (CH3), 32.46
(CH2), 22.96 (CH2).
M−1 cm−1; [Cu2(mXHI)(azido)2]2+ ε278 = 3090 M−1 cm−1, ε396
2110 M−1 cm−1, ε630 = 370 M−1 cm−1.
=
Titration of [Cu2(mXHI)]4+ with Hydroxide. The binding of
hydroxide to [Cu2(mXHI)]4+ was studied spectrophotometrically by
adding concentrated methanol solutions of sodium hydroxide to
solutions of the complexes dissolved in MeOH/MeCN, 9:1 (v/v). All
measurements were performed in a thermostated optical cell, with
magnetic stirring, at 20
0.1 °C. In all cases, it was found that
binding of the anion is fast, so it was not necessary to incubate the
mixtures before the spectroscopic measurements were performed.
Titration of 1.0 mL of [Cu2(mXHI)]4+ solution (3.82 × 10−4 M) was
performed by the addition of successive and equal amounts (9.8 μL)
of a hydroxide solution (0.01 M), until further addition of hydroxide
did not produce appreciable spectral changes.
The [Cu2(mXHI)]4+ complex with a 2:1 excess of hydroxide was
studied by mass spectrometry (ESI), showing a [M+2Cu2++2CH3O−]
+ 498.5 (detected as double charged ion) peak. The bis-methoxo
species probably spontaneously formed from the bis-μ-hydroxo during
the mass spectra acquisition in the methanolic solution. The same
coordination change was already observed in a parent dicopper(II)
complex.49
Synthesis of Dimethyl 2,2′-((1,3-phenylenebis(methylene))bis-
(((1-methyl-1H-imidazol-2-yl)methyl)azanediyl))bis(3-(1-benz yl-
1H-imidazol-4-yl)propanoate) (mXHI, 7). Compound 6 (430 mg,
0.70 mmol) was dissolved in dichloroethane (5 mL) under an inert
atmosphere. A solution of 1-methyl-2-imidazole carboxaldehyde (186
mg, 1.73 mmol) in dichloroethane (1 mL) was slowly added. The
reaction was stirred for 30 min at room temperature. Solid STABH
(440 mg, 2.10 mmol) was added, and the suspension was stirred at
room temperature for 8 h. The same amount of aldehyde (1.73
mmol) and an equivalent amount of STABH were added again to the
reaction mixture, which was left under stirring overnight. ESI-MS
monitoring revealed incomplete reduction of the bis-imine
intermediate, and thus another addition of equimolar amounts of
the aldehyde and reducing agent (1.73 mmol) was made. The
reaction was then allowed to react for 24 h at room temperature and
was stopped by removing the solvent by rotary evaporation. The
resulting solid residue was suspended with dichloromethane and
centrifuged. After centrifugation, the solution was recovered and
evaporated to isolate the crude product as a yellow oil. This product
was purified by chromatography on silica using a mixture of ethyl
acetate−methanol−aqueous ammonia (80:20:3, v/v) as eluent, giving
NMR Spectra of [Cu2(mXHI)]4+-Hydroxo Species. An NMR
spectrum was recorded of a solution containing 3.1 mg of mXHI,
dissolved in 600 μL of CD3OD. To this solution 2.8 mg of copper(II)
trifluoromethanesulfonate (OTf) was added, and a new spectrum was
recorded. Two additional spectra were recorded after two additions of
1.9 μL solution of 2 M NaOD. The spectra in the presence of
copper(II) were recorded using a spectral width of 187 ppm, centered
at 30 ppm; 800 spectra were acquired with a 0.3 s delay.
General Procedure for Preparation of DBU Salts of
Different Substrates. The substrate is dissolved in methanol, and
0.9 equiv of DBU are added to the solution. The reaction is kept
under stirring for half an hour, and then the solvent is removed by
rotary evaporation, affording the desired salt.
Synthesis of D-Dopa Methyl Ester. D-Dopa was suspended in
methanol and cooled in an ice bath under argon flux. SOCl2 (10
equiv) was added slowly, and, then, the reaction was stirred at room
temperature overnight under an inert atmosphere. The solvent was
evaporated, affording the pure product as dihydrochloride in
quantitative yield.
