(O-benzoylsalicylato)copper(II) complexes as synthetic enzyme-product models for flavonol 2,4-dioxygenase

Article abstract of DOI:10.1016/j.inoche.2005.11.011

Mononuclear complexes of the type [Cu^II(BPEA)(O-bs)], [Cu ^II(BnBPA)(O-bs)]ClO₄, and [Cu^II(Bn-6Me ₂BPA)(O-bs)]ClO₄ [O-bsH = O-benzoylsalicylic acid, BPEAH = N,N-bis(2-pyridylmethyl)amino-2-ethanoic acid, BnBPA = N-benzyl-N,N-bis(2- pyridylmethyl)amine, Bn-6Me₂BPA = N-benzyl-N,N-bis(6-methyl-2- pyridylmethyl)amine] were synthesized as synthetic enzyme-depside complexes, and characterized by spectroscopic measurements and X-ray crystal analysis. The oxygenation of flavonol using the above-mentioned complexes as catalyst results in the oxidative cleavage of the heterocyclic ring to give a O-benzoylsalicylic acid and CO as a mimic of quercetinase action.

Full text of DOI:10.1016/j.inoche.2005.11.011

Inorganic Chemistry Communications 9 (2006) 251–254
www.elsevier.com/locate/inoche
(O-Benzoylsalicylato)copper(II) complexes as synthetic
enzyme-product models for flavonol 2,4-dioxygenase
a
b
a,b,
c
c
*
´
´
´
´
, Marius Reglier , Michel Giorgi
Jozsef Kaizer , Szabina Goger , Gabor Speier
a
Research Group for Petrochemistry, Hungarian Academy of Sciences at University of Veszpre´m, 8201 Veszpre´m, Hungary
b
Department of Organic Chemistry, University of Veszpre´m, Wartha V. u. 1., 8201 Veszpre´m, Hungary
Laboratoire de Cristallochimie et Laboratoire de Bioinorganique Structurale, Universite´ Paul Ce´zanne Aix-Marseille III,
ˆ
F.S.T. Saint-Je´rome, Service 432, Avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France
c
Received 8 November 2005; accepted 13 November 2005
Available online 22 December 2005
Abstract
Mononuclear complexes of the type [CuII(BPEA)(O-bs)], [CuII(BnBPA)(O-bs)]ClO4, and [CuII(Bn-6Me2BPA)(O-bs)]ClO4 [O-bsH =
O-benzoylsalicylic acid, BPEAH = N,N-bis(2-pyridylmethyl)amino-2-ethanoic acid, BnBPA = N-benzyl-N,N-bis(2-pyridylmethyl)-
amine, Bn-6Me2BPA = N-benzyl-N,N-bis(6-methyl-2-pyridylmethyl)amine] were synthesized as synthetic enzyme-depside complexes,
and characterized by spectroscopic measurements and X-ray crystal analysis. The oxygenation of flavonol using the above-mentioned
complexes as catalyst results in the oxidative cleavage of the heterocyclic ring to give a O-benzoylsalicylic acid and CO as a mimic of
quercetinase action.
Ó 2005 Elsevier B.V. All rights reserved.
Keywords: Copper; Carboxylates; O-Benzoylsalicylic acid; Flavonol; Dioxygenation
To date, four prokaryotic dioxygenases are known,
which catalyze an oxidative C–C bond cleavage with incor-
poration of two O atoms into, and release of CO from their
substrate [1–4]. Flavonol 2,4-dioxygenase (FDO), which
catalyzes the cleavage of flavonol (1) to phenolic carboxylic
acid ester (2) and carbon monoxide was produced by the
copper-containing fungi like Aspergillus flavus [5], Aspergil-
lus niger [6], Aspergillus japonicus [7] and the iron-contain-
ing protein Yxag from Bacillus subtilis [8] (Eq. (1)). The
crystal structure from A. japonicus has recently been
solved. The native structure shows an active site with a
copper atom coordinated by three histidine residues at
formation). The co-existence of these two conformations of
the native enzyme is also present in the EPR spectrum,
which exhibits a mixed signal [9]. Upon anaerobically bind-
ing the substrate, the structure (major conformation)
changes to a pentacoordinated square pyramidal geometry
by replacement of the water and additional binding of a
glutamate and the substrate. Two histidines, glutamate,
and the O3 atom of the substrate form the equatorial base,
and the third histidine is the apical ligand. In the course of
model studies of FDO numerous copper complexes of fla-
vonol and O-benzoylsalicylic acid have been prepared and
characterized [10–14].
