Bio-inspired flavonol and quinolone dioxygenation by a non-heme iron catalyst modeling the action of flavonol and 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases

Article abstract of DOI:10.1016/j.jinorgbio.2011.11.013

The mononuclear complex, Fe^III(O-bs)(salen) (salenH₂ = 1,6-bis(2-hydroxyphenyl)-2,5-diaza-hexa-1,5-diene; O-bsH = O-benzoylsalicylic acid) was synthesized as synthetic enzyme-depside complex, and characterized by spectroscopic methods and X-ray crystal analysis. The dioxygenation of flavonol (flaH) and 3-hydroxy-4-quinolone (quinH₂) derivatives in the presence of catalytic amounts of Fe^III(O-bs)(salen) results in the oxidative cleavage of the heterocyclic ring to give the corresponding O-benzoylsalicylic and anthranilic acid derivatives with concomitant release of carbon monoxide. These reactions can be regarded as biomimetic functional models with relevance to the iron-containing flavonol and the cofactor-independent 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases.

Full text of DOI:10.1016/j.jinorgbio.2011.11.013

Journal of Inorganic Biochemistry 108 (2012) 15–21
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Bio-inspired flavonol and quinolone dioxygenation by a non-heme iron catalyst
modeling the action of flavonol and 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases
József S. Pap a, Andrea Matuz a, Gábor Baráth a, Balázs Kripli a, Michel Giorgi b,
Gábor Speier a, József Kaizer a,
⁎
a
Department of Chemistry, University of Pannonia, 8201 Veszprém, Hungary
Aix-Marseille Université, FR1739, Spectropole, Campus St. Jérôme, Avenue Escadrille Normandie-Niemen, 13397 Marseille cedex 20, France
b
a r t i c l e i n f o
a b s t r a c t
The mononuclear complex, FeIII(O-bs)(salen) (salenH2=1,6-bis(2-hydroxyphenyl)-2,5-diaza-hexa-1,5-diene;
O-bsH=O-benzoylsalicylic acid) was synthesized as synthetic enzyme-depside complex, and characterized by spec-
troscopic methods and X-ray crystal analysis. The dioxygenation of flavonol (flaH) and 3-hydroxy-4-quinolone
(quinH2) derivatives in the presence of catalytic amounts of FeIII(O-bs)(salen) results in the oxidative cleavage of
the heterocyclic ring to give the corresponding O-benzoylsalicylic and anthranilic acid derivatives with concomitant
release of carbon monoxide. These reactions can be regarded as biomimetic functional models with relevance to the
iron-containing flavonol and the cofactor-independent 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases.
© 2011 Elsevier Inc. All rights reserved.
Article history:
Received 30 August 2011
Received in revised form 27 October 2011
Accepted 15 November 2011
Available online 29 November 2011
Keywords:
Iron complexes
Flavonols
Quinolones
Dioxygenation
Kinetics and mechanism
1. Introduction
the signaling molecules in the quorum sensing sytem of the opportunis-
tic pathogen Pseudomonas aeruginosa, and therefore was named
Ring cleavage is the crucial step in the microbial degradation of aro-
matic and heterocyclic compounds. Flavonoids (flaH, 1,2) are polyphe-
nolic pigments found in higher plants and some fungi (Scheme 1) [1,2].
Current interest in the compounds of flavonoid group is explained by
their varied biological activity, with antioxidant, antiradical, antimicro-
bial, hepatoprotective, anti-inflammatory and other interesting proper-
ties [3–7]. In a clinical study, the risk of coronary heart disease was
shown to correlate inversely with the extent of flavonoid intake [8].
2-Methylquinoline utilization by the soil bacterium Arthrobacter nitro-
guajacolicus Rü61a involves two initial hydroxylation steps to form
2-methyl-3-hydroxy-4(1H)-quinolone (2Me-quinH2, 3) [9]. Pseudomo-
nas putida 33/1 utilizes 3-hydroxy-4(1H)-quinolone (quinH2, 4) as sole
source of carbon, nitrogen, and energy [10]. These substrates with the
2-phenyl derivatives (PhquinH2, 5 and 1Me-PhquinH2, 6) structurally
are isoelectronic with flavonols. 2-Alkyl-4(1H)-quinolones are known
as secondary metabolites of fluorescent pseudomonads, exhibiting anti-
microbial activity primarily towards Gram-positive bacteria [11].
