Carboxylate-enhanced reactivity in the oxygenation of copper flavonolate complexes

Article abstract of DOI:10.1016/S1381-1169(03)00456-4

The oxygenolytic cleavage of flavonolate coordinated to copper(II) with the auxiliary ligand 3,3′-iminobis(N,N-dimethyl-propylamine), [Cu ^II(fla)(idpa)]ClO₄, is enhanced by the addition of acetate and benzoate. Monodentate coordinati

Full text of DOI:10.1016/S1381-1169(03)00456-4

Journal of Molecular Catalysis A: Chemical 206 (2003) 83–87
Carboxylate-enhanced reactivity in the oxygenation
of copper flavonolate complexes
Éva Balogh-Hergovicha, József Kaizera, Gábor Speiera,b,∗
a
Research Group for Petrochemistry, Hungarian Academy of Sciences, Veszprém 8200, Hungary
b
Department of Organic Chemistry, University of Veszprém, Wartha V. u. 1, Veszprém 8200, Hungary
Received 12 June 2003; received in revised form 12 June 2003; accepted 13 June 2003
Abstract
The oxygenolytic cleavage of flavonolate coordinated to copper(II) with the auxiliary ligand 3,3ꢀ-iminobis(N,N-dimethyl-
propylamine), [CuII(fla)(idpa)]ClO4, is enhanced by the addition of acetate and benzoate. Monodentate coordination of the
flavonolate forced by the carboxylate ligands is believed to be the reason for the higher reaction rates.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Quercetinase; Model; Dioxygenation; Copper; Dioxygen
1. Introduction
closing their molecular structures by X-ray diffractom-
etry [8–13]. Oxygenation of these (flavonolato)copper
complexes led to the (O-benzoylsalicylato)copper
complexes and CO in almost all cases mimicking
the enzyme reaction [14–20]. However, the rates of
the reactions were rather slow and higher temper-
atures were necessary. Recent characterization of
the enzyme quercetinase from Aspergillus japoni-
cus by X-ray crystallography has shown that in the
coordination environment of copper(II) at the ac-
tive site beside histidine carboxylate coordination
of glutamate is also possible [21]. This fact has
prompted us to check at model reactions whether
carboxylate coordination enhances the reaction rates
of the ring scission reaction of flavonol. We used
the formerly studied (flavonolato)copper(II) com-
plex [CuII(fla)(idpa)]ClO4 (fla = flavonolate; idpa =
3,3ꢀiminobis(N,N-dimethylpropylamine)) (3) [13] for
the oxygenation reactions by adding carboxylic acids
or carboxylate anions in order to see whether these
coordinate to the copper(II) ion or enhance the reac-
tivity of the complex.
Quercetin 2,3-dioxygenase is a copper-containing
metalloenzyme, which catalyzes the oxygenolytic
cleavage of the heterocyclic ring of quercetin at the
C2–C3 carbon bonds yielding the depside (carboxylic
acid ester) and carbon monoxide (Eq. (1)) [1–4]. The
substrate is believed to bind to the copper(II) ion
=
through its 3OH and 4C O groups [5–7].
Model studies on copper flavonolate complexes
with various ligands have established this binding
mode of the substrate quercetin to copper(II) by dis-
∗
Corresponding author. Tel.: +36-88-422-022/4657;
fax: +36-88-427-492.
E-mail address: speier@almos.vein.hu (G. Speier).
1381-1169/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S1381-1169(03)00456-4