˚
˚
2.09–2.16 A and a water molecule at 2.21 A (major confor-
mation); an additional coordination is present, in which a
˚
glutamate residue is coordinated at 2.10 A, while the water
˚
molecule is found at a larger distance of 2.41 A (minor con-
*
Corresponding author. Tel.: +36 88 422 022/4657; fax: +36 88 427 492.
ð1Þ
E-mail address: speier@almos.vein.hu (G. Speier).
1387-7003/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2005.11.011

252
J. Kaizer et al. / Inorganic Chemistry Communications 9 (2006) 251–254
In this communication, we present the synthesis and
structural characterization of new (O-benzoylsalicy-
lato)copper(II) complexes with a tetradentate ligand N,N-
bis(2-pyridylmethyl)amino-2-ethanoic acid (BPEAH), and
tridentate ligands N-benzyl-N,N-bis(2-pyridylmethyl)amine
(BnBPA) and N-benzyl-N,N-bis(6-methyl-2-pyridylmethyl)-
amine (Bn-6Me2BPA) which can serve a good mimics for
the enzyme-depside complexes (Fig. 1). These are formu-
lated as CuII(BPEA)(O-bs) (3), [CuII(BnBPA)(O-bs)]ClO4
(4), and [CuII(Bn-6Me2BPA)(O-bs)]ClO4 (5). The determi-
nation of their structure in most cases was carried out by
X-ray single crystal study. In addition, their IR and UV–
Vis analysis and dioxygenase-like activity in the oxygena-
tion of flavonol was also examined in order to see whether
the carboxylate coordinate to the copper(II) ion or enhance
the reactivity of the complex.
Complex 3 isolated as a blue solid is stable in air and
analyzed satisfactorily for C, H, N [15]. The infrared (IR)
spectrum of the complex shows bands corresponding to
the coordinated O-benzoylsalicylate at 1731 m(CO), and
1571, 1344 m(CO2) cmꢀ1. The difference between the asym-
metric and symmetric stretching frequencies of this carb-
Fig. 2. Molecular structure of CuII(BPEA)(O-bs) (3) with crystallographic
numbering. Relevant bond lengths (A) and angles (°): Cu(1)–O(1)
˚
1.972(2), Cu(1)–O(3) 2.015(2), Cu(1)–N(2) 2.016(3), Cu(1)–N(3) 2.047(2),
Cu(1)–N(1) 2.264(3), C(14)–O(1) 1.274(4), O(2)–C(14) 1.228(4), O(3)–
C(15) 1.225(4), O(4)–C(15) 1.238(4), O(5)–C(22) 1.348(4), O(5)–C(21)
1.405(4), O(6)–C(22) 1.192(4), O(1)–Cu(1)–O(3) 91.41(8), O(1)–Cu(1)–
N(2) 145.68(10), O(3)–Cu(1)–N(2) 100.91(9), O(1)–Cu(1)–N(3) 84.80(9),
O(3)–Cu(1)–N(3) 176.17(9), N(2)–Cu(1)–N(3) 81.98(10), O(1)–Cu(1)–N(1)
94.10(10), O(3)–Cu(1)–N(1) 102.50(9), N(2)–Cu(1)–N(1) 113.86(10), N(3)–
Cu(1)–N(1) 78.42(9), C(14)–O(1)–Cu(1) 115.33(18), C(15)–O(3)–Cu(1)
118.79(19).