2-Heptyl-3-hydroxy-4(1H)-quinolone was later found to act as one of
Pseudomonas quinolone signal (PQS) [12]. PQS is involved in the regula-
tion of the biosynthesis of many virulence factors, and it affects biofilm
development and cellular fitness. It also acts as an iron trap, sequester-
ing iron from the environment [13,14].
Flavonols are readily degraded by microorganisms. The copper- and
iron-containing flavonol 2,4-dioxygenases (quercetinases) catalyze the
oxidative degradation of flavonol(s) to depside(s) (phenolic carboxylic
acid ester(s)) and carbon monoxide by fungi like Aspergillus flavus [15],
Aspergillus niger [16], Aspergillus japonicus [17] and the protein YxaG
from Bacillus subtilis [18–20] (Eq. (1)).
ð1Þ
Ring opening reaction of 2-methyl-3-hydroxy-4(1H)-quinolone (3)
and 3-hydroxy-4(1H)-quinolone (4) to N-acetylanthranilic (8) and N-
formylanthranilic acid (9), including CO release, is catalyzed by 1H-3-
⁎
Corresponding author. Fax: +36 88624469.
E-mail address: kaizer@almos.vein.hu (J. Kaizer).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.11.013

16
J.S. Pap et al. / Journal of Inorganic Biochemistry 108 (2012) 15–21
Scheme 1. Structure and substitution pattern of the applied model substrates.
hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) and 1H-3-hydroxy-4-
oxoquinaldine 2,4-dioxygenase (HDO) (Eq. (2)) [11,21–25].
and carboxylate-enhanced dioxygenation was investigated in detail. It
was shown 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) flavo-
nolate complexes [32]. Evidence for direct electron transfer from the
flavonolate ligand to dioxygen to form superoxide radical anion was
proved by the superoxide scavenger nitroblue tetrazolium (NBT) [32].
The base-catalyzed dioxygenation of 1H-3-hydroxy-4-quinolone de-
rivatives leads to the corresponding cleavage products derived from ei-
ther an endoperoxide or a 1,2-dioxetane intermediate. A persistent 1H-
2-phenyl-3-oxy-4-quinolone radical could also be detected by EPR [33].
In this study we present the synthesis and structural characteriza-
tion of the first iron-containing synthetic enzyme-depside complex,
FeIII(O-bs)(salen) (10), that highly selectively catalyzes the dioxygena-
tion of flavonol and quinolone derivatives, mimicking the action of
2,4-dioxygenases.
ð2Þ
Model studies of FDOs have demonstrated that autoxidations of
potassium, zinc, iron, manganese, and copper flavonolates, mainly at
elevated temperatures, have resulted in enzyme-like products
[26–31]. Mononuclear iron(III) flavonolate complex FeIII(fla)(salen)
was synthesized as synthetic enzyme-substrate complex, and its direct
2. Experimental
Table 1
Summary of the crystallographic data and structure parameters for FeIII(O-bs)(salen).
2.1. Materials
Formula weight
Crystal system
Crystal description
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α (°)
β (°)
1198.85
Monoclinic
Prism
All manipulations were performed under a pure argon atmosphere
using standard Schlenk-type inert-gas techniques unless otherwise stat-
ed. Solvents used for the reactions were purified by literature methods
and stored under argon. Flavonol [34], 4′-methyl- [35], 4′-methoxy-
[34], 4′-chloro- [35], 4′-cianoflavonol [35], 1-methyl-2-phenyl-3-
hydroxy-4-quinolone (6) [36], 2-phenyl-3-hydroxy-4(1H)- quinolone
[36], 4′-bromo-, 4′-methoxy-, 4′-nitro-2-phenyl-3-hydroxy-4(1H)- quin-
olone [36], 2-methyl-3-hydroxy-4(1H)-quinolone [36], and O-benzoylsa-
licylic acid were prepared according to published procedures [37].