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84
2. Experimental
absorbance of the reaction mixture at 426.5 nm (λmax
of a typical band of [CuII(fla)(idpa)]ClO4). The argon
was then replaced by dioxygen, and the consumption
of [CuII(fla)(idpa)]ClO4 was analyzed periodically.
Experimental conditions are summarized in Table 1.
The O2 concentration was calculated from literature
data [23] taking into account the partial pressure of
DMF [24] and assuming the validity of Dalton’s law.
The rate of consumption was independent of the stir-
ring rate, excluding eventual diffusion control effects.
All manipulations were carried out using standard
Schlenk technique under purified argon [22]. Anhy-
drous DMF was purchased from Aldrich and used as
supplied. [CuII(fla)(idpa)]ClO4 was prepared as de-
scribed earlier [13]. Gaseous dioxygen from Messer-
griesheim was 99.6% and passed through P2O5 and
Blaugel in order to remove traces of water and other
impurities. Electronic spectra were recorded on a
Shimadzu UV-160A spectrophotometer using quartz
cells.
3. Results and discussion
2.1. Oxygenation of [CuII(fla)(idpa)]ClO4
The X-ray structure determination of quercetin
2,3-dioxygenase revealed that the type copper(II) cen-
ter at the active site in the enzyme isolated from A.
japonicus displays two distinct geometries; a distorted
tetrahedral coordination, formed by three histidine
and a water molecule, and a distorted trigonal bipyra-
midal environment, which additionally comprises a
carboxylate coordination of glutamate. In the course
of our earlier studies on quercetin 2,3-dioxygenase
model systems we prepared (flavonolato)copper(II)
complexes (fla = flavonolate) with the auxiliary lig-
and 3,3ꢀ-iminobis(N,N-dimethylpropylamine) (idpa).
The molecular structure of [CuII(fla)(idpa)]ClO4, has
been determined by X-ray diffractometry to have a dis-
torted trigonal bipyramidal geometry and the kinetics
of its oxygenation has been disclosed to give a second
order rate law [13,19]. The tridentate N3-ligand idpa
seemed to model the histidine environment leaving
[CuII(fla)(idpa)]ClO4 (0.294 g, 0.5 mmol) in DMF
(50 cm3) was treated with dioxygen (0.1 MPa) at
100 ◦C for 8 h, the solution was cooled to room tem-
perature and diethyl ether was layered to afford blue
powder of [CuII(O-bs)(idpa)]ClO4 (0.183 g, 62%) [13].
2.2. Kinetic measurements
Reactions of the flavonolato copper complex with
O2 were performed in DMF solution. [CuII(fla)(idpa)]-
ClO4 was dissolved under argon in a thermostatically
controlled reaction vessel with an inlet for taking
samples with a syringe, and connected to a mercury
manometer to maintain a constant pressure. The solu-
tion was then heated to 100 ◦C, a sample was taken by
syringe, and the initial concentration of complex was
determined by UV/Vis spectroscopy measuring the
Table 1
Kinetic data for the oxygenation of [CuII(fla)(idpa)]ClO4 in DMF solution in the presence of carboxylatesa
Expriment no.
[Cu]
[Additive]
104kꢀ
Rb (%)
kobs
(×104; mol dm−3
)
(×103; mol dm−3
)
(×103; s−1
)
(×102; mol dm−3
)
1
2
3
4
5
6
7
8
9
5.23
5.16
5.06
5.34
5.17
5.30
5.16
5.48
5.31
–
0.52
1.83
1.88
3.01
3.47
3.19
3.39
1.21
0.47
99.09
98.28
99.32
99.42
99.23
99.61
99.63
99.72
99.65
0.67
2.38
2.45
3.91
4.51
4.14
4.40
1.57
0.61
0.03
0.29
0.11
0.14
0.16
0.11
0.10
0.01
0.01
Na(OAc), 1.63
Na(OAc), 1.87
Na(OAc), 5.43
Na(OAc), 10.46
Na(OAc), 15.55
Na(OAc), 20.67
Na(OBenz), 5.46
HOAc, 5.31
a Conditions: 50 cm3 DMF, T = 100 ◦C, [O2] = 7.7 × 10−3 mol dm−3
.
b Correlation coefficients of least-squares regressions.