1
mXHI as a colorless oil (Rf = 0.23, 430 mg, 78%). H NMR (400
MHz, CDCl3) δ 7.42 (s, 2H), 7.29 (t, J = 5.0 Hz, 6H), 7.12.7.05 (m,
4H), 7.05−6.90 (m, 4H), 6.80 (d, J = 2.9 Hz, 2H), 6.59 (s, 2H), 6.40
(s, 2H), 4.96 (d, J = 2.8 Hz, 4H), 3.86−3.70 (m, 8H), 3.63 (t, J = 4.0
Hz, 6H), 3.51 (dd, J = 13.8, 2.3 Hz, 2H), 3.20−2.85 (m, 10H). 13C
NMR (101 MHz, CDCl3) δ 172.8 (q), 145.2 (q), 139.3 (q), 139.1
(q), 136.8 (CH), 136.6 (q), 130.3 (CH), 129.3 (CH), 128.7 (CH),
128.3 (CH), 128.3 (CH), 127.7 (CH), 126.7 (CH), 122.1 (CH),
116.9 (CH), 63.0 (CH), 55.3 (CH2), 51.8 (CH2), 51.2 (CH3), 47.9
(CH2), 32.9 (CH3), 28.4 (CH2). MS (ESI) [M+H+] + 809 (m/z).
Synthesis of [Cu2(mXHI)](ClO4)4. To a solution of mXHI (10 mg,
1.24 × 10−5 mol) in methanol (0.6 mL) copper(II) perchlorate
hexahydrate (9.1 mg, 2.47 × 10−5 mol) dissolved in the same volume
Catalytic Oxidation of o-Catechols. The catecholase activity of
[Cu2(mXHI)]4+ was studied through the catalytic oxidation of L- and
D-dopa, L- and D-dopaOMe, R- and S-(+)-norepinephrine. The
kinetic studies of the oxidation of the o-diphenols were performed
spectrophotometrically by the method of initial rates by monitoring
the aminochrome absorption band with time, at 475 nm for L-/D-
dopa, 468 nm for L-/D-dopaOMe, and 480 nm for R-/S-
norepinephrine. The solvent used was a 10:1 (v/v) mixture of
methanol/aqueous acetate buffer (50 mM, pH 5.1) saturated with
atmospheric oxygen. The experiments were performed at a constant
temperature of 20 0.1 °C, varying the substrate concentration (from
5 × 10−5 to 1 × 10−3 M) and using a 5 × 10−5 M concentration of
G
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[Cu2(mXHI)]4+. A stock solution of 5 × 10−4 M [Cu2(mXHI)]-
(ClO4)4 was prepared, by dissolving 3.3 mg of complex in 5 mL of
methanol, and used for the kinetic measurements with all the
substrates, to warrant reproducibility of the experiments. The rate
data are expressed as v/[catalyst] = [(ΔA/s)/εaminochrome]/[complex],
where ΔA/s is the absorbance change with time upon formation of
the aminochrome, and εaminochrome is taken as 3600 M−1 cm−1.57
Further experimental details are reported in the Supporting
Information.
Low-Temperature Oxidation of N-Acetyl-L/D-tyrosine ethyl
ester phenolate Salts. The TBA salt of N-acetyl-L/D-tyrosinate
ethyl ester was prepared by reaction of the phenol (0.33 mmol) with a
slight defect of tetrabutylammonium hydroxide (0.3 mm, 0.9 equiv)
(in CH2Cl2 (2 mL). After evaporation of the solvent, the resulting oil
was treated with 2 mL of toluene, and the mixture was rotary
evaporated again to remove traces of moisture, yielding a colorless oil.