Diazomethane was freshly prepared according to the literature in ether
and immediately used for the methylation reactions [38]. The complex
FeIII(salen)Cl was synthesized as it was published earlier [39]. All other
P2/n
22.5007(3)
10.1645(1)
25.5072(4)
90.00
107.537(1)
90.00
5562.57(13)
4
1.432
0.4×0.25×0.25
0≤h≤30
0≤k≤13
−34≤l≤32
203(2)
MoKα(λ=0.71073)
0.592
2496
γ (°)
Volume (Å3)
Z
Calculated density (g cm−1
Crystal size (mm3)
Index ranges
)
Table 2
Selected bond lengths (Å) and bond angles (deg) for FeIII(O-bs)(salen).
Temperature (K)
Radiation
N(1)–Fe(1)
O(1)–Fe(1)
O(3)–Fe(1)
O(4)–C(17)
O(2)–C(16)
C(23)–O(5)
C(24)–O(6)
O(1)–Fe(1)–N(2)
O(4)–Fe(1)–O(3)
Fe(1)–O(4)–C(17)
2.131(3)
1.897(2)
2.156(2)
1.266(4)
1.317(4)
1.401(4)
1.203(4)
161.33(12)
61.06(9)
91.29(19)
N(2)–Fe(1)
O(2)–Fe(1)
O(4)–Fe(1)
O(3)–C(17)
O(1)–C(1)
O(5)–C(24)
C(24)–C(25)
O(4)–Fe(1)–N(1)
O(2)–Fe(1)–O(3)
Fe(1)–N(2)–C(9)
2.086(3)
1.912(2)
2.106(2)
1.256(4)
1.324(4)
1.363(4)
1.481(6)
142.59(10)
152.81(10)
113.1(2)
Absorption coefficient (mm−1
F (0 0 0)
)
Reflections collected
Observed reflections
[I>2σ(I)]
Goodness-of-fit
Final R indices
13979
8858
1.028
R1 =0.0711a, wR2 =0.1463b
R1 =0.1258, wR2 =0.1766
R indices (all data)
a
R1 =Σ||Fo|−|Fc||/Σ|Fo|.bwR2 =[Σw(Fo2 −Fc2)2/Σw(Fo2)2]1/2
.

J.S. Pap et al. / Journal of Inorganic Biochemistry 108 (2012) 15–21
17
Fig. 1. Molecular structure of 10. Ellipsoids are plotted at the 50% probability level.
chemicals were commercial products and were used without further
purification.
and Ag/AgCl with 3 M KCl (reference). The potentials were referenced
vs. the ferrocene/ferrocenium (Fc/Fc+) redox couple. CVs were mea-
sured in argon saturated DMF using 0.1 M tetrabuthylammonium per-
chlorate (TBAP) salt as electrolyte. The crystal evaluations and
intensity data collections for FeIII(O-bs)(salen) (10), were performed
on a Bruker–Nonius Kappa CCD single-crystal diffractometer using Mo
Kα radiation (λ=0.71073 Å) at 203(2) K. Crystallographic data and
details of the structure determination are given in Table 1, whereas
elected bond lengths and angles are listed in Table 2. SHELX-97 [40]
was used for structure solution and full matrix least squares refinement
on F2. CIF files are available in the CCDC database (CCDC 824209).
2.2. Analytical and physical measurements
Infrared spectra were recorded on an Avatar 330 FT-IR Thermo
Nicolet instrument using samples in KBr pellets. UV–vis spectra were
recorded on an Agilent 8453 diode–array spectrophotometer using
quartz cells (data are summarized in chapter 2.3). GC analyses were
performed on a Hewlett Packard 5830A gas chromatograph equipped
with a flame ionization detector and a CP SIL 8CB column. Microanaly-
ses were done by the Microanalytical Service of the University of Panno-
2.3. Synthesis of FeIII(O-bs)(salen) (10)
nia. Cyclic voltammograms (CV) were taken on
potentiostat with VoltaMaster 4 software for data process. The elec-
trodes were as follows: glassy carbon (working), Pt wire (auxiliary),
a VoltaLab 10
A
solution of 0.57 g (1.6 mmol) of FeIII(salen)Cl and 0.38 g
(1.6 mmol) of O-bsH in 10 mL MeOH was stirred at room temperature
Fig. 2. Dependence of the initial rates on the initial concentration of 2a (Table S1,
exp. 4–7) and 5a (Table S2, exp. 4–7).
