Manganese and iron flavonolates as flavonol 2,4-dioxygenase mimics

Article abstract of DOI:10.1039/b711864c

Mononuclear manganese(II) and iron(III) flavonolates were synthesized as synthetic enzyme-substrate complexes, and their oxygenation reactions as biomimetic functional models with relevance to flavonol 2,4-dioxygenases are briefly described. The Royal Soc

Full text of DOI:10.1039/b711864c

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Manganese and iron flavonolates as flavonol 2,4-dioxygenase mimics{
Jo´zsef Kaizer,a Ga´bor Bara´th,a Jo´zsef Pap,a Ga´bor Speier,*a Michel Giorgib and Marius Re´glierc
Received (in Cambridge, UK) 2nd August 2007, Accepted 28th September 2007
First published as an Advance Article on the web 11th October 2007
DOI: 10.1039/b711864c
we report details for the synthesis and characterization of
MnII(fla)2(py)2 and FeIII(49OMefla)3 complexes in order to obtain
information of the possible binding of the substrate to the metal
ion and to use them in oxygenation reactions to study the
oxidative ring splitting of the heterocyclic ring in the substrate
compared to the copper-containing models.
Mononuclear manganese(II) and iron(III) flavonolates were
synthesized as synthetic enzyme-substrate complexes, and their
oxygenation reactions as biomimetic functional models with
relevance to flavonol 2,4-dioxygenases are briefly described.
Flavonols (flaH) are widely distributed in vascular plants,1 and
flavonoids form active constituents of a number of herbal and
traditional medicines.2 Flavonol 2,4-dioxygenase (FDO), which
catalyses the oxidative degradation of flavonols to a depside
(phenolic carboxylic acid esters) with concomitant evolution of
carbon monoxide, was first recognized more than four decades ago
in species of Aspergillus grown on rutin, and quercetinases from
Aspergillus flavus,3 Aspergillus niger,4 and Aspergillus japonicus5
have been characterized, and the crystal structure of quercetinase
Because of the oxidation state of manganese in the manganese-
containing flavonol 2,4-dioxygenase is believed to be two, the
preparation and characterization of a manganese(II) flavonolate
seemed to be of great interest. Complex MnII(fla)2(py)2 (1) was
isolated as a yellow solid in y80% yield by the reaction of
Mn(ClO4)2?6H2O, flavonol, and pyridine (py) in the presence of
triethylamine at room temperature in methanol under argon.
Compound 1 shows a characteristic p–p* transition in the visible
region at 432 nm due to the coordinated flavonolate ligand.11
A
5
from Aspergillus japonicus has been reported [eqn. (1)].
strong IR band at 1542 cm21 assigned to n(CO), showing a
decrease of 60 cm21 [n(CO) = 1602 cm21] compared to flavonol,
arise as a result of the formation of a five-membered ring.12
A
ð1Þ
strong band around 1600 cm21 and a weak band at 1003 cm21 are
seen due to the coordinated pyridines [n(CLN)]. The molecular
structure and atom numbering scheme is depicted in Fig. 1.{ The
manganese ion, which lies on an inversion center, has a slightly
distorted tetragonal-bipyramidal geometry, which possesses high
symmetry with trans coordination of the flavonolate ligands in the
basal plane and the two pyridines in apical positions.
The diffraction studies showed that the enzyme forms homo-
dimers, and each unit is mononuclear, with a type 2 copper center.