The TBA salt of N-acetyl-L/D-tyrosine ethyl ester (1 × 10−5 mol) and
mXHI (5 × 10−7 mol) were dissolved in dry acetone (50 mL) and
cooled at −80 °C in a cryostat, and the solution was degassed with 5
vacuum/argon purge cycles. [Cu(CH3CN)4]PF6 (1 ×10−6 mol)
dissolved in dry degassed acetone was added to the previously
described mixture, and oxidation was initiated by purging with pure
oxygen the solution. The reaction was quenched after 16 h with 2 mL
of HClO4 1 M. The organic portion was evaporated by rotary
evaporation, and the aqueous phase was extracted three times with
dichloromethane. The combined organic phase was dried over
sodium sulfate, filtered, and evaporated to give the crude product.
Catalytic Sulfoxidation of Thioanisole. To a 10 mM solution
of thioanisole in 1 mL of 9:1 methanol/acetate buffer 50 mM (pH
5.1), a methanolic solution of [Cu2(mXHI)]Cl4 (final concentration
10 μM) was added, followed by the addition of the appropriate
volume of a solution of hydroxylamine hydrochloride in acetate buffer
pH 5.1. The reaction mixture was stirred overnight at room
temperature. In a series of experiments, the concentration of
hydroxylamine was changed from 0.3 to 10 mM. For the high-
performance liquid chromatography (HPLC) analysis, 100 μL of the
reaction mixture was withdrawn, and a 1 μL sample of a 0.05 M
solution of p-nitroacetophenone in water was added as internal
standard. The resulting mixture was analyzed by HPLC, using an
Ascentis Express C18 (100 × 4.6 mm) column, with elution gradient
from acetonitrile/0.1% trifluoroacetic acid (TFA) to water/0.1% TFA
and flux of 0.8 mL/min. Retention times: methylphenylsulfoxide: 9.5
min; p-nitroacetophenone: 12.5 min; thioanisole: 14.2 min. For the
determination of the enantiomeric excess, the reaction was quenched
by the addition of 1 mL of 1 M solution of HClO4. After the
evaporation of the methanolic portion, the residual water was
extracted with 3 × 3 mL of dichloromethane. The collected organic
extracts were dried over sodium sulfate, filtered, and evaporated
(thioanisole is partially lost during rotary evaporation). The crude
product was analyzed by HPLC, on a chiral column Lux 5 μ amylose-
2 (250 × 4.60 mm), with 85:15 hexane/2-propanol as eluent (0.8
mL/min flux). Retention times of the sulfoxide enantiomers are 25
and 27 min for the (S) and (R) sulfoxides, respectively.14
Determination of H2O2 Accumulated during the Kinetic
Oxidation of Catechols. 3,5-Ditertbutyl catechol was chosen as
representative catecholic substrate. The catecholase activity was
followed spectrophotometrically, monitoring the increase of a band at
412 nm, attributed to the 3,5-ditertbutyl quinone (ε = 1550 M−1
cm−1) in a 10:1 (v/v) mixture of methanol/aqueous acetate buffer
(50 mM, pH 5.1). The reaction was quenched as the absorbance of
the quinone reached 0.4. After it was diluted with an equal volume of
distilled water, the solution was extracted three times with CH2Cl2,
and the aqueous layer was treated with H2SO4 to pH 2. Then, 1 mL of
the solution was mixed with 1 mL of a solution containing 50 g/L of
KI and a catalytic concentration of HRP (∼1 × 10−8 M), to accelerate
the oxidation of I− promoted by H2O2. The absorbance value at 353
−
nm (ε = 26 000 M−1 cm−1), attributed to the formation of I3 , was
measured. The experiment was performed with two concentrations of
catechol (2 × 10−4 M and 1 × 10−3 M), and in both cases the
determined H2O2 corresponded to 6% of the obtained quinone.
Catalase Activity of [Cu2(mXHI)](ClO4)4. A 0.1 M solution of
H2O2 in methanol was prepared and divided into two. One part was
used as blank, while the other one was incubated for 2 h in the
presence of [Cu2(mXHI)](ClO4)4. The solution (5 μL) was added to
a solution (2 mL) containing 5 g/L of KI and a catalytic amount of
HRP (∼1 × 10−8 M). Then, it was brought to pH 2 with H2SO4 and
incubated again for 15 min. Measurements of absorbance at 353 nm
showed no significant differences between the blank and the solution
containing the copper complex.
