Reversible dioxygen binding and phenol oxygenation in a tyrosinase model system

Article abstract of DOI:10.1002/(SICI)1521-3765(20000204)6:3<519::AID-CHEM519>3.0.CO;2-I

The complex [Cu₂(L-66)]^2- (L-66 = a,a'-bis{bis[2-(1'-methyl-2'- benzimidazolyl)ethyl]amino}-m-xylene) undergoes fully reversible oxygenation at low temperature in acetone. The optical [λ(max) = 362 (ε 15000), 455 (ε 2000), and 550 nm

Full text of DOI:10.1002/(SICI)1521-3765(20000204)6:3<519::AID-CHEM519>3.0.CO;2-I

FULL PAPER
Reversible Dioxygen Binding and Phenol Oxygenation in a
Tyrosinase Model System
Laura Santagostini,[b] Michele Gullotti,[b] Enrico Monzani,[a] Luigi Casella,*[a]
ReneÂe Dillinger,[c] and Felix Tuczek[c]
Abstract: The complex [Cu2(L-66)]2‡
(L-66 a,a'-bis{bis[2-(1'-methyl-2'-
studied at 788C, gave the rate constant
performs the regiospecific ortho-hy-
droxylation of 4-carbomethoxypheno-
late to the corresponding catecholate
and the oxidation of 3,5-di-tert-butyl-
1
ˆ
k1 ˆ 1.1m 1 s , for adduct formation, and
1
benzimidazolyl)ethyl]amino}-m-xylene)
undergoes fully reversible oxygenation
at low temperature in acetone. The optical
[lmax ˆ 362 (e 15000), 455 (e 2000), and
k
ˆ 7.8  10 5 s , for dioxygen release
1
from the Cu2O2 complex. From these
values, the O2 binding constant K ˆ
catechol to the quinone at
608C.
1
1.4 Â 104 m at 788C could be deter-
Therefore, [Cu2(L-66)]2‡ is the first
synthetic complex to form a stable
dioxygen adduct and exhibit true tyro-
sinase-like activity on exogenous phe-
nolic compounds.
1
550 nm (e 900m 1 cm )] and resonance
mined. The [Cu2(L-66)(O2)]2‡ complex
1
Raman features (760 cm , shifted to
1
719 cm with 18O2) of the dioxygen
Keywords: copper
´ oxidations ´
adduct [Cu2(L-66)(O2)]2‡ indicate that
it is a m-h2:h2-peroxodicopper(ii) com-
plex. The kinetics of dioxygen binding,
oxygenations ´ peroxo complexes ´
Raman spectroscopy
several systems.[6±11] These Cu2O2 cores contain either end-on
m-1,2 or side-on m-h2:h2 peroxide coordination, or have a bis-m-
oxo arrangement. Some of them have been shown to be
capable of performing hydroxylation reactions at C H bonds
of the ligand,[6, 8±10] or radical reactions at C H bonds of
exogenous substrates, including phenols.[11] These radical
reactions are reminiscent of the nonphysiological, poorly
efficient enzymatic activity exhibited by molluscan hemocya-
nin,[12a] while tyrosinase catalysis proceeds through nonradical
pathways.[12b] We previously reported conversion of phenolic
substrates to catechols by the complex [Cu2(L-66)]2‡ (L-66 ˆ
a,a'-bis{bis[2-(1'-methyl-2'-benzimidazolyl)ethyl]amino}-m-
xylene) and dioxygen at 408C or room temperature.[13] Full
incorporation of 18-oxygen from 18O2 into the product was
demonstrated.[13b] Here we show that [Cu2(L-66)]2‡ undergoes
fully reversible oxygenation at 808C (Scheme 1), and that
the resulting m-peroxodicopper(ii) adduct is involved in the
regiospecific ortho-hydroxylation of an exogenous, electron-
Introduction
Tyrosinase is a nearly ubiquitous copper-containing mono-
oxygenase that has both catecholase and cresolase activity.[1]
It is responsible for browning reactions that occur to perform
diverse physiological functions in different organisms.[2]
Tyrosinase reversibly binds dioxygen,[3] and the spectral
characteristics of oxytyrosinase bear strong similarities to
those of the oxy form of the dioxygen carrier hemocyanin:[4]
absorption bands at 350 nm (e ꢀ 20000m 1 cm ) and 590 nm
1
(e ꢀ 1000m 1 cm ), an O O stretching frequency of
1
1
ꢀ750 cm , and a Cu Cu distance of 3.6 Š, as shown by
X-ray crystallography of hemocyanin.[5] These features are
associated with a Cu2O2 core containing a side-on bridged (or
m-h2:h2) peroxide moiety.[5] Dioxygen binding to biomimetic
CuI complexes, to form Cu2O2 adducts, has been reported for
[a] Prof. L. Casella, Dr. E. Monzani
Á
Dipartimento di Chimica Generale, Universita di Pavia
Via Taramelli 12, 27100 Pavia (Italy)
Fax : (‡ 39)0382-528544
E-mail: bioinorg@unipv.it.
