Diethanolamine Pd(II) complexes in bioorganic modeling as model systems of metallopeptidases and soybean lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2009.07.003

The reaction of PdCl₂ with diethanolammonium chloride (DEAxHCl), in the molar ratio 1:2, affords the [HDEA]₂[PdCl₄] complex (1). The hydrolytic activity of the novel Pd(II) complex 1 was tested in reaction with N-acetylate

Full text of DOI:10.1016/j.bioorg.2009.07.003

Bioorganic Chemistry 37 (2009) 162–166
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Diethanolamine Pd(II) complexes in bioorganic modeling as model systems
of metallopeptidases and soybean lipoxygenase inhibitors
Zorica D. Petrovi c´ a, ,aDimitra Hadjipavlou-Litina , Eleni Pontiki , Dušica Simijonovi c´ ,
b,*
b
a
*
Vladimir P. Petrovic´
a
Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovi ´c a 12, P.O. Box 60, 34000 Kragujevac, Serbia
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2009
Available online 18 July 2009
The reaction of PdCl2 with diethanolammonium chloride (DEAxHCl), in the molar ratio 1:2, affords the
[
HDEA]
with N-acetylated L-histidylglycine dipeptide (AcHis-Gly). Complex 1, as well as earlier prepared trans-
PdCl (DEA) ] complex (2), and DEA, as their precursor, were tested for their in vitro free radical scaveng-
2 4
[PdCl ] complex (1). The hydrolytic activity of the novel Pd(II) complex 1 was tested in reaction
[
2
2
Keywords:
Palladium(II) complex
Structure
Hydrolytic activity
Soybean lipoxygenase inhibitors
Free radical scavenger activity
ing activity. UV absorbance-based enzyme assays were done in order to evaluate their inhibitory activity
of soybean lipoxygenase (LOX). Also, assays with superoxide anion radical were done. The scavenging
activities of the complexes were measured and compared with those of their precursors and caffeic acid.
Complex 2 exhibits the highest antioxidant activity and the highest inhibitory effect against the soybean
LOX.
Ó 2009 Elsevier Inc. All rights reserved.
1
. Introduction
It was shown that some of palladium(II) complexes, as model
products and the propagation of inflammation [19,20]. Linoleic
acid is the primary substrate in the reaction of dioxigenation of
polyunsaturated fatty acids catalyzed by plant LOX, while the
mammalian isozymes mainly catalyze the metabolism of arachi-
donic acid [21]. In mammalian cells, LOXs play an essential role
in the biosynthesis of many bioregulatory molecules such as leu-
kotrienes, lipoxins and hepoxylines. These compounds are media-
tors in the pathophysiology of variety of diseases, for example
bronchial asthma, psoriasis and inflammation [22]. The fatty acid
hydroperoxides, generated in the reaction of dioxigenation of
polyunsaturated fatty acids catalyzed by LOX, significantly con-
tribute to the creation of atherosclerotic lesions [23]. These com-
pounds have critical influence on the development of several
human cancers [24]. For these reasons, extensive research was
done to find efficient inhibitors of LOX activity [21,25–33]. Most
of these studies have used readily obtainable soybean lipoxyge-
nase, which is a homologue of mammalian lipoxygenase and well
examined [34,35]. Most of the lipoxygenases inhibitors are anti-
oxidants or free radical scavengers, since lipoxygenation occurs
via a carbon-centered radical.
systems in bioorganic chemistry, can be interesting as artificial
metallopeptidases, inhibitors of enzymes, and free radical
scavengers.
Interest in the study of interactions of platinum(II) and palla-
dium(II) complexes with sulfur- and histidine-containing peptides
and proteins became of capital importance after the discovery that
their aqua complexes can be promising reagents for the selective
hydrolytic cleavage of the above mentioned peptides [1–12]. Fur-
thermore, it was found that some of palladium complexes can be
enzyme inhibitors. These compounds inhibit various enzymes such
are RNA polymerase [13], topoisomerase III, reverse transcriptase
(
by free radical generation) [14], leukemia virus reverse transcrip-
tase [15], cellobiohydrolase [16], and soybean LOX [11]. Some pal-
ladium complexes have been evaluated for their anti-inflammatory
and antioxidant activities in vitro, whereby palladium complexes
exhibited higher activity compared to the activity of the ligand
[
12,17,18].