Oxidation of 3,5-Ditertbutylphenolate. A 25 mL sample of 5
× 10−5 M mXHI and 1 × 10−3 M DBU salt of 3,5-diterbutylphenolate
in freshly distilled acetone was cooled in cryostat at −40 °C. The
solution was degassed with four vacuum/argon cycles, and then 1 mL
of a solution of [Cu(CH3CN)4]PF6 was added to obtain a final
copper(I) concentration of 1 × 10−4 M. The reaction was started with
the addition of pure oxygen (1 atm) and left under stirring overnight.
The reaction was quenched at low temperature with the addition of 1
mL of 1 M HClO4. The organic phase was evaporated, and then the
aqueous phase was extracted three times with dichloromethane. The
combined organic extract was dried over sodium sulfate, filtered, and
evaporated to give the crude product.
ASSOCIATED CONTENT
* Supporting Information
■
S
Hydroxylation of β-Naphthol. A solution of mXHI (5 × 10−5
M) and the DBU salt of β-naphthol (1 × 10−3 M) was prepared in
degassed acetone (20 mL), maintaining an argon atmosphere with the
use of a Schlenk line. A concentrated solution of [Cu(CH3CN)4]PF6
in freshly distilled and degassed acetone was added (1 × 10−4 M final
concentration) to the naphtholate solution, with a gastight syringe.
The reaction was initiated by the addition of dioxygen, and the
mixture was stirred at room temperature overnight, turning from
colorless to pale gold yellow. The solvent was removed by rotary
evaporation, and then the residue was dissolved in dichloromethane,
and a few drops of distilled water containing a small amount of
ethylenediaminetetraacetic acid (EDTA) were added, the latter
serving to remove copper(II) from the reaction mixture. The organic
phase was separated and evaporated to give the crude product.
Purification by liquid chromatography, using silica gel (230−400 Å
mesh) and hexane/ethyl acetate 7:3 as eluent, gave the purified
product. 1H NMR (400 MHz, acetone-d6) δ 8.82 (s, 1H), 8.13 (d, J =
7.3 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.77
(d, J = 7.6 Hz, 1H), 7.56 (dd, J = 11.6, 7.4 Hz, 2H), 7.44−7.21 (m, J
= 7.1 Hz, 4H), 6.85 (d, J = 7.6 Hz, 1H), 6.46 (s, 1H).
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b00473.
Characterization of [Cu2(mXHI)]4+ complex by UV−
vis, CD, and NMR; kinetic profiles of catechols
oxidation reactions; dependence of reaction rates and
UV spectrum on the acetate buffer concentration;
characterization of phenol oxidation products; titration
of [Cu2(mXHI)]4+ with N-acetyl-L/D-tyrosine methyl
esters; experimental details (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: enrico.monzani@unipv.it.
■
ORCID
Luigi Casella: 0000-0002-7671-0506
Enrico Monzani: 0000-0002-8791-6446
Notes
The experiment of incorporation of labeled oxygen was performed
in the same conditions as above, but using 18O2 that was added with a
gastight syringe.
The authors declare no competing financial interest.
H
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(18) Santagostini, L.; Gullotti, M.; Pagliarin, R.; Monzani, E.;
Casella, L. Enantio-differentiating catalytic oxidation by a biomimetic
trinuclear copper complex containing L-histidine residues. Chem.
Commun. 2003, 17, 2186−2187.
(19) Santagostini, L.; Gullotti, M.; Pagliarin, R.; Bianchi, E.; Casella,
L.; Monzani, E. Functional mimics of copper enzymes. Synthesis and
stereochemical properties of the copper(II) complexes of a
trinucleating ligand derived from L-histidine. Tetrahedron: Asymmetry
1999, 10, 281−295.
(20) Gullotti, M.; Santagostini, L.; Pagliarin, R.; Palavicini, S.;
Casella, L.; Monzani, E.; Zoppellaro, G. Ligand binding, conforma-
tional and spectroscopic properties, and biomimeticmonooxygenase
activity by the trinuclear copper−PHI complex derived from L-
histidine. Eur. J. Inorg. Chem. 2008, 2008, 2081−2089.