oxylato group [Dm = mas(CO2) ꢀ ms(CO2)] is 227 cmꢀ1
,
rendering these to a monodentate carboxylate bonding
mode [16,17]. The absorption bands at 1601, 1480 and
1421 cmꢀ1 can be rendered to the pyridine skeleton of the
N,N-bis(2-pyridylmethyl)amino-2-ethanoic acetate. Bands
corresponding to m(CO2) for the BPEAH ligand appear
at 1629 and 1445 cmꢀ1 [18]. The electronic spectrum of
complex 3 shows an absorption band at 914 nm due to
d–d transition. A higher energy band at 290 nm is associ-
ated with the ligand-to-metal charge transfer transition.
Suitable crystals of 3 were obtained by slow diffusion of
diethyl ether into acetonitrile solution of the complex at
20 °C. The molecular structure of CuII(BPEA)(O-bs) [19]
as well as selected bond lengths and angles is shown in
Fig. 2. The molecule is monomeric in the solid state. The
overall geometry around the five-coordinate copper ion is
described as a distorted square planar geometry as judged
by the method of Addison and co-workers [22] (s = 0.51).
Two nitrogen atoms of the tetradentate ligand BPEAH
and two oxygen atoms of the monodentate carboxylate
groups occupy basal positions. The Cu(1)–O(3) bond is
The IR spectra of the complexes [CuII(BnBPA)(O-
bs)]ClO4 and [CuII(Bn-6Me2BPA)(O-bs)]ClO4 show princi-
pal bands corresponding to m(CO) at 1731 and 1735 cmꢀ1
,
respectively [23,24]. Bands corresponding to m(CO2) for the
two complexes appear at 1559 and 1374, and 1570 and
1375 cmꢀ1. The differences between the asymmetric and
symmetric stretching frequencies of the carboxylato groups
are 185 and 195 cmꢀ1, rendering these to a bidentate car-
boxylate bonding mode. Absorptions at 1589, 1474, 1449
(4) and 1592, 1472, 1450 cmꢀ1 (5) can be assigned to the
pyridine skeleton. The infrared spectra of the complexes
show m(Cl–O) absorptions around 1100 cmꢀ1. Bands at
682 (4) and 736 nm (5) are associated with d–d transitions
of the complexes.
Suitable crystals of 5 were obtained by slow diffusion of
diethyl ether into ethanol solution of the complex at 20 °C.
The molecular structure of [CuII(Bn-6Me2BPA)(O-
bs)]ClO4 [25] as well as selected bond lengths and angles
is shown in Fig. 3.
˚
0.043 A longer than the Cu(1)–O(1) bond. The third nitro-
gen atom of N,N-bis(2-pyridylmethyl)amino-2-ethanoic
acid ligand is in the apical position. The Cu–N and Cu–
O distances in the basal plane are shorter than the Cu–N
distance in the apical position.
N
N
N
N
N
OH
N
N
O
N
N
BPEAH
BnBPA
Bn-6Me2BPA
Fig. 1. Structures of the used ligands.

J. Kaizer et al. / Inorganic Chemistry Communications 9 (2006) 251–254
253
fact that there is no difference on the catalytic activity of
the complexes CuII(BPEA)(O-bs) (3) and [CuII(BnB
PA)(O-bs)]ClO4 (4) suggests a role for the glutamate in
the enzyme and the glioxylate linkage in the model reaction
in the deprotonation of the substrate and protonation of
the forming depside. It can be seen that in the case of
[CuII(Bn-6Me2BPA)(O-bs)]ClO4 (5) the conversion is rela-
tively low, which can be explained by steric effect of the
methyl-substitution. These reactions resemble the enzyme
action on the flavonol to give the cleavage product as
shown earlier (Eq. (1)).
Acknowledgements
Financial support of the Hungarian National Research
Fund (OTKA T-043414) and Budakonzum Ltd. are grate-
fully acknowledged.