With the availability of the sequence and structural parameters for
Aspergillus japonicus quercetinase, homologous enzymes were
sought from other species. A BLAST search conducted against
the sequence of Aspergillus japonicus identified the YxaG protein
from Bacillus subtilis, as the protein with the highest degree of
similarity.6 Recent studies has been described the protein YxaG as
an iron-containing flavonol 2,4-dioxygenase,7 which can function
with a number of different divalent metals such as copper, cobalt
and manganese, although manganese(II) appeared to be the
preferred cofactor for this enzyme.8 Since flavonol 2,4-dioxy-
genases are metal-containing enzymes, metal complexes of copper
have been used in model reactions.9 Autooxidation reactions of
potassium and zinc flavonolates have also resulted in enzyme-like
products, and some efforts have been made to elucidate the
mechanism of the reaction.10 Since there are no manganese- or
iron-containing model systems in the literature, in this paper
The manganese–oxygen bond distances are in the range of
II
2.127–2.184 A, somewhat longer than those in Cu (fla)2 (1.901–
˚
9
1944 A). The O2–C1 distance is shorter while the O3–C9 distance
˚
is longer than those in the uncoordinated flavonol [1.357(3) and
aDepartment of Chemistry, University of Pannonia, 8201, Veszpre´m,
Hungary. E-mail: speier@almos.vein.hu; Fax: +36 88 624 469;
Tel: +36 88624 657
bSpectropoˆle, Universite´ Paul Ce´zanne Aix-Marseille III F.S.T. Saint-
Jeroˆme, Service 432 Avenue Escadrille Normandie-Niemen, 13397,
Marseille Cedex 20, France
˚
Fig. 1 The molecular structure of 1 with selected bond distances (A) and
angles (u): Mn1–O2 2.1274(16), Mn1–O3 2.1839(18), Mn1–N1 2.348 (2),
O1–C15 1.351(3), O1–C2 1.374(3), O2–C1 1.302(3), O3–C9 1.257(3),
C1–C9 1.460(3), C1–C2 1.385(3), C10–C15 1.393(3), C9–C10 1.437(4);
O2–Mn1–O3 76.81(6), O2–Mn1–N1 90.99(7), N1–Mn1–N1* 180.0.
*Symmetry code: (1 2 x, 1 2 y, 1 2 z). Ellipsoids are shown at the
50% probability level.
cBiosciences, Universite´ Paul Ce´zanne Aix-Marseille III F.S.T. Saint-
Jeroˆme, Service 432 Avenue Escadrille Normandie-Niemen, 13397,
Marseille Cedex 20, France
{ Electronic supplementary information (ESI) available: Kinetic measure-
ments data and mass spectra for compounds 1 and 2. See DOI: 10.1039/
b711864c
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Chem. Commun., 2007, 5235–5237 | 5235

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1.232(3) A].13 Due to coordination to the manganese ion there are
˚
Table 1 Kinetic data for the oxygenation of metal flavonolates
DH#/ DS#/
also changes in the bond lengths of the pyranone ring. The O1–C2
˚
˚
ka/M21 s21 kJ mol21 J mol21 K21
[1.374(3) A] and C10–C15 [1.393(3) A] bond lengths become
longer, and the C1–C9 bond length [1.460(3) ] is somewhat shorter,
which may be assigned to delocalization of the p-system over the
whole molecule.
2
0.08
0.50
49
40
53
—
2137
2144
2138
—
This work
This work
1
CuII(fla)2
0.0087
9
—
CuII(fla)2(py)n 0.04
Tris(ethoxo)iron(III)
reacted
with
49-methoxyflavonol
a
(49MeOflaH) in acetonitrile at room temperature to give pure,
homoleptic tris(49-methoxyflavonolato)iron(III) (2) in 85% yield. It
shows absorption in the visible region at 411 nm, and n(CO)
absorption band at 1547 cm21 in the IR spectrum assignable to the
flavonolate ligand, A shoulder at 680 and a maximum at 530 nm
characteristic of an octahedral arrangement around the ferric ion,
In DMF at 100 uC.
product permits identification of the place of the dioxygen
incorporation. As a result of oxygenations, carried out under an
atmosphere containing y60% 18,18O2, the 18O-benzoylsalicylic
acid derivative gave a molecular ion at m/z 260 (256 + 4), showing
that both 18O atoms of 18,18O2 are incorporated into the carboxylic
acid from the molecular oxygen, and the gas phases showed only
the presence of unlabeled CO [eqn. (2)]. The relative abundances of
m/z 260 to that at m/z 256 parallel the 18,18O2 enrichments used in
the experiments.