(21) Mutti, F. G.; Gullotti, M.; Casella, L.; Santagostini, L.; Pagliarin,
R.; Andersson, K. K.; Iozzi, M. F.; Zoppellaro, G. A new chiral, poly-
imidazole N8-ligand and the related di- and tri-copper(II) complexes:
synthesis, theoretical modelling, spectroscopic properties, and
biomimetic stereoselective oxidations. Dalton Trans. 2011, 40,
5436−5457.
(22) Mimmi, M. C.; Gullotti, M.; Santagostini, L.; Battaini, G.;
Monzani, E.; Pagliarin, R.; Zoppellaro, G.; Casella, L. Models for
biological trinuclear copper clusters. Characterization and enantiose-
lective catalytic oxidation of catechols by the copper(II) complexes of
a chiral ligand derived from (S)-(−)-1,1′-binaphthyl-2,2′-diamine.
Dalton Trans. 2004, 14, 2192−2201.
ACKNOWLEDGMENTS
■
The authors thank the Univ. of Pavia for support. The
interuniversity consortium CIRCMSB is also acknowledged by
the authors.
REFERENCES
■
(1) Hamann, J. N.; Herzigkeit, B.; Jurgeleit, R.; Tuczek, F. Small-
molecule models of tyrosinase: From ligand hydroxylation to catalytic
monooxygenation of external substrates. Coord. Chem. Rev. 2017, 334,
54−66.
(2) Hoffmann, A.; Citek, C.; Binder, S.; Goos, A.; Rubhausen, M.;
̈
́
́
Troeppner, O.; Ivanovic-Burmazovic, I.; Wasinger, E. C.; Stack, T. D.
P.; Herres-Pawlis, S. Catalytic phenol hydroxylation with dioxygen:
extension of the tyrosinase mechanism beyond the protein matrix.
Angew. Chem., Int. Ed. 2013, 52, 5398−5401.
(3) Esguerra, K. V. N.; Fall, Y.; Lumb, J. P. A biomimetic catalytic
aerobic functionalization of phenols. Angew. Chem., Int. Ed. 2014, 53,
5877−5881.
̈
(4) Wilfer, C.; Liebhauser, P.; Hoffmann, A.; Erdmann, H.;
Grossmann, O.; Runtsch, L.; Paffenholz, E.; Schepper, R.; Dick, R.;
́ ́
Bauer, M.; Durr, M.; Ivanovic-Burmazovic, I.; Herres-Pawlis, S.
̈
Efficient biomimetic hydroxylation catalysis with a bis(pyrazolyl)-
imidazolylmethane copper peroxide complex. Chem. - Eur. J. 2015, 21,
17639−17649.
(5) Schottenheim, J.; Fateeva, N.; Thimm, W.; Krahmer, J.; Tuczek,
F. Catalytic conversion of monophenols to ortho-quinones in a
tyrosinase-like fashion: Towards more biomimetic and more efficient
model systems. Z. Anorg. Allg. Chem. 2013, 639, 1491−1497.
(6) Hamann, J. N.; Tuczek, F. New catalytic model systems of
tyrosinase: fine tuning of the reactivity with pyrazole-based N-donor
ligands. Chem. Commun. 2014, 50, 2298−2300.
(23) Gullotti, M.; Santagostini, L.; Pagliarin, R.; Granata, A.; Casella,
L. Synthesis and characterization of new chiral octadentate nitrogen
ligands and related copper(II) complexes as catalysts for stereo-
selective oxidation of catechols. J. Mol. Catal. A: Chem. 2005, 235,
271−284.
(24) Mutti, F. G.; Zoppellaro, G.; Gullotti, M.; Santagostini, L.;
Pagliarin, R.; Andersson, K. K.; Casella, L. Biomimetic modelling of
copper enzymes: synthesis, characterization, EPR analysis and
enantioselective catalytic oxidations by a new chiral trinuclear
copper(II) complex. Eur. J. Inorg. Chem. 2009, 2009, 554−566.