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85
6
+ NaOAc
+ NaOBenz
+ HOAc
5
4
3
2
1
no additive
0
20
40
60
80
100
t / min
Fig. 1. Influence of additives on the oxygenation of [CuII(fla)(idpa)]ClO4 (3). Conditions: 50 cm3 DMF,
T
=
100 ◦C,
[O2] = 7.7 × 10−3 mol dm−3, (×) 0.026 mmol 3, (᭺) 0.026 mmol 3 + 0.26 mmol HOAc, (᭛) 0.027 mmol 3 + 0.273 mmol NaOBenz, (᭜)
0.026 mmol 3 + 0.272 mmol NaOAc (experiments 1, 9, 8, and 4; Table 1).
one or two coordination sites free for the coordination
of solvent and/or the substrate flavonolate. In order
to mimic the carboxylate coordination in the model
system, we added sodium acetate to the complex
[CuII(fla)(idpa)]ClO4 and looked at the kinetics of the
reaction.
The explanation of the larger reaction rates of
the oxygenation of [CuII(fla)(idpa)]ClO4 in the
presence of added acetate or benzoate can be ex-
plained as shown in the Scheme 1. In complex
[CuII(fla)(idpa)]ClO4 (3) the geometry around the
Experiments showing the time course of the oxy-
genation reactions of [CuII(fla)(idpa)]ClO4 (3) and
those with added sodium acetate, sodium benzoate,
and acetic acid are shown in Fig. 1. It can be seen
that the addition of acetic acid did not have any
effect on the reaction rate of the oxygenation. The
addition of sodium acetate and benzoate, however,
accelerate the reaction rates compared to the reaction
with no additives. If we alter the ratio of sodium
acetate to the complex 3 we can see that at higher
acetate concentration the oxygenation reaction pro-
ceeds faster (Fig. 2). The second order reaction rate
constants (kobs) are compiled in Table 1. The kinetic
date demonstrate clearly that the addition of acetic
acid does not have any influence on the reaction rate
of the oxygenation of [CuII(fla)(idpa)]ClO4, however
carboxylate addition (acetate, benzoate) enhances the
reaction rates. Higher carboxylate concentration re-
sults in higher reaction rates. At a 20:1 acetate to
(flavonolato)copper(II) complex ratio leads to almost
one order of magnitude higher reaction rate.
O
O
R
O
C
O
R
O
O
O
O
O
O
N
Me
Me
Me
Me
Me
Me
Me
Me
N
Cu
N
N
Cu
NH
NH
3
5
O2
-CO
O2 -CO
O
O
O
O
O
O
O
C
R
Me
Me
O
O
N
O
Me
Me
Me
Me
Me
Me
Cu
N
N
Cu
N
NH
NH
a) R = Me
b) R = Ph
6
4
Scheme 1.

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6
5
4
3
2
1
0
10
20
30
40
t / min
50
60
70
Fig. 2. Time course for the oxygenation of [CuII(fla)(idpa)]ClO4 (3) in the presence of sodium acetate. Conditions: 50 cm3 DMF, T = 100 ◦C,
[O2] = 7.7 × 10−3 mol dm−3, (᭹) 0.026 mmol 3, (᭛) 0.025 mmol 3 + 0.093 mmol NaOAc, (᭜) 0.026 mmol 3 + 0.272 mmol NaOAc, (×)
0.025 mmol 3 + 0.523 mmol NaOAc (experiments 1, 3, 4, and 5; Table 1).
copper(II) ion can be regarded as a distorted trigonal
bipyramidal with a τ value of 0.61 [13]. The two ni-
trogen atoms of the N3-ligand and one oxygen atom
of the flavonolate ligand occupy basal positions. The
remaining N-atom of the N3-ligand and the other
oxygen atom of the flavonolate ligand lie in apical
positions. The flavonolate ligand in 3 is bidentate,
however on addition of the carboxylate ions to the
complex they coordinate to the copper(II) in a mon-
odentate fashion just displacing one of the oxygen
atoms of the flavonolate ligand. According to that 3 is
converted to 5, where the flavonolate ligand becomes
monodentate. This may be the reason for the en-
hanced reactivity towards dioxygen. So 5 is converted
to 6 by incorporating both O-atoms of dioxygen into
the substrate with the concomitant release of carbon
monoxide, just in agreement with the enzyme reac-
tion. Seemingly, carboxylate coordination, as found
in the structure of quercetin 2,3-dioxygenase from
A. japonicus, is necessary for the enzyme reaction
in order to elicit reasonable reaction rates. On the
bases of these experiments we believe to have the
first evidence that carboxylate coordination plays an
important role in the enzyme reaction, and these re-
actions may be regarded as the first best mimicking
models for the enzyme. Further work is in progress to
work out structural and functional models of quercetin
2,3-dioxygenase in light of the recent knowledge of
its structure.
Acknowledgements
We thank the Hungarian National Research Fund
OTKA # T 03400 for financial support of this work.
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