6
4
6
4
can be assigned to the A1g A T1g and A1g A T2g transition,
respectively. The crystal structure of 2, shown in Fig. 2{ together
with selected data, shows a distorted octahedral geometry around
the iron(III) center, with all coordination sites being occupied by
the bidentate 49-methoxyflavonolate ligands. The iron–oxygen
˚
bond distances are in the range of 1.955–2.109 A, somewhat longer
than those in CuII(fla)2, but somewhat shorter than those in
MnII(fla)2(py)2. Complex 2 crystallizes as two independent
molecules with each Fe cation lying on a three-fold axis.
After the characterization of the prepared complexes, their
flavonol 2,4-dioxygenase activity was examined. The manganese
and iron flavonolate complexes 1 and 2, in DMF solutions are
stable under anaerobic conditions and oxidized upon addition of
dioxygen at 95 uC. The CO content was determined in both cases
by GC-MS (80–90%). The formation of O-benzoylsalicylic acid
from flavonol requires dioxygen but no apparent dioxygen uptake
was observed since the absorption of dioxygen and the liberation
of carbon monoxide compensate each other. The GLC-MS
analysis of the residue of the hydrolyzed complexes, after treatment
with ethereal diazomethane, showed the presence of the
O-benzoylsalicylic acid methylester. Provided that a mixture of
18,18O2 and 16,16O2 is used in the oxygenation, the labelling of the
ð2Þ
Reactions of 1 and 2 with dioxygen were performed in DMF
solutions at 85–120 uC, and the concentration change of 1 and 2
was followed by electronic spectroscopy measuring the absorbance
of the reaction mixture at 432 and 411 nm. Kinetic studies on the
oxygenation of the manganese and iron flavonolate complexes
established second-order overall rate expressions –d[1 or 2]/dt = [1
or 2][O2], and both reactions were entropy driven (Table 1)
indicating that the rate-determining step is bimolecular.
As a conclusion it can be said that in the enzyme-like oxygena-
tion of the coordinated flavonolate ligand of manganese(II) and
iron(III) (1, 2), the formation of endoperoxide (3) in bimolecular
reactions can be assumed, and the unique decomposition of this
species accompanied by loss of carbon monoxide results in the
corresponding O-benzoylsalicylate complexes (4) as a good mimic
of the enzyme action. On the basis of the k values (compared to
our earlier copper-containing systems), it can be said that the
reactivity order is Fe . Mn . Cu. Work is still in progress on
model studies to disclose more details of this cleavage reaction.
We thank the Hungarian Research Fund (K-67871), Ce´lker and
Budaconsum Ltd for financial support.
Notes and references
{ Intensity data were measured on a Bruker-Nonius Kappa CCD single-
crystal diffractometer, using graphite-monochromated Mo-Ka radiation
˚
(l = 0.71073 A) and w scan technique at 293 K. The structures were solved
by direct and difmap methods (SIR92),14 and refined on F2 by using full-
matrix least-squares methods.15
Crystal data: Compound 1: C40H28MnN2O6, Mw = 687.58, triclinic,
¯
˚
space group P1, a = 8.310(6), b = 10.095(6), c = 11.167(5) A, a =
3
˚
113.361(4), b = 97.542(4), c = 106.255(4)u, V = 793.82(8) A , Z = 1, Dc =
Fig. 2 The molecular structure of one of the two independent co-
˚
1.438 g cm23, m(Mo-Ka) = 4.70 cm21, 2882 reflections measured, 223
crystallized molecules of 2 with selected bond distances (A) and angles (u):
parameters refined on F2 using 2667 unique reflections to final indices
Fe1–O1 2.109(8), Fe1–O2 1.955(7), O1–C1 1.306(12), O2–C9 1.299(13),
O3–C7 1.302(13), O3–C8 1.373(13), C1–C9 1.367(14), C1–C2 1.490(15),
C2–C7 1.454(16), C8–C9 1.404(15); O2–Fe1–O1 80.0(3), O1–C1–C9
120.1(9). Ellipsoids are shown at the 50% probability level.
R [F2 . 2s(F2)] = 0.0444, wR = 0.1354, w = 1/[s2(Fo ) + (0.0912P)2
+
2
0.3239P], P = (Fo + 2Fc2)/3.