(25) Perrone, M. L.; Salvadeo, E.; Lo Presti, E.; Pasotti, L.; Monzani,
E.; Santagostini, L.; Casella, L. A dinuclear biomimetic Cu complex
derived from L-histidine: synthesis and stereoselective oxidations.
Dalton Trans. 2017, 46, 4018−4029.
(7) Hamann, J. N.; Rolff, M.; Tuczek, F. Monooxygenation of an
appended phenol in a model system of tyrosinase: implications on the
enzymatic reaction mechanism. Dalton Trans. 2015, 44, 3251−3258.
(8) Trammell, R.; Rajabimoghadam, K.; Garcia-Bosch, I. Copper-
promoted functionalization of organic molecules: from biologically
relevant Cu/O2 model systems to organometallic transformations.
Chem. Rev. 2019, 119, 2954−3031.
(9) Esguerra, K. V. N.; Lumb, J.-P. Cu(III)-mediated aerobic
oxidations. Synthesis 2019, 51, 334−358.
(26) Mirica, L. M.; Ottenwaelder, X.; Stack, T. D. P. Structure and
spectroscopy of copper-dioxygen complexes. Chem. Rev. 2004, 104,
1013−1046.
(10) Askari, M. S.; Esguerra, K. V. N.; Lumb, J.-P.; Ottenwaelder, X.
A biomimetic mechanism for the copper-catalyzed aerobic oxygen-
ation of 4-tert-butylphenol. Inorg. Chem. 2015, 54, 8665−8672.
(11) Esguerra, K. V. N.; Lumb, J.-P. Selectivity in the aerobic
dearomatization of phenols: total synthesis of dehydronornuciferine
by chemo- and regioselective oxidation. Angew. Chem., Int. Ed. 2018,
57, 1514−1518.
(27) Hatcher, L. Q.; Karlin, K. D. Ligand influences in copper-
dioxygen complex-formation and substrate oxidations. Adv. Inorg.
Chem. 2006, 58, 131−184.
(28) Reedijk, J.; Poeppelmeier, K. Comprehensive inorganic chemistry
II; Elsevier Ltd., 2013.
(29) Serrano-Plana, J.; Costas, M.; Company, A. Building complexity
in O2-binding copper complexes. Site-selective metalation and
intermolecular O2-binding at dicopper and heterometallic complexes
derived from an unsymmetric ligand. Inorg. Chem. 2014, 53, 12929−
12938.
(30) Serrano-Plana, J.; Garcia-Bosch, I.; Miyake, R.; Costas, M.;
Company, A. Selective ortho-hydroxylation-defluorination of 2-
fluorophenolates with a bis(μ-oxo)dicopper(III) species. Angew.
Chem., Int. Ed. 2014, 53, 9608−9612.
(31) Itoh, S.; Fukuzumi, S. Monooxygenase activity of type 3 copper
proteins. Acc. Chem. Res. 2007, 40, 592−600.
(32) Casella, L.; Monzani, E.; Gullotti, M.; Cavagnino, D.; Cerina,
G.; Santagostini, L.; Ugo, R. Functional modeling of tyrosinase.
Mechanism of phenol ortho-hydroxylation bydinuclear copper
complexes. Inorg. Chem. 1996, 35, 7516−7525.
(33) Santagostini, L.; Gullotti, M.; Monzani, E.; Casella, L.;
Dillinger, R.; Tuczek, F. Reversible dioxygen binding and phenol
oxygenation in a tyrosinase model system. Chem. - Eur. J. 2000, 6,
519−522.
(34) Palavicini, S.; Granata, A.; Monzani, E.; Casella, L.
Hydroxylation of phenolic compounds by a peroxodicopper(II)
(12) Huang, Z.; Kwon, O.; Huang, H.; Fadli, A.; Marat, X.; Moreau,
M.; Lumb, J.-P. A bioinspired synthesis of polyfunctional indoles.
Angew. Chem., Int. Ed. 2018, 57, 11963−11967.