The lipoxygenases are non-heme iron containing enzymes
In the present study, a new diethanolammonium–tetrachlorido-
which catalyze the oxidative metabolism of fatty acids, and are
useful target for the design and development of new drugs that
substantially inhibit the generation of the final inflammatory
palladate(II) complex (1) ([HDEA]
2 4
[PdCl ], where DEA is diethanol-
amine), was synthesized and employed to study its hydrolytic
activity in the reaction with N-acetylated L-histidylglycine dipep-
tide (AcHis-Gly). New complex 1 and earlier prepared diethanola-
2 2
mine complex trans-[PdCl (DEA) ] complex (2), as well as
diethanolamine, precursor of these compounds, were tested for
their LOX inhibitory activity and free radical scavenging activity.
*
Corresponding authors.
E-mail addresses: zorica@kg.ac.rs (Z.D. Petrovi c´ ), hadjipav@pharm.auth.gr
(
D.Hadjipavlou-Litina).
0
045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.07.003

Z.D. Petrovi ´c et al. / Bioorganic Chemistry 37 (2009) 162–166
163
Spectral characterization of the complex [HDEA]
NMR spectrum (200 MHz, D O): d = , t, 3.24 (4H, –CH
J = 5.2 Hz), 3.86 (4H, –CH
2
[PdCl
4
]: 1
H
2
. Materials and methods
The compounds D O, DNO
2
2
–NH
2
3
, and PdCl
2
were obtained from Al-
2
–OH, t, J = 5.2 Hz) ppm; IR (KBr):
ꢀ
1
drich Chemical Co. All common chemicals were of reagent grade.
Dipeptide L-histidylglycine (His-Gly), soybean lipoxygenase, lino-
leic acid sodium salt, NADH, and nitrotetrazolium blue (NBT) were
obtained from Sigma Chemical Co. N-methylphenazonium-methyl
v
= 323, 698, 958, 1065, 1088, 1405, 1574, 2874, 3299, 3343 cm
.3. Soybean lipoxygenase inhibition study in vitro
In vitro study was evaluated as reported previously [44]. The
.
3
2 2 2
sulfate and diethanolamine (DEA; NH(CH CH OH) ) were pur-
chased from Fluka. The terminal amino group in His-Gly was acet-
ylated by standard method to obtain AcHis-Gly [1].
tested compounds dissolved in ethanol were incubated at room
3
temperature with sodium linoleate (0.1 mM) and 0.2 cm of en-
ꢀ4
All pH measurements were made at 25 °C. The pH meter (Iskra
MA 5704) was calibrated with Fischer certified buffer solutions of
pH 4.00 and 7.00. The results were not corrected for the deuterium
isotope effect. Reactions of AcHis-Gly with palladium(II) complex
zyme solution (1/9 ꢁ 10 w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded
and compared with the appropriate standard inhibitor.
in D
2
O solutions were followed by 1H NMR spectroscopy using a
3.4. Assay of superoxide anion radical scavenging activity [44,45]
Varian 200 MHz spectrometer. The palladium(II) complex 1 and
peptide were mixed in an NMR tube in molar ratio 1:2. The pH
was 2.0. The internal reference was TSP (sodium trimethylsilylpro-
pane-3-sulfonate). The IR spectra were recorded on a Perkin-Elmer
Spectrum One FT-IR spectrometer using the KBr pellet technique.
Elemental microanalyses for carbon, hydrogen, and nitrogen were
performed at the Faculty of Chemistry, Belgrade University.