References
Fig. 3. Molecular structure of [CuII(Bn-6Me2BPA)(O-bs)]ClO4 (5) with
crystallographic numbering. Hydrogen atoms are omitted for clarity.
Relevant bond lengths (A) and angles (°): Cu(1)–O(1) 1.9835(19), Cu(1)–
N(3) 1.995(2), Cu(1)–N(2) 2.004(2), Cu(1)–N(1) 2.041(2), O(1)–C(22)
1.266(3), C(22)–O(2) 1.242(3), O(4)–C(29) 1.213(5), O(1)–Cu(1)–N(3)
98.03(9), O(1)–Cu(1)–N(2) 98.62(9), N(3)–Cu(1)–N(2) 163.34(9), O(1)–
Cu(1)–N(1) 151.30(8), N(3)–Cu(1)–N(1) 82.83(9), N(2)–Cu(1)–N(1)
82.83(9), C(22)–O(1)–Cu(1) 101.16(17).
˚
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¨
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all geometry around the six-coordinate copper ion is
described as a distorted elongated octahedral geometry.
Three nitrogen atoms of the tridentate ligand Bn-6Me2
BPA and one of the oxygen atom of the bidentate carbox-
ylate group occupy basal positions. The another oxygen
[7] F. Fusetti, K.H. Schro¨ter, R.A. Steiner, P.I. van Nort, T. Pijning,
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Egmond, M. Huber, Eur. J. Biochem. 12 (2002) 2971.
˚
atom of the depside jCu(1)–O(2), 2.488 Aj and one of the
´
´
´
[10] E. Balogh-Hergovich, J. Kaizer, G. Speier, G. Argay, L. Parkanyi, J.
˚
oxygen of the perchlorate anion jCu(1)–O(5), 2.496 Aj are
Chem. Soc., Dalton Trans. (1999) 3847.
in the apical positions.
´
´
´
[11] E. BaBalogh-Hergovich, J. Kaizer, G. Speier, V. Fulo¨p, L. Parkanyi,
¨
Inorg. Chem. 38 (1999) 3787.
The reaction between flavonol and dioxygen in the pres-
ence of catalytic amounts of copper(II) carboxylates (3, 4,
and 5) were performed in DMF solutions and examined at
100 °C with a ratio of 1:20 between initial concentration of
copper complexes and flavonol (1). We found that the oxy-
genation of flavonol results in oxidative cleavage of the het-
erocyclic ring to give O-benzoylsalicylic acid (2) and CO as
products [26]. The oxygenation reaction in all cases were
selective, no other products were obtained (Table 1). A
comparison of their activities was made through the deter-
mination of the conversion of the reactions (Table 1). The
´
[12] E. Balogh-Hergovich, J. Kaizer, G. Speier, G. Huttner, A. Jacobi,
Inorg. Chem. 39 (2000) 4224.
[13] J. Kaizer, J. Pap, G. Speier, L. Parkanyi, Eur. J. Inorg. Chem. (2004)
´
´
2253.
´
[14] E. Balogh-Hergovich, J. Kaizer, J. Pap, G. Speier, G. Huttner, L.
Zsolnai, Eur. J. Inorg. Chem. (2002) 2287.