[b] Dr. L. Santagostini, Prof. M. Gullotti
Á
Dipartimento C.I.M.A., Universita di Milano
Centro CNR, Via Venezian 21, 20133 Milano (Italy).
[c] R. Dillinger, Dr. F. Tuczek[‡]
Institut für Anorganische Chemie und Analytische Chemie
Universität Mainz, Staudinger Weg 9, 55099 Mainz (Germany).
[ ] Current address: Institut für Anorganische
‡
Universität Kiel, Otto-Hahn-Platz 6/7, 24098 Kiel (Germany)
Scheme 1. The reversible oxygenation of [Cu2(L-66)]2‡
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519

L. Casella et al.
FULL PAPER
poor phenol to catechol [Eq. (1)] and oxidation of an
electron-rich catechol to quinone [Eq. (2)].[13, 14] All these
features are characteristic of tyrosinase.[1] Therefore, this
system represents the first true tyrosinase model.
Results and Discussion
Oxygenation of [Cu2(L-66)]2‡ carried out at about 808C in
acetone yields an intensely brown solution with lmax ˆ 362 (e
1
15000), 455 (e 2000), and 550 nm (e 900m 1 cm ) (Fig-
ure 1).[15] The stoichiometry of oxygen binding (Cu:O2 ˆ 2:1)
Figure 2. Resonance Raman spectra of a <1mm solution of [Cu2(L-
66)(O2)]2‡ in acetone including 18O2 data. A) lexc. ˆ 350.7 nm, 1000 s,
808C. B) Temperature cycle
80 ! 45 ! 808C (lexc. ˆ 350.7 nm,
100 s spectra). C) Difference spectrum after O2 addition showing the O O
1
1
stretching vibration at 760 cm and 719 cm for 16O2 and 18O2, respec-
tively (lexc. ˆ 415.4 nm, 1000 s, 808C). * ˆ solvent peaks.
peaks could be detected with this excitation wavelength.
However, when excited at l ˆ 415.4 nm an additional peak is
Figure 1. Optical spectra developed on oxygenating [Cu2(L-66)]2‡ in
acetone at 788C.
1
1
observed at 760 cm , which shifts to 719 cm upon 18O2
substitution (Figure 2C). Owing to its isotopic shift
1
(41 cm ), this peak is assigned to the O O stretch. The low
indicates that
a
peroxodicopper(ii) complex, [Cu2(L-
frequency of this vibration, combined with the UV-visible
spectral data, proves the presence of side-on (m-h2:h2)-
coordinated peroxide in [Cu2(L-66)(O2)]2‡.[16] Also typical
for this binding geometry are the peaks at 256, 283, and
66)(O2)]2‡, is formed. The oxygenation reaction can be fully
reversed at low temperature upon application of a vacuum,
the process being accelerated by mild warming of the solution.