Thesuperoxide-producingsystemwassetupbymixingphenazine
methylsulfate (PMS), NADH and air-oxygen. The production of super-
oxide was estimated by the nitroblue tetrazolium method. The
reaction mixture containing investigated compound, 3
lM PMS,
78
l
M NADH, and 25 M NBT in 19 M phosphate buffer pH 7.4
l
l
was incubated for 2 min at room temperature and the absorption
measured at 560 nm against a blank containing PMS. The tested com-
pounds were preincubated for a blank containing PMS. The tested
compounds were preincubated for 2 min before adding NADH.
2.1. Computational method
All calculations were conducted using Gaussian03 [36] with the
B3LYP hybrid functional [37–39]. The triple split valence basis set
-311G(d,p) was used for C, H, O, N, and Cl [40], whereas LANL2D-
4
. Results and discussion
6
Z + ECP [41] was employed for the Pd center. Geometrical parame-
ters of all investigated species were optimized in vacuum.
Vibrational analysis was performed for all structures. All calculated
structures were verified to be local minima (all positive eigen-
values) for ground state structures by frequency calculations. The
natural bond orbital analysis [42,43] (Gaussian NBO version) was
performed for all structures.
4
.1. Structure examination of [HDEA]
2
[PdCl
4
] complex (1)
The structure of complex [HDEA]
2
[PdCl
4
] was examined using
DFT method. The optimized structure of complex is presented in
Fig. 1. [PdCl
2ꢀ
4
]
anion exhibits square planar coordination, where
the four chlorine anions lie in the equatorial plane, whereas the
two protonated diethanolamine cations form hydrogen bonding
with chlorido ligands. Cis- and trans-chlorido ligands form with pal-
ladium bond angles of 90° and 180°, respectively. The Pd–Cl bond
length is equal to 2.40 Å, whereas the distances between chlorido li-
gands and hydrogens bonded to nitrogen lie in the range of 2.10–
2.15 Å. The NBO analysis of the complex reveals covalent Pd–Cl
3
. Synthesis of the diethanolammonium–tetrachloridopalladate
(
II) complex, [HDEA] [PdCl
2
4
]
3.1. Synthesis of the diethanolammonium chloride, [HDEA]Cl
2
5.80
bonds, with hybrid compositions of 0.41(sp d)Pd + 0.91(sp
Cl
) .
Diethanolamine hydrochloride was prepared by slowly dropping
hydrochloric acid to diethanolamine dichlormetane solution.
Hydrochloric acid was added excessively to 5% of the stoichiometric
amount. Resulting solution was mixed during 2 h at room tempera-
ture. Finally, volatile component was evaporated in vacuo. Prepared
ionic liquid is colourless viscous liquid whose boiling point is 112 °C.
1
Spectral characterization of the ionic liquid [HDEA]Cl: H NMR
spectrum (200 MHz, D
ppm; 3.891(4H, –CH –OH, t, J = 5.0 Hz) ppm. IR (film):
31, 949, 1039, 1064, 1408, 2852, 3336, 3384 cm
2
O): d = 3.266 (4H, –CH
2
–NH
2
, t, J = 5.0 Hz)
2
v
= 325,
ꢀ
1
6
.
2 4
3.2. Synthesis of the [HDEA] [PdCl ] complex
The [HDEA]
PdCl and two equivalents of diethanolamine hydrochloride,
according to the procedure published earlier [11]. In the course
of 3 h, the reaction of 0.1774 g (0.001 mol) of PdCl dissolved in
5 mL of water with 0.283 g (0.002 mol) of diethanolamine hydro-
2 4
[PdCl ] complex was synthesized starting from
2
2
1
chloride, at 50–60 °C, afforded an orange–brown solution which
was left at room temperature for 2 days. The precipitated brown
crystals were filtered off, washed with ethanol, air-dried and
showed a melting point of 115–116 °C. Yield 0.446 g (97%). Calcu-
2 4 8 22 4 2 4
lated for [HDEA] [PdCl ] = C H O N Cl Pd (FW = 460.42): C,
2
0.95; N, 6.09; H, 5.21%; found: C, 21.01; N, 6.13; H, 5.18%.
Fig. 1. Optimized geometry of complex 1 with delineated LUMO map.