Fig. 3. Dependence of the initial rates on the concentration of FeIII(O-bs)(salen) for 2a
(Table S1, exp. 4, 8–10) and 5a (Table S2, exp. 4, 8–10).

18
J.S. Pap et al. / Journal of Inorganic Biochemistry 108 (2012) 15–21
Fig. 5. CVs of anions from 2a (top), 5a (bottom) and 4′H-Phquinox (dashed line), 1 mM in
DMF containing ~1 mM ferrocene as internal standard, at 100 mV/s scan rate. Voltammo-
grams are plotted against the Ag/AgCl reference electrode.
Fig. 4. Dependence of the initial rates on the dioxygen concentration for 2a (Table S1,
exp. 4, 11–12) and 5a (Table S2, exp. 4, 11–12).
under argon. After 30 min 220 μL (1.6 mmol) of triethylamine was
added drop-wise. The mixture was stirred for 1 h, the solvent was
evaporated in a vacuum, and the residue was washed with cold metha-
nol and diethylether and vacuum-dried. Yield: 0.39 g (69%). Anal. Calcd
for C30H23N2O6Fe: C, 63.96; H, 4.12; N, 4.97. Found: C, 63.78; H, 4.17; N,
4.85. FT-IR bands (KBr pellet, cm−1): 3188 m, 3010 m, 2925 m, 1731 s,
1629 s, 1597 s, 1512 s, 1445 m, 1385 m, 1327 w, 1294 m, 1200 m,
1034 s, 905 m, 923 759 s, 618 m, 425 w. Crystals suitable for X-ray
structural determination were obtained from dichloromethane.
concentration of flavonol or 2-phenyl-3-hydroxy-4(1H)-quinolone
was determined by measuring the absorbance of the reaction mixture
at 343 or 372 nm [log ε 4.24 or log ε 3.96]. Experimental conditions
are summarized in Tables S1 and S2. The dioxygen concentration in
DMF was calculated from literature data taking into account the partial
pressure of DMF [41] and assuming the validity of Dalton's law [42].
3. Results and discussion
3.1. Synthesis and characterization of the FeIII(O-bs)(salen) (10)
2.4. Catalytic oxygenation of flavonol, or 2-phenyl-3-hydroxy-4(1H)-quino-
lone catalyzed by FeIII(O-bs)(salen)
As a synthetic enzyme-depside model (O-benzoylsalicylato)iron(III)
complex was prepared by the reaction of equimolar amounts of FeIII(sa-
len)Cl and O-benzoylic acid in the presence of triethylamine in metha-
nol. Complex 10 isolated as a brown solid is stable in air and analyzed
satisfactorily for C, H, N. The infrared (IR) spectrum of the complex
shows ν(CO) and ν(CO2) bands corresponding to the coordinated O-
benzoylsalicylate at 1731, and 1544, 1385 cm−1, respectively. The dif-
ference between the asymmetric and symmetric stretching
frequencies of this carboxylato group [Δν=νas(CO2)−νs(CO2)] is
159 cm−1, rendering these to a bidentate bonding mode [43,44].
Crystals of complex 10 of X-ray quality were obtained from dichloro-
methane. Crystallographic characterization has shown that the geome-
try around the six-coordinated iron(III) center is distorted octahedral,
and that the O-benzoylsalicylate anion is coordinated as a bidentate
ligand with a strongly twisted conformation of the salen ligand. A
view of the coordination geometry is given in Fig. 1. The Fe(1)\O(3)
bond is only 0.040 Å longer than the Fe(1)\O(4) bond.
100 mg (0.42 mmol) of flavonol (2-phenyl-3-hydroxy-4(1H)-quino-
lone) and 9 mg (0.017 mmol) of FeIII(O-bs)(salen) were dissolved and
stirred at 100 °C in 5 mL DMF for 15 h under dioxygen atmosphere.
Diazomethane solution (2 mL) was added to 0.2 mL of the reaction
mixture at r.t. and the conversion of flavonol into O-benzoylsalicylic
acid
(75%),
or
2-phenyl-3-hydroxy-4(1H)-quinolone
into
N-benzoylanthranilic acid (64%) was determined by GC as methylated
derivatives in the presence of benzoin as internal standard. The formation
of CO was proved by GC analysis of the gas phase (~70 and ~60%,
respectively).