After several oxygenation/deoxygenation cycles at 808C no
appreciable loss of absorption intensity from the oxygen
adduct is observed. The pattern of three UV-visible bands
developing upon oxygenation of [Cu2(L-66)]2‡ bears resem-
blance with that of the dioxygen adducts formed at low
temperature by the dinuclear complexes [Cu2(R-
XYL)]2‡(R ˆ H)[8b] and [Cu2(NnPY2)]2‡[8c] and has been at-
tributed to peroxide !CuII LMCT transitions of a side-on (m-
h2:h2)-bridging peroxide unit. The resonance Raman spectra
of [Cu2(L-66)(O2)]2‡ at 808C in acetone exhibit the follow-
ing features: with an excitation wavelength of l ˆ 350.7 nm
1
314 cm , which do not shift upon 18O2 substitution (Fig-
ure 2A); in resonance Raman spectra of hemocyanin these
features have been assigned to Cu (Ligand)N stretching
vibrations.[17] An alternative assignment of the peak at
283 cm 1 to the symmetric Cu2O2 core stretch, which primarily
involves copper motion, has been suggested.[16b]
The dioxygen binding process undergone by [Cu2(L-66)]2‡
occurs in a single observable step; no evidence was found for
an intermediate mononuclear copper(ii)-superoxide species.
The rate constant for adduct formation determined at 788C
1
was k1 ˆ 1.1m 1 s , and that of release of O2 from the Cu2O2
1
1
four peaks are observed at 214, 256, 283 and 314 cm , the
strongest one at 283 cm (Figure 2A). Upon warming, these
peaks gradually decrease in intensity and have essentially
disappeared at 458C. After subsequent cooling to 808C
the peaks reappear, demonstrating again the reversible
formation of the peroxo complex (Figure 2B). No further
complex k ˆ 7.8  10 5 s . From these values the binding
1
1
constant for dioxygen at 788C is determined to be K ˆ (k1/
k ) ˆ 1.4  104 m . This value is about three orders of
1
1
magnitude smaller than the equilibrium constant reported
for the oxygenation of [Cu2(H-XYL)]2‡ in dichlorometh-
ane.[8a] The difference is almost entirely due to the lower value
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Tyrosinase Model System
519±522
of k1, possibly because the larger benzimidazole groups slow
down the conformational rearrangement of the complex
leading to the dioxygen adduct. The different bite angle of the
five-membered benzimidazole ring, with respect to the six-
membered pyridine ring, may also cause some difference in
the stereochemistry of the two peroxo complexes. However,
whereas the [Cu2(H-XYL)]2‡ complex does not form a stable
dioxygen adduct and undergoes a fast ligand hydroxylation
reaction,[18] replacement of the pyridine donors of [Cu2(H-
XYL)]2‡ with benzimidazoles in [Cu2(L-66)]2‡ disables the
endogenous ligand hydroxylation. This is a necessary con-
dition to perform the regiospecific ortho-hydroxylation of an
exogenous phenolate.[13a]
Figure 4. Difference spectra recorded during the reaction of [Cu2(L-
66)(O2)]2‡ and 3,5-di-tert-butylcatechol (50 mol equiv) at
608C in
acetone.
When sodium 4-carbomethoxyphenolate is added to the
solution of [Cu2(L-66)(O2)]2‡ at 808C (excess O2 removed)
no change of the UV-visible spectrum occurs. Upon raising
the temperature to 608C the intensity of the peroxide !CuII
CT band at 362 nm diminishes to about 50%, due to thermal
decay of the dioxygen adduct. In addition, a slow reaction
takes place, as indicated by a further decrease in intensity of
the 362 nm band and appearance of a new absorption feature
near 340 nm (Figure 3). The spectrum that results after
and slow rate of the hydroxylation at this temperature, side
reactions between phenolate, catecholate ions, and acetone
decrease the yield of catechol (about 20% with respect to the
starting [Cu2(L-66)]2‡ complex , i.e. about 40% with respect
to [Cu2(L-66)(O2)]2‡, after 2 h). In contrast, clean oxygen-
ation of the same substrate by [Cu2(L-66)]2‡ and dioxygen was
observed in acetonitrile at 408C.[13a] However, under those
conditions the dioxygen adduct does not build up. Moreover,
hydroxylation was achieved by exposing the CuI-phenolate
adduct to dioxygen;[13] without substrate the reaction between
[Cu2(L-66)]2‡ and dioxygen at or near room temperature
results in the di-m-hydroxo-bridged dicopper(ii) complex
[Cu2(L-66)(OH)2]2‡.[19] In contrast, here the phenolate has
been added after oxygenation of the CuI complex at low
temperature, demonstrating conversion of the substrate into
catechol in the presence of the peroxo complex. In this way,
the [Cu2(L-66)]2‡ complex is shown to be the first small-
molecule system to form a stable dioxygen adduct and to
mediate the hydroxylation of exogeneous phenols, in analogy
to tyrosinase.
