One metal-two pathways to the carboxylate-enhanced, iron-containing quercetinase mimics

Article abstract of DOI:10.1039/b903224j

Mononuclear iron(iii) flavonolate was synthesized as synthetic enzyme-substrate complex, and its direct and carboxylate-enhanced dioxygenation as biomimetic functional models with relevance to flavonol 2,4-dioxygenase are briefly described.

Full text of DOI:10.1039/b903224j

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One metal–two pathways to the carboxylate-enhanced, iron-containing
quercetinase mimicsw
a
a
a
b
c
+
Gabor Barath, Jozsef Kaizer,* Gabor Speier,* Laszlo Parkanyi, Erno Kuzmann
´
´
and Attila Vertes
´
´
´
´
´
´
c
´
Received (in Cambridge, UK) 16th February 2009, Accepted 17th April 2009
First published as an Advance Article on the web 12th May 2009
DOI: 10.1039/b903224j
Mononuclear iron(III) flavonolate was synthesized as synthetic
enzyme–substrate complex, and its direct and carboxylate-
enhanced dioxygenation as biomimetic functional models with
relevance to flavonol 2,4-dioxygenase are briefly described.
Aspergillus japonicus identified the YxaG protein from Bacillus
subtilis, as the protein with the highest degree of similarity.7 Recent
studies has been described the YxaG as an iron-containing
flavonol dioxygenase with three histidines, one glutamate and
one water around the metal center, and their arrangement is
similar to that of the Aspergillus japonicus dioxygenase.8 It was
also found that the packing of the modeled quercetin in the Yxag
structure appears to be less tight than in the Aspergillus japonicus.
The synthesis of low molecular weight metal complexes
mimicking dioxygenase activity has been challenging for
bioinorganic chemists and for many years efforts have been
made to obtain catalytically active compounds. Autoxidations
of potassium, zinc, iron, manganese and copper flavonolates,
mainly at elevated temperatures, have resulted in enzyme-like
products, and efforts have also been made to elucidate the
mechanisms of these reactions.9
Flavonol 2,4-dioxygenases (FDO) are enzymes that catalyze the
oxidative cleavage of the O-heterocycle of polyphenolic flavonols
(flaH), which represent a major class of flavonoids.1 These
compounds are important dietary components and have attracted
considerable attention owing to their antioxidizing properties.2 In
the enzymatic process, two C–C bonds are broken, producing
more easily degradable depsides (phenolic carboxylic acid esters)
and concomitantly carbon monoxide is released (eqn (1)).
ð1Þ
The stability of the metal flavonolates above can be explained
by the chelation and formation of a stable five-membered ring in
the flavonolate complexes. It can be assumed that the coordination
mode of the substrate in the enzymatic and model systems could
give rise to differences in the degradation rates. In this paper, we
report the synthesis and characterization of a FeIII(fla)(salen)
[salenH2 = 1,6-bis-(2-hydroxyphenyl)-2,5-diaza-hexa-1,5-diene]
complex as a synthetic model for the iron-containing Yxag
dioxygenase, and its direct and carboxylate-enhanced dioxygena-
tion, respectively. We will show that bulky carboxylates as
coligands dramatically enhance the reaction rate, which can be
explained by two different mechanisms, caused by the formation
of more reactive monodentate iron(III) flavonolate complexes.
Complex FeIII(fla)(salen) was synthesized by the reaction
between FeIII(salen)(Cl) and flavonol in the presence of triethyl-
amine at room temperature in MeOH. The crystal structure of
FeIII(fla)(salen), shown in Fig. 1z together with selected data,
shows a distorted octahedral geometry around the iron(III) center,
and that the flavonolate anion is coordinated as a bidentate ligand
with a strongly twisted conformation of the salen ligand. The
FDO enzymes from various species of Aspergillus, Aspergillus
flavus,3 Aspergillus niger,4 and Aspergillus japonicus5 have been
characterized, and were found to contain a type II copper ion
at the active site. The crystal structure of quercetinase from
Aspergillus japonicus shows that the copper center has two alter-
native conformations, the main form (B70%) being pseudotetra-
hedral (three histidine residues and a water molecule), and the
minor form (B30%) having a mixed trigonal bipyramidal/square
pyramidal geometry (side chain of glutamate additionally coordi-
nates the metal).5 Interestingly, an X-ray structure of Aspergillus
japonicus anaerobically complexed with the natural substrate
quercetin indicated that flavonols bind to the copper ion in a
monodentate fashion through their O3 atom.6 In this geometry,
the carbonyl O4 of the flavonol molecule cannot approach the
metal, so the substrate chelation (bidentate coordination),
generally observed in flavonolato complexes, doesn’t occur.