[15] Synthesis of [CuII(BPEA)(O-bs)] (3): Cu(OMe)2 (0.126 g, 1 mmol), O-
benzoylsalicylic acid (0.242 g, 1 mmol) and N,N-bis(2-pyridylm-
ethyl)amino-2-ethanoic acid (0.256 g, 1 mmol) were dissolved in
10 cm3 of acetonitrile under argon and two equivalent of NEt3
(0.202 g, 2 mmol) was dropped slowly into the solution. After 10 h
stirring, the product was collected by filtration, washed with diethyl
ether, dried in vacuum and then recrystallized from ethanol by ether
diffusion (0.31 g, 55%). M.p. 165 °C. IR (KBr) cmꢀ1: 3061 vw, 3029
vw, 2950 vw, 2880 vw, 1731 s, 1629 vs, 1601 s, 1572 m, 1480 m, 1445
m, 1421 m, 1344 s, 1271 s, 1252 m, 1193 m, 1157 m, 1090 m, 1059 m,
1025 m, 756 m, 703 m, 750 m, 707 m, 658 w. UV–Vis (DMF) kmax
(loge/dm3 molꢀ1 cmꢀ1): 270 (3.72); 914 (1.50). Anal. Calc. for
C28H23N3O6Cu: C, 59.94; H, 4.13; N, 7.49%. Found: C, 60.11; H,
4.08; N, 7.41%.
Table 1
Cu-catalyzed oxygenation of flavonol
Catalyst
[Cu]a
[FlaH]a Conversionb TN
(mmol) (mmol) (%)
CuII(BPEA)(O-bs) (3)
0.05
0.05
1
1
1
90
91
69
9.0
9.1
6.9
[CuII(BnBPA)(O-bs)]ClO4 (4)
[16] G. Christou, S.P. Perlepes, E. Libby, K. Folting, J.C. Huffman, R.J.
Webb, D.N. Hendrickson, Inorg. Chem. 29 (1990) 3657.
[17] I. Chadjistamatis, A. Terzis, C.P. Raptopoulou, S.P. Perlepes, Inorg.
Chem. Commun. 6 (2003) 1365.
[CuII(Bn-6Me2BPA)(O-bs)]ClO4 (5) 0.05
a
In 15 ml DMF under O2 at 100 °C.
By glc (benzoin as an internal standard).
b

254
J. Kaizer et al. / Inorganic Chemistry Communications 9 (2006) 251–254
[18] K.-Y. Choi, Y.-M. Jeon, H. Ryu, J.-J. Oh, H.-H. Lim, M.-W. Kim,
Polyhedron 23 (2004) 903.
[24] Synthesis of [CuII(Bn-6Me2BPA)(O-bs)]ClO4 (5): Cu(ClO4)2 Æ 6H2O
(0.371 g, 1 mmol), O-benzoylsalicylic acid (0.242 g, 1 mmol) and N-
benzyl-N,N-bis(6-methyl-2-pyridylmethyl)amine (0.381 g, 1 mmol)
were dissolved in 10 cm3 of methanol under argon and one equivalent
of NEt3 (0.101 g, 1 mmol) was dropped slowly into the solution. After
8 h stirring, the product was collected by filtration, washed with
diethyl ether, dried in vacuum and then recrystallized from ethanol by
ether diffusion (0.40 g, 55%). M.p. 119 °C. IR (KBr) cmꢀ1: 3140 vw,
2970 vw, 2921 vw, 2880 vw, 1735 s, 1612 vs, 1592 w, 1570 m, 1472 m,
1450 m, 1375 s, 1344 vs, 1269 s, 1258 s, 1201 m, 1117 vs, 1094 vs, 1025
m, 851 w, 791 w, 750 m, 707 m, 625 s. UV–Vis (DMF) kmax (loge/dm3
[19] Crystal data for 3: C28H23CuN3O6, 561.03 g molꢀ1, monoclinic, P21/c,
˚
˚
˚
a = 9.0410(1) A, b =17.5690(3)A, c =16.0680(3)A, b =103.3540(7)°,
Z = 4, V = 2483.25(7) A , l(MoKa) = 0.929 mmꢀ1, dcalc = 1.501 g
3
˚
cmꢀ3, T = 293(2) K, F(000) = 1156. The intensity data were collected
with a Nonius Kappa CCD single-crystal diffractometer using Mo Ka
radiation (k = 0.71073). Reflections collected = 4735, reflections
total = 4735, reflections unique = 4275. The structure was refined to
R = 0.0461 (0.0520) and wR2 = 0.1240 for the reflections with
I > 2r(I) (all data) and max. resd. density = 0.611 e Aꢀ3. The com-
˚
puter program used was SHELXL97 [20]. The structure was solved by
direct and difmap methods (SIR92) [21]. CCDC reference number:
288962.
molꢀ1 cmꢀ1): 271 (3.93); 736 (2.16). Anal. Calc. for C35H32
-
N3O8ClCu: C, 58.25; H, 4.47; N, 5.82%. Found: C, 58.46; H, 4.51;
N, 5.75%.