Experimental Section
Figure 3. Changes in the absorption spectra for the reaction of [Cu2(L-
66)(O2)]2‡ with sodium 4-carbomethoxyphenolate at low temperature. In
order of decreasing absorbance: a) [Cu2(L-66)(O2)]2‡ after the addition of
phenolate at 808C; b) after equilibration at 608C; c) after 1 h, d) 2 h,
e) 3 h, f) 4 h, and g) 5 h reaction at 608C.
The ligand L-66 and the corresponding dinuclear CuI and CuII complexes
were prepared as described before.[13] Low-temperature UV/Vis spectra
were recorded by using a HP 8452A diode array spectrophotometer in
conjunction with a custom-designed immersible fiber-optic quartz probe
(5 mm path length, Hellma) fitted to a Schlenk vessel with gas and vacuum
inlets. The stoichiometry of oxygen binding to [Cu2(L-66)]2‡ was deter-
mined by assaying the hydrogen peroxide produced by the addition of
excess trifluoroacetic acid to the oxygenated solution at 788C. A small
amount of this acidified solution was added to an aqueous phosphate buffer
solution (100mm) at pH 7 that contained an excess of 2,2'-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) and horseradish peroxidase
several hours and which exclusively exhibits the 340 nm band
is analogous to that observed for the catecholate adduct of
[Cu2(L-66)]4‡ in acetonitrile,[13a] and thus strongly suggests
conversion of phenol into catechol mediated by the copper-
dioxygen adduct. The further oxidation of catechol to quinone
is demonstrated by the reaction of 3,5-di-tert-butylcatechol
with [Cu2(L-66)(O2)]2‡ at 608C (excess O2 removed) (Fig-
ure 4). The decrease of the copper-peroxide band at 362 nm
and the development of the quinone band at about 420 nm
.
‡
(40nm). The peroxidase reaction produces the ABTS radical cation,
characterized by intense absorption bands at 414 nm (e 3.6 Â
104 m 1 cm
)
[20] and 660 nm (e 1.47 Â 104 m 1 cm 1).[21] The yield of hydrogen
1
peroxide found considering a 2Cu:1O2 stoichiometry was >90%. The
dicopper(ii) complex of L-66 gives no interference in this assay.
Raman spectra were recorded on a home-built spectrometer at Universität
Mainz and consisted of an Acton SpectraPro-500i double monochromator
equipped with 3600, 1800, and 600 groove per mm gratings and with a
nitrogen-cooled, back-illuminated CCD camera (PI Instruments, 1100PB,
1100 Â 330 PI Chip). The 3600 groove per mm grating was used for all
1
follow the same time profile (kobs ˆ 2.8  10 3 s and 2.6 Â
1
10 3 s , respectively). The process shown corresponds to one
catalytic cycle (2 catechol:1 O2).[14]
The conversion of 4-carbomethoxyphenolate to catecholate
by [Cu2(L-66)(O2)]2‡ at 608C is also proven by workup of
the reaction mixture followed by chemical analysis. Due to the
thermal instability of [Cu2(L-66)(O2)]2‡ at 608C (see above)
‡
measurements. Excitation source was a Spectra Physics 2080 Kr Laser
(350.7 nm, 415.4 nm). Typically spectra were run with laser powers of
10 mWand acquisition times of 100 s and 1000 s. Spectral resolution was set
to <3.5 cm 1. Low-temperature measurements were carried out in a liquid-
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L. Casella et al.