2.5. Kinetic measurements
The FeIII(O-bs)(salen)-catalyzed oxygenation of flavonol or
2-phenyl-3-hydroxy-4(1H)-quinolone was followed by UV–vis spec-
troscopy. In a typical experiment flavonol or 2-phenyl-3-hydroxy-
4(1H)-quinolone and FeIII(O-bs)(salen) was dissolved under argon at-
mosphere in a thermostated reaction vessel with an inlet for taking
samples with a syringe, and connected to mercury manometer to regu-
late constant dioxygen pressure. The solution was then heated to appro-
priate temperature and the argon was replaced with dioxygen. The
3.2. Catalytic dioxygenation of flavonol and 3-hydroxy-quinolone derivatives
catalyzed by FeIII(O-bs)(salen) (10)
The dioxygenation reaction of flaH and PhquinH2 with dioxygen in
the presence of a catalytic amount of FeIII(O-bs)(salen) was performed
in DMF solution and examined at 100 °C with a ratio of 1:25 between
initial concentration of the iron complex and the corresponding
substrate (2a or 5a). We have found that the dioxygenation of 2a and
5a results in oxidative cleavage of the heterocyclic ring to give
O-benzoylsalicylic acid (75%) and N-benzoylanthranilic acid (64%)
with concomitant release of carbon monoxide. The dioxygenation reac-
tion in both cases was selective, no other products were obtained.
Detailed kinetic measurements were carried out for the 2a and 5a
dioxygenation in DMF solution and examined in the temperature range
from 353 to 383 K with a ratio between initial concentrations of FeIII(O-
bs)(salen) and the substrate in the range 1:20–40. Experiments were
Table 3
Kinetic data for the FeIII(O-bs)(salen)-catalyzed dioxygenation of flavonol and 3-hidroxy-
quinolone derivatives in DMF.
104ka M−2 s−1
EA≠ kJ mol−1
ΔH≠ kJ mol−1
ΔS≠ J mol−1 K-1
σ
2a
3
5a
6
4.82 0.15
2.65 0.09
1.36 0.04
1.63 0.06
53
–
2
50
–
2
−27
–
1
0.37
–
60
58
4
4
57
55
4
4
−20
−20
2
1
0.75
–
a
In DMF at 110 °C.

J.S. Pap et al. / Journal of Inorganic Biochemistry 108 (2012) 15–21
19
Table 4
Electrochemical data for 4′R-flaH and 4′R-PhquinH2 derivatives and 4′H-Phquinox in DMF vs. the ferrocene internal standard ([E]=mV).
4′R-flaH
2a
2a
2f
2b
2e
2d
2c
a
E°′pa1
E°′pc1
−326
–
−225
–
−593
–
−300
–
−378
–
−446
–
a
4′R-PhquinH2
5a
5b
5c
5d
a
E°′pa1
−527
−693
−292
–
−559
−716
−321
−510
−648
–
−449
−605
–
a
E°′pc1
–
E°′pa2
−291b
a
a
5 mV.
4′H-Phquinox
b
.
also carried out at different dioxygen concentrations. Experimental condi-
tions are summarized in Tables S1 and S2. Catalytic reactions were fol-
lowed by UV–vis spectroscopy by determining the substrate
concentration as a function of time under constant dioxygen pressure.
The variation of the absorbance was determined for 2a and 5a at 343
and 372 nm, respectively. The measured absorption spectra for the cata-
lytic dioxygenation of flavonol at 333 K are shown in Fig. S1, with time
range being 2.5 h. Direct electron transfer from the flavonolate ligand to
dioxygen to form superoxide radical anion was proved by the reduction
of nitroblue tetrazolium (NBT) as superoxide scavenger to blue formazan.
By the addition of NBT to the reaction mixture, the yellow color turns to
intense green and new intense band at 520 nm arises. This absorption
has been assigned to formazan signaling the presence of superoxide
anion [45].