[25] Crystal data for 5 Æ 0.5(C2H5)O: C37H37ClCuN3O8.5, 758.69 g molꢀ1
,
[20] G.M. Sheldrick, SHELXL97. Program for the Refinement of Crystal
Structures, University of Go¨ttingen, Germany, 1997.
[21] A. Altamore, G. Cascarano, C. Giacovazzo, A. Guagliardi,
M.C. Burla, G. Polidori, M.J. Camalli, J. Appl. Cryst. 27
(1994) 435.
˚
˚
monoclinic, P21/c, a = 8.6330(2) A, b = 15.4140(2) A, c = 29.1940
3
˚
˚
(4) A, b = 95.7240(6)°, Z = 4, V = 3865.45(12) A , l(Mo Ka) =
0.687 mmꢀ1, dcalc = 1.304 g cmꢀ3, T = 293(2) K, F(000) = 1576. The
intensity data was collected with a Nonius Kappa CCD single-crystal
diffractometer using Mo Ka radiation (k = 0.71073). Reflections
collected = 14692, reflections total = 7906, reflections unique = 5614.
The structure was refined to R = 0.0506 (0.0819) and wR2 = 0.1415
for the reflections with I > 2r(I) (all data) and max. resd. den-
[22] A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Verschoor, J.
Chem. Soc., Dalton Trans. (1984) 1349.
[23] Synthesis of [CuII(BnBpa)(O-bs)]ClO4 (4): Cu(ClO4)2 Æ 6H2O (0.371 g,
1 mmol), O-benzoylsalicylic acid (0.242 g, 1 mmol) and N-benzyl-
N,N-bis(2-pyridylmethyl)amine (0.289 g, 1 mmol) were dissolved in
10 cm3 of methanol under argon and one equivalent of NEt3 (0.101 g,
1 mmol) was dropped slowly into the solution. After 8 h stirring, the
product was collected by filtration, washed with diethyl ether, dried in
vacuum and then recrystallized from ethanol by ether diffusion
(0.28 g, 40%). M.p. 189 °C. IR (KBr) cmꢀ1: 3067 vw, 2921 vw, 2820
vw, 1731 s, 1610 vs, 1589 w, 1559 m, 1474 m, 1449 m, 1374 s, 1344 vs,
1258 s, 1201 m, 1193 vs, 1092 vs, 1029 m, 753 m, 706 m, 625 m. UV–
Vis (DMF) kmax (loge/dm3 molꢀ1 cmꢀ1): 270 (3.85); 682 (2.41). Anal.
Calc. for C33H28N3O8ClCu: C, 57.15; H, 4.07; N, 6.06%. Found: C,
56.86; H, 4.01; N, 6.12%.
sity=0.332 e Aꢀ3. The computer program used were SHELXL97 [20].
˚
The structure was solved by direct and difmap methods (SIR92) [21].
CCDC reference number: 288963.
[26] Copper-catalyzed oxygenation of flavonol: flavonol (0.238 g, 1 mmol)
and the corresponding copper(II) O-benzoylsalicylate (0.05 mmol)
were dissolved and stirred at 100 °C in 15 cm3 DMF for 20 h under
dioxygen atmosphere. Diazomethane solution (2 cm3) (in diethyl
ether) was added to 0.5 cm3 of the reaction mixture at r.t. and the
conversion of flavonol into O-benzoylsalicylic acid was determined by
GC as the methylated derivative in the presence of benzoin (internal
standard).