57Mossbauer spectrum of the complex exhibits a dominant
A
BLAST search conducted against the sequence of
¨
doublet with isomer shift, d = 0.49 mm sꢀ1 and quadrupole
splitting, DEQ = 1.44 mm sꢀ1, indicating a high spin Fe(III)
compound.10 This is consistent with the structure of the
complex where iron is surrounded by ligands resulting a con-
siderable asymmetric charge distribution reflected by the obtained
quadrupole splitting value. FeIII(fla)(salen) shows absorption
in the visible region at 407 nm due to the flavonolate
ligand.11 Strong IR band at 1549 cmꢀ1 assigned to n(CO),
showing a decrease of 53 cmꢀ1 compared to flavonol [n(CO) =
1602 cmꢀ1], arise as a results of the formation of a five-membered
ring.12
a Department of Chemistry, University of Pannonia,
´
8201 Veszprem Hungary. E-mail: speier@almos.vein.hu;
Fax: +36 88 624 469; Tel: +36 88624 657
b Chemical Research Center, Institute of Structural Research,
Hungarian Academy of Sciences, P.O. Box 17, H-1525 Budapest,
Hungary
c Laboratory of Nuclear Chemistry, Chemical Research Center,
Hungarian Academy of Sciences, Eotvos University, Budapest,
Hungary
¨
¨
w Electronic supplementary information (ESI) available: Kinetic
measurements data, mass and Mossbauer spectra for Fe(fla)(salen).
¨
CCDC 719842. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/b903224j
ꢁc
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Fig. 1 The molecular structure of FeIII(fla)(salen) with selected bond
distances (A) and angles (1): Fe2–O1 1.899(4), Fe2–O1a 1.935(4),
Fe2–O3 1.955(4), Fe2–O2 2.139(4), Fe2–N1 2.141(5), Fe2–N1a
2.080(5), O2–C9 1.272(7), O3–C10 1.318(7), C9–C10 1.432(8), C9–C17
1.414(8), C10–C11 1.363(8), C11–O4 1.370(7), C12–O4 1.354(7), N1–C1
1.462(8), N1–C2 1.273(8), N1a–C1a 1.283(8), N1a–C2a 1.283(8),
O1a–Fe2–O2 161.72(16), O3–Fe2–N1 158.85(19), O1–Fe2–N1a
158.29(19). Ellipsoids are shown at the 50% probability level.
Fig. 2 (a) Steric effects on the reaction rate for the dioxygenation of
FeIII(fla)(salen) in the presence of 10 equiv. acetates in DMF at 100 1C
(correlation between the number of phenyl substituents of acetates and
the relative rates) (b) Substituent effects on the rate constants for the
dioxygenation of FeIII(40R0-fla)(salen) in DMF at 100 1C.
various coligands, such as acetate (CH3CO2ꢀ), phenyl
(PhCH2CO2ꢀ), diphenyl (Ph2CHCO2ꢀ) or triphenyl acetate
(Ph3CCO2ꢀ) (Fig. 2). For example, addition of 10 equiv. of the
Complex FeIII(fla)(salen) is inert to dioxygen in its solid form,
and even in solution at ambient conditions. At an elevated
temperature (100–120 1C) the dioxygenation reaction proceeds
reasonably fast in DMF. The CO content was determined by
GC-MS. The GLC-MS analysis of the residue of the hydrolyzed
complex, after treatment with ethereal diazomethane, showed
the presence of the O-benzoylsalicylic acid methylester. Kinetic
experiments were performed in DMF solutions at 90–105 1C.
The dioxygenation reactions, followed by measuring the absor-
bance decrease of the p–p* transition at 407 nm arising from the
coordinated flavonolate ligand, afforded a time profile indicating
an exponential decay of FeIII(fla)(salen). A straight line was
obtained in a plot of the reaction rate versus the initial concen-
tration of FeIII(fla)(salen) and dioxygen to establish a rate law of
ꢀd[FeIII(fla)(salen)]/dt = k[FeIII(fla)(salen)][O2] with kinetic
ꢀ
bulky Ph3CCO2 to FeIII(fla)(salen) accelerated its decay by
two order of magnitude (Vr = 171) at 100 1C, and the reaction
above can take place even at ambient temperatures (20 1C).
The GLC-MS analysis of the hydrolyzed complexes, after
treatment with ethereal diazomethane, showed the presence of
the O-benzoylsalicylic acid methylester. Provided 18,18O2 and
16,16O2 is used in the dioxygenation, the labeling of the product
permits identification of the place of the dioxygen incorporation.
As a results of dioxygenations, carried out under an atmosphere
containing B40% 18,18O2, the 18O-benzoylsalicylic acid derivative
gave a molecular ion at m/z 260 (256 + 4), showing that both 18
O
atoms of 18,18O2 are incorporated into the carboxylic acid from
molecular oxygen and the gas phase showed only the presence of
unlabeled CO. The kinetics of the carboxylate-enhanced dioxy-
genation of FeIII(fla)(salen) measured at 40 1C (Fig. 3) resulted in
a rate equation with first order dependence on FeIII(fla)(salen),
parameters: k = (2.07 ꢂ 0.12) ꢃ 10ꢀ2 Mꢀ1
s =
ꢀ1, DH#
76 kJ molꢀ1, DS# = ꢀ94 J molꢀ1 Kꢀ1 at 373.16 K (Table 1).