The observed initial rates for 2a and 5a are compiled in Tables S1 and
S2. When dioxygenase activity was studied, flavonol was the more reac-
tive substrate. The estimated initial rates (under the same conditions)
were V0=1.33×10−8 Ms−1, and V0=0.39×10−8 Ms−1, respectively,
for 2a and 5a. Therefore, the flavonol is three-times more reactive than
the 2-phenyl-3-hydroxy-4(1H)-quinolone, toward dioxygen. In order
to determine the rate-dependence on the various reactants catalytic
reactions were performed at different substrate, catalyst and dioxygen
concentrations (Tables S1 and S2). Initial reaction rates plotted versus
the initial concentration of substrate (Fig. 2), catalyst (Fig. 3) and dioxy-
gen (Fig. 4) could be fitted with straight lines for both cases establishing
a rate law of –d[2a or 5a]/dt=kobs[2a or 5a][FeIII(O-bs)(salen)][O2],
Activation parameters calculated for 2a, 5a and 6 from temperature-
dependence of the reaction rates are EA≠=53 2 kJ mol−1
ΔH≠=50 2 kJ mol−1 EA≠=60
4 kJ mol−1 ΔH≠=57 4 kJ mol−1 ΔS≠=−20 2 J mol−1 K−1
and EA≠=58 4 kJ mol−1 ΔH≠=55 4 kJ mol−1
,
,
ΔS≠=−27 1 J mol−1 K−1
;
,
,
;
,
,
ΔS≠=−20
1 J mol−1 K−1, respectively (Table 3). These values are very similar to
each other supporting similar, associative-type mechanism (Fig. S2)
for the investigated substrates.
3.3. Electrochemical properties of flavonol and 2-phenyl-3-hydroxy-4(1H)-
quinolone derivatives
Cyclic voltammetric measurements on the flavonol derivatives in
the presence of base have been performed earlier and the irreversible
oxidations were associated with the low stability of the generated flavo-
noxyl radical (4′R-fla•) [31]. A correlation between the rate of oxygena-
tion for the potassium flavonolates and their measured Epa values was
also established. However, no further details were given on the possible
chemical changes accompanying this irreversible electrochemical tran-
sition. Now, we have measured the CVs of the various derivatives
shown Scheme 1 in DMF under argon, in the presence of an equivalent
amount of potassium tert-butoxide. In the voltammogram of 4′H-fla−
(Fig. 5, top) an irreversible, one electron oxidation is present
at −326 mV vs. ferrocene. This transition has been assigned as the
one electron oxidation of 4′H-fla− to 4′H-fla• in the previous study.
Very similar CVs were taken with each derivative (potential values are
listed in Table 4). The flavonoxyl radical once generated, readily
undergoes chemical transformation. We presume that the electrochem-
ical generation of the radical leads to the rapid formation of a dimeric
product (Scheme 2) that has been described earlier [46]. This can be
accounted for the irreversibility of the electrochemical transition, e.g.
from which
a mean value of the kinetic constants of (4.82
0.15)×104 M−2 s−1 (2a), and (1.36 0.04)×104 M−2 s−1 (5a), at
333 K were obtained, respectively.
Reaction rate on the catalytic dioxygenation of the 1- and
2-substituted 3-hydroxy-4-quinolone derivatives (1Me-PhquinH (6);
2Me-quinH2 (3)) under identical conditions were also determined in
order to elucidate the role of electronic effects in the reaction rates
(Table 3). On the basis of comparison of rate constants at 333 K, these
substrates are more reactive than 5a, suggesting a substrate dependent
reactivity order of 2a>6>3>5a.
the absence of E°′pc1
.
In contrast with those of the 4′R-fla− compounds, the CV of 4′H-
PhquinH− (Fig. 5, bottom) exhibits one reversible transition followed
by an irreversible oxidation. This behavior is observed for each deriva-
tive, although the second oxidation peak is present only as a shoulder
Scheme 2. Formation of the dimeric product upon one-electron oxidation of 4'R-flaH derivatives.