(13) Gamba, I.; Palavicini, S.; Monzani, E.; Casella, L. Catalytic
sulfoxidation by dinuclear copper complexes. Chem. - Eur. J. 2009, 15,
12932−12936.
(14) Perrone, M. L.; Lo Presti, E.; Dell’Acqua, S.; Monzani, E.;
Santagostini, L.; Casella, L. Synthesis, characterization, and stereo-
selective oxidations of the dinuclear copper(II) complex derived from
a chiral diamino-m-xylenetetra(benzimidazole) ligand. Eur. J. Inorg.
Chem. 2015, 2015, 3493−3500.
(15) Presti, E. L.; Monzani, E.; Santagostini, L.; Casella, L. Building
biomimetic model compounds of dinuclear and trinuclear copper
clusters for stereoselective oxidations. Inorg. Chim. Acta 2018, 481,
47−55.
(16) Moore, K.; Vigee, G. S. The synthesis of binuclear copper(II)
complexes and the study of their catecholase activity. Inorg. Chim.
Acta 1982, 66, 125−130.
(17) Allen, S. E.; Walvoord, R. R.; Padilla-Salinas, R.; Kozlowski, M.
C. Aerobic copper-catalyzed organic reactions. Chem. Rev. 2013, 113,
6234−6458.
I
DOI: 10.1021/acs.inorgchem.9b00473
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
complex: further insight into the mechanism of tyrosinase. J. Am.
Chem. Soc. 2005, 127, 18031−18036.
of the copper catalyst to achieve control of oxidase vs oxygenase
activity. J. Am. Chem. Soc. 2008, 130, 12232−12233.
(35) Battaini, G.; Granata, A.; Monzani, E.; Gullotti, M.; Casella, L.
Biomimetic oxidations by dinuclear and trinuclear copper complexes.
Adv. Inorg. Chem. 2006, 58, 185−233.
(53) Verma, P.; Weir, J.; Mirica, L.; Stack, T. D.P. Tale of a twist:
magnetic and optical switching in copper(II) semiquinone complexes.
Inorg. Chem. 2011, 50, 9816−9825.
́
(54) Huang, Z.; Askari, M. S.; Esguerra, K. V. N.; Dai, T.-Y.; Kwon,
O.; Ottenwaelder, X.; Lumb, J.-P. A bio-inspired synthesis of
oxindoles by catalyticaerobic dual C−H functionalization of phenols.
Chem. Sci. 2016, 7, 358−369.
(55) Huang, Z.; Lumb, J.-P. A catalyst-controlled aerobic coupling of
ortho-quinones and phenols applied to the synthesis of aryl ethers.
Angew. Chem., Int. Ed. 2016, 55, 11543−11547.
(56) Jain, R.; Cohen, L. A. Regiospecific alkylation of histidine and
histamine at N-1 (τ). Tetrahedron 1996, 52, 5363−5370.
(57) Granata, A.; Monzani, E.; Bubacco, L.; Casella, L. Mechanistic
insight into the activity of tyrosinase from variable-temperature
studies in an aqueous/organic solvent. Chem. - Eur. J. 2006, 12,
2504−2514.
(36) Company, A.; Palavicini, S.; Garcia-Bosch, I.; Mas-Balleste, R.;
Que, L.; Rybak-Akimova, E. V.; Casella, L.; Ribas, X.; Costas, M.
Tyrosinase-like reactivity in a Cu(III)2(mu-O)2 species. Chem. - Eur. J.
2008, 14, 3535−3538.
(37) Garcia-Bosch, I.; Company, A.; Frisch, J. R.; Torrent-Sucarrat,
M.; Cardellach, M.; Gamba, I.; Guell, M.; Casella, L.; Que, L.; Ribas,
̈
X.; Luis, J. M.; Costas, M. O2 activation and selective phenolate ortho
hydroxylation by an unsymmetric dicopper mu-eta1:eta1-peroxido
complex. Angew. Chem., Int. Ed. 2010, 49, 2406−2409.