The influence of the 40-substituted groups on the reaction rate
showed a linear Hammett plot with a reaction constant of r =
ꢀ0.54, indicating that the electron-releasing groups result in
remarkable increase (ca. 2.5-fold) in the reaction rates (Fig. 2).
Besides the electronic factors, the steric effect was also
investigated on the dioxygenation reaction. We have found
that the rate of dioxygenolysis is dramatically enhanced by
dioxygen and triphenyl acetate (k0 = (5.02 ꢂ 0.35) ꢃ 102 Mꢀ2 sꢀ1
,
DH# = 35 kJ molꢀ1, DS# = ꢀ120 J molꢀ1 Kꢀ1 at 313.16 K). The
main mechanistic difference between the direct and carboxylate-
enhanced dioxygenation of FeIII(fla)(salen) is that in the latter case
there is an electron transfer from FeIꢀII(fla)(salen) to dioxygen
ꢄ
resulting in the formation of free O2 (ca. 25%), which was
proved by the test for free O2 ꢀ with nitroblue tetrazolium (NBT),
ꢄ
Table 1 Kinetic data for the dioxygenation of FeIII(fla)(salen)
Vra
DH#/kJ molꢀ1
DS#/J molꢀ1 Kꢀ1
r
Fe(40R0-fla)(salen)(L)
R0 = H; L = -;
—
—
76
—
—
—
—
—
—
—
35
—
—
ꢀ0.54
1.00
0.61
1.40
2.45
19
30
86
ꢀ94
R0 = Cl; L = -;
—
—
—
—
R0 = OMe; L = -;
R0 = NMe2; L = -;
R0 = H; L = CH3CO2
—
—
—
—
—
—
—
—
ꢀ
;
ꢀ
R0 = H; L = PhCH2CO2ꢀ
;
;
R0 = H; L = Ph2CHCO2
ꢀ
R0 = H; L = Ph3CCO2ꢀ
;
;
171
—
—
—
R0 = H; L = Ph3CCO2
ꢀ120b
ꢀ0.78b
a
b
In DMF at 100 1C. At 40 1C.
ꢁc
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In conclusion, we have shown a significant steric effect on
the oxidative decay of FeIII(fla)(salen) on addition of a bulky
carboxylate such as triphenyl acetate, which accelerates the decay
of FeIII(fla)(salen) by as much as two orders of magnitude. This
probably renders a monodentate coordination of the flavonol to
the iron, which is less stable than the bidentate coordination and
the higher electron density on C2 makes electron transfer from the
flavonolate to O2 easier. On adding bulky carboxylates the reaction
proceeds even at room temperature, which is an unprecedented
model. These observations lend strong credence to postulated
mechanism of copper and iron-containing dioxygenases.
Financial support of the Hungarian National Research
Fund (OTKA K67871 and K75783), COST and Budaconsum
Ltd is gratefully acknowledged.
Fig. 3 (a) (A) Visible spectral change for the decay of FeIII(salen)(fla)
ꢀ
in the presence of 10 equiv. Ph3CCO2 in DMF at 40 1C, (B) in the
presence of NBT. (b) Time-dependent conversion of FeIII(fla)(salen)
under the condition described above, monitored at 407 nm.
Notes and references
z Intensity data were measured on a Rigaku R-Axis Rapid single-
where the reduction of the added dye to the blue diformazan took
place (Fig. 3).
crystal diffractometer, using
radiation (l
a graphite-monochromated Mo-Ka
= 0.71075 A) and f scan technique at 293 K.
SHELX-9713 was used for structure solution, and full matrix least
squares refinement on F2.14 CCDC 719842 (Fe(fla)(salen)).w
Crystal data: Compound Fe(fla)(salen): C31H23FeN2O5, Mw
=
559.37, triclinic, space group P 1, a = 10.505(9), b = 12.060(11),
c = 13.489(12) A, a = 63.650(12), b = 67.833(17), g = 85.29(2)1, V =
1411(2) A3, Z = 2, Dc = 1.317 g cmꢀ3, m(Mo-Ka) = 0.576 cmꢀ1
,
12 569 reflections measured, 2536 parameters refined on F2 using 3583
unique reflections to final indices R[F2 4 2s(F2)] = 0.0773, wR =
0.1571, w = 1/[s2(F02) + (0.0709P)2 + 2.7386P], P = (F02 + 2Fc2)/3.
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¨
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flavonolatoiron complexes (eqn (2)). An analogous reaction path-
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This journal is The Royal Society of Chemistry 2009
3632 | Chem. Commun., 2009, 3630–3632