FULL PAPER
helium flow cryostat with
a
specially designed quartz sample rod
[5] K. A. Magnus, B. Hazes, H. Ton-That, C. Bonaventura, J. Bonaven-
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(CryoVac). The sample rod contained vacuum and gas inlets as well as a
silicon septum through which solutions could be injected under inert
atmosphere. The CuI complex [Cu2(L-66)]2‡ was prepared directly in the
sample rod under Ar and after cooling to 808C, oxygen was bubbled
through the solution for ꢁ 15 min, generating the brown peroxo complex in
solution. Concentrations of this species were estimated to be <1mm. 18O-
substitution experiments were performed by oxygenating the [Cu2(L-66)]2‡
solution with 18O2 gas (99%) obtained from Chemotrade.
Â
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1299 ± 1308.
The kinetics of dioxygen binding by [Cu2(L-66)]2‡ were studied at 362 nm,
upon injecting a small volume of a concentrated solution of the CuI
complex in acetone into a large volume of the solvent saturated with
dioxygen at low temperature. The kinetic constant kobs was obtained from
fitting of the data of absorbance versus time at 788C to the equation for
the reversible oxygenation equilibrium (even though at 788C oxygen-
ation of the complex is nearly complete), Equations (3) ± (5), in which
[CuI2]0 is the initial concentration of [Cu2(L-66)]2‡
.
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k1
„
CuI2 ‡ O2
Cu2O2
(3)
(4)
(5)
k
Â
1
Y.-M. Neuhold, T. Palanche, J. A. Halfen, V. G. Young, Jr., T. A.
0
k1‰O2Š‰CuI2Š
Kaden, L. Que, Jr., A. D. Zuberbühler, W. B. Tolman, Inorg. Chem.
1997, 36, 6343 ± 6356.
[Cu2O2] ˆ
{1 exp[ (k1[O2] ‡ k 1)t]}
k1‰O2Š ‡ k
1
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kobs ˆ k1[O2] ‡ k
1
The solubility of dioxygen in acetone at low temperature was taken from
the literature.[9b] The deoxygenation of [Cu2(L-66)(O2)]2‡ is slow at low
temperature and was followed spectrally after applying a vacuum to the
oxygenated solution and purging with an inert gas. Under these conditions,
deoxygenation proceeded until the system equilibrated to the residual
dioxygen pressure. To achieve full deoxygenation, application of a second
vacuum/purge cycle would be necessary, but since this process causes base
line problems in the optical readings we limited the spectral observation to
the first cycle.
The phenol oxygenation experiments were carried out under Ar by adding
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a
solution of sodium 4-carbomethoxyphenolate (0.024 mmol) in dry
acetone (1 mL) to [Cu2(L-66)(O2)]2‡ (0.024 mmol, excess O2 removed) in
the same solvent (40 mL) at 808C. After 15 min, the reaction was allowed
to proceed at 608C. Samples (10 mL) of the solution were withdrawn and
immediately quenched in H2SO4 (0.2m, 1 mL). The mixture was taken to
dryness, and the residue was extracted with chloroform and analyzed by
1H NMR and HPLC (after addition of
a standard) as described
previously.[13, 14] Total recovery of catechol plus unreacted phenol did not
exceed 50%. By-products from side reactions are evident in the NMR
spectra and HPLC traces.
[15] For solubility reasons, the low-temperature oxygenation experiments
could be carried out only in dilute acetone solution. The solubility of
the complex in solvents like dichloromethane or THF is extremely
low, while in propionitrile no appreciable dioxygen adduct formation
was observed at low temperature.
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10421 ± 10431.
Acknowledgments
This work was supported by the Italian MURST and by DFG (Tu58/7-1).
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Received: November 5, 1999 [F2119]
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