(38) Abdel-Magid, A. F.; Mehrman, S. J. A. A review on the use of
sodium triacetoxyborohydride in the reductive amination of ketones
and aldehydes. Org. Process Res. Dev. 2006, 10 (5), 971−1031.
(39) Casella, L.; Gullotti, M. Coordination modes of histidine. 5.
Copper(II) complexes of L-N.tau.-methylhistidine and L-N.alpha.,
N.alpha.-dimethylhistidine in aqueous solution. Inorg. Chem. 1983, 22,
242−249.
(40) Fawcett, T. G.; Bernarducci, E. E.; Krogh-Jespersen, K.;
Schugar, H. J. Charge-transfer absorptions of copper(II)-imidazole
and copper(II)-imidazolate chromophores. J. Am. Chem. Soc. 1980,
102, 2598−2604.
(41) Bernarducci, E.; Schwindinger, W. F.; Hughey, J. L.; Krogh-
Jespersen, K.; Schugar, H. J. Electronic spectra of copper(II)-
imidazole and copper(II)-pyrazole chromophores. J. Am. Chem. Soc.
1981, 103, 1686−1691.
(42) Casella, L.; Gullotti, M. Coordination modes of histidine. 4.
Coordination structures in the copper(II)-L-histidine (1:2) system. J.
Inorg. Biochem. 1983, 18, 19−31.
(43) Casella, L.; Carugo, O.; Gullotti, M.; Garofani, S.; Zanello, P.
Hemocyanin and tyrosinase models. Synthesis, azide binding, and
electrochemistry of dinuclear copper(II) complexes with poly-
(benzimidazole) ligands modeling the met forms of the proteins.
Inorg. Chem. 1993, 32, 2056−2067.
(44) Mason, S. F Molecular optical activity and the chiral
discriminations; Cambridge University Press, 1982.
(45) Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in
solution. Determination of equilibrium constants with the HYPER-
QUAD suite of programs. Talanta 1996, 43, 1739−1753.
(46) Monzani, E.; Quinti, L.; Perotti, A.; Casella, L.; Gullotti, M.;
Randaccio, L.; Geremia, S.; Nardin, G.; Faleschini, P.; Tabbì, G.
Tyrosinase models. Synthesis, structure, catechol oxidase activity, and
phenol monooxygenase activity of a dinuclear copper complex derived
from a triamino pentabenzimidazole ligand. Inorg. Chem. 1998, 37,
553−562.
(47) Granata, A.; Monzani, E.; Casella, L. Mechanistic insight into
the catechol oxidase activity by a biomimetic dinuclear copper
complex. JBIC, J. Biol. Inorg. Chem. 2004, 9, 903−913.
(48) Herlinger, E.; Jameson, R. F.; Linert, W. Spontaneous
autoxidation of dopamine. J. Chem. Soc., Perkin Trans. 2 1995, 2,
259−263.
(49) Battaini, G.; Casella, L.; Gullotti, M.; Monzani, E.; Nardin, A.;
Perotti, A.; Randaccio, L.; Santagostini, L.; Heinemann, F. W.;
Schindler, S. Structure and reactivity studies on dinuclear copper
complexes of the ligand α,α’-bis{bis[1-(1′-methyl-2′-benzimidazolyl)-
methyl]amino}-m-xylene. Eur. J. Inorg. Chem. 2003, 2003, 1197−
1205.
(50) Holz, R. C.; Bradshaw, J. M.; Bennett, B. Synthesis, molecular
structure, and reactivity of dinuclear copper(II) complexes with
carboxylate-rich coordination environments. Inorg. Chem. 1998, 37,
1219−1225.
(51) Pievo, R.; Gullotti, M.; Monzani, E.; Casella, L. Tyrosinase
catalyzes asymmetric sulfoxidation. Biochemistry 2008, 47, 3493−
3498.
(52) Hewgley, J. B.; Stahl, S. S.; Kozlowski, M. C. Mechanistic study
of asymmetric oxidative biaryl coupling: evidence for self-processing
J
DOI: 10.1021/acs.inorgchem.9b00473
Inorg. Chem. XXXX, XXX, XXX−XXX