Compound 2: C48H33FeO12, Mw = 857.59, trigonal, space group P31c,
2
3
˚
˚
a = 15.488(5), b = 15.488(5), c = 19.997(5) A, c = 120(4)u, V = 4154.2(2) A ,
5236 | Chem. Commun., 2007, 5235–5237
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Z = 4, Dc = 1.371 g cm23, m(Mo-Ka) = 4.29 cm21, 2081 reflections
measured, 367 parameters refined on F2 using 1820 unique reflections to
final indices R [F2 . 2s(F2)] = 0.0725, wR = 0.1901, w = 1/[s2(Fo2) +
8 M. R. Schaab, B. M. Barney and W. A. Francisco, Biochemistry, 2006,
45, 1009.
´
9 E. Balogh-Hergovich, J. Kaizer, G. Speier, G. Argay and L. Pa´rka´nyi,
´
J. Chem. Soc., Dalton Trans., 1999, 3847; E. Balogh-Hergovich,
J. Kaizer, G. Speier, V. Fu¨lo¨p and L. Pa´rka´nyi, Inorg. Chem., 1999,
´
38, 3787; E. Balogh-Hergovich, J. Kaizer, G. Speier, G. Huttner and
´
A. Jacobi, Inorg. Chem., 2000, 39, 4224; E. Balogh-Hergovich, J. Kaizer,
J. Pap, G. Speier, G. Huttner and L. Zsolnai, Eur. J. Inorg. Chem., 2002,
´
2287; J. Kaizer, E. Balogh-Hergovich, M. Czaun, T. Csay and G. Speier,
Coord. Chem. Rev., 2006, 250, 2222.
10 L. Barha´cs, J. Kaizer and G. Speier, J. Org. Chem., 2000, 65, 3449;
L. Barha´cs, J. Kaizer and G. Speier, J. Mol. Catal. A: Chem., 2001, 172,
117.
11 L. Jurd and T. X. Geissman, J. Org. Chem., 1956, 21, 1395.
12 L. M. Bellamy, Ultrarot Spectrum und Chemische Konstitution,
Dr. Dietrich Steinkopf Verlag, Darmstadt, 1966, p. 112.
13 M. C. Etter, Z. Urbanczyk-Lipkowska, S. Baer and P. F. Barbara,
J. Mol. Struct., 1986, 144, 155.
14 A. Altamore, G. Cascarano, C. Giacovazzo, A. Guagliardi, M. C. Burla,
G. Polidori and M. Camalli, J. Appl. Crystallogr., 1994, 27, 435.
15 G. M. Sheldrick, SHELXL-97: Program for the Refinement of Crystal
Structures, University of Go¨ttingen, Germany, 1997.
(0.1136P)2 + 9.3277P], P = (Fo + 2Fc2)/3.
2
CCDC 651207 (1) and 651208 (2). For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b711864c
1 E. Wollenweber, in Flavonoids: Advances in Research, ed. J. B. Harborne
and T. J. Mabry, Chapman & Hall, London, New York, 1982.
2 E. Wollenweber, Prog. Clin. Biol. Res., 1988, 45, 2850.
3 T. Oka and F. J. Simpson, Biochem. Biophys. Res. Commun., 1971, 43, 1.
4 H. K. Hund, J. Breuer, F. Lingens, J. Huttermann, R. Kappl and
S. Fetzner, Eur. J. Biochem., 1999, 263, 871.
5 F. Fusetti, K. H. Schroter, R. A. Steiner, P. I. van Noort, T. Pijning,
H. J. Rozeboom, K. H. Kalk, M. R. Egmond and B. W. Dijkstra,
Structure, 2002, 10, 259.
6 L. Bowater, S. A. Fairhurst, V. J. Just and S. Bornemann, FEBS Lett.,
2004, 557, 45; B. M. Barney, M. R. Schaab, R. LoBrutto and
W. Francisco, Protein Expression Purif., 2004, 35, 131.
7 B. Gopal, L. L. Madan, S. F. Betz and A. A. Kossiakoff, Biochemistry,
2005, 44, 193.
This journal is ß The Royal Society of Chemistry 2007
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