20
J.S. Pap et al. / Journal of Inorganic Biochemistry 108 (2012) 15–21
Scheme 3. Proposed products in the one-electron oxidation steps of 4'R-PhquinH derivatives.
of E°′pa1 when R_Br, or NO2 thus the potential was not determined in
these two cases. The potential data and assignments are listed in
Table 4. The observed current peaks are associated with the E°′pa1 and
E°′pa2 transitions (Scheme 3). The presence of a reverse reduction
peak (E°′pc1) is associated with the higher stability of 4′R-PhquinH•
radicals that do not undergo chemical changes in the time-scale of the
CV experiment. This can be supported by the comparison of E°′pa1
values of the corresponding 4′R-fla− and 4′R-PhquinH− (R_H, or
OCH3) derivatives: the lower potential indicates higher stability for
the electrochemically generated radical species in the latter case. The
origin of the irreversible E°′pa2 transition was probed by taking the
voltammogram of 4′R-Phquinox (Fig. 5. dashed line) [47] in the oxida-
tion direction from −300 to 500 mV vs. the Ag/AgCl electrode. The
sole peak that is present coincides with E°′pa2 therefore we tentatively
assign this peak as the one-electron process shown in Scheme 3.
The substitution at the 4′R position had a systematic effect on the
E°′pa1 potentials that show correlation with Hammett's σ (Fig. 6). This
effect can be expected to influence the reaction rates for the oxygena-
tion of these compounds in the presence of the iron complex (Fig. 7).
The influence of the 4′R groups on the reaction rate of the catalytic diox-
ygenation of 4′R-flaH (2) and 4′R-PhquinH2 (5) showed a linear
Hammett plot with a reaction constant of ρ=−0.37 and −0.75, re-
spectively, indicating that the electron-releasing groups result in re-
markable increase in the reaction rates (Fig. S3). These results suggest
that the dioxygen species at the transition state should be electrophilic.
depside model, highly selectively and efficiently catalyzes the dioxygen-
olytic cleavage of the heterocycle ring of flavonols and 3-hydroxy-4(1H)-
quinolone derivatives to give the corresponding O-benzoylsalicylic and
anthranilic acid derivatives with concomitant release of carbon monox-
ide and thus serve as one of the first biomimetic functional models
with relevance to the iron-containing flavonol and the cofactor-
independent 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases. The relative
reactivity order of the substrates are flaH (2a)>1Me-PhquinH (6)
>2Me-quinH2 (3)>PhquinH2 (5a). The values of the activation param-
eters, are very similar to each other, supporting similar, associative-type
mechanism (Fig. S2) for the investigated substrates. A reaction mecha-
nism that fits the chemical, spectroscopic, kinetic and thermodynamic
data is shown in Scheme 4. In summary: FeIII(O-bs)(fla)(salen)-cata-
lyzed dioxygenation of flavonol and 3-hidroxy-quinolone derivatives in
DMF has a single electron transfer (SET) mechanism in which the depro-
tonated substrate with increased basicity reduces the molecular oxygen,
initiating further steps including a fast consecutive formation of the
trioxametallocycle, which reacts by a nucleophilic addition on the C4 car-
bon to give the endoperoxide intermediates, that lead to the enzyme-like
products. It can be also said that the forming carboxylates such as O-
benzoylsalicilate, and N-anthranilate derivatives as coligands dramati-
cally enhance the reaction rate, which can be explained by the formation
of more reactive monodentate iron substrate complexes, which is neces-
sary to the intermolecular electron transfer.
Acknowledgments
4. Conclusions
In this study we have demonstrated that the complex FeIII(O-bs)(sa-
len), which is the first synthetic iron-containing 2,4-FDO enzyme-
The authors are grateful for the financial support of the grant
TAMOP-4.2.1/B-09/1/KONV-2010-0003: Mobility and Environment:
Fig. 6. Correlation between the E°′ potentials and the Hammett's σ for 2 and 5.
Fig. 7. Dependence of rate constant k, on the E°′pa1 for 2 and 5.

J.S. Pap et al. / Journal of Inorganic Biochemistry 108 (2012) 15–21
21
Scheme 4. General mechanism proposal for the catalytic dioxygenation of the various substrates.
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Researches in the fields of motor vehicle industry, energetics and
environment in the Middle- and West-Transdanubian Regions of
Hungary. The project is supported by the European Union and
co-financed by the European Regional Development Fund. Financial
support of the Hungarian National Research Fund (OTKA K67871,
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