1
64
Z.D. Petrovi ´c et al. / Bioorganic Chemistry 37 (2009) 162–166
Lower occupancy in all
r
Pd–Cl orbitals (1.90) is due to donation of
results confirms the predicted structure of complex 1. These results
were used in order to describe the structure–activity relationship
*
density from each bonding orbital to the trans-
r
r
Pd–Cl antibond-
ing orbital, in accord with the usual chemical picture of delocalized
(SAR) of complex compound 1.
chemical systems.
The vibrational analysis of complex 1 was performed. There are
characteristic vibrations at 3740 cm (stretching vibrations of OH
4.2. Hydrolytic reaction of [HDEA] [PdCl ] complex with AcHis-Gly
2
4
ꢀ
1
ꢀ
1
groups of protonated diethanolamine), 3128 and 3042 cm
2 2
The Pd(II)-diethanolamine complex trans-[PdCl (DEA) ] [11]
þ
ꢀ1
(
stretching vibrations of NH2 ions), 1623 cm
(deformational
showed selective hydrolytic activity in the reaction with N-acetylated
L-histidylglycine dipeptide (AcHis-Gly). We supposed that novel
Pd(II)-diethanolamine complex 1 can be artificial metallopeptidase,
also. To confirm our presumption, we performed the reaction of this
complex and dipeptide AcHis-Gly (molar ratio 1:2) at pH 2.0 and
þ
ꢀ1
vibrations of NH2 ions), 3011 cm (stretching vibrations of CH
groups), and 1505 (deformational vibrations of CH groups). These
values are significantly different from the experimental values of
2
2
ꢀ1
3
343, 3051, 2874, 1574, 2912, and 1455 cm . It is well known that
1
the computed values of vibrational frequencies contain systematic
error due to neglect of electron correlation. There is also a contri-
bution to the error due to the fact that the calculated frequencies
come from treating the potential energy surface near the station-
ary point as a harmonic oscillator. In reality, the potential energy
surface near the stationary point is generally anharmonic. This re-
sults in overestimating the vibrational frequencies values, on aver-
age, by anywhere between 0% and 20%, implying that they need to
be scaled to provide satisfactory approximation of experimental
vibrational frequencies. By applying a scaling factor for B3LYP/6-
60 °C inD O, andstudiedby HNMR spectroscopy. Inour experiment,
2
under the above mentioned conditions, only one complex and selec-
tive cleavage of peptide bond were observed after 15 h. The NMR
spectrashoweddecreasingofthesingletat4.00(duetomehylenegly-
cine protons of the non-hydrolyzed substrate) and increasing of the
singletat3.77 ppmfortheseprotonsinfreeglycine. After43 hofheat-
ing the reaction mixture at 60 °C, the intensity of singlet at 3.77 ppm
was not changed, Graphic 1. Addition of amino-acid glycine to the
reaction mixture caused an increase of the signal at 3.77 ppm. Under
these reaction conditions free acetic acid was not detected by NMR
spectroscopy during investigated reaction time.
3
11G(d,p) of 0.967 ± 0.021 [46] to the computed values of vibra-
tional frequencies for complex 1, the agreement with experimental
data is improved. The only exception is the stretching vibration of
OH groups of protonated diethanolamine, which can be attributed
to association of complex via hydrogen bonds crosswise OH
groups. Such agreement between experimental and computational
4
.3. Soybean lipoxygenase inhibition study in vitro [44]
In continuation of our study on biological significant derivatives
of histidine, this part of our work is devoted to the study of in vitro
Graphic 1. Parts of 1H NMR spectra for the hydrolytic reaction of AcHis-Gly with [HDEA]
2 4 2
[PdCl ] complex as function of time at pH = 2 and 60 °C in D O as solvent. The
chemical shifts are in ppm relative to TSP. Resonance are indicated as follows: (a) methylene glycine protons of the starting dipeptide; (b) protons of the protonated
diethanolamine units of the [HDEA] [PdCl ] complex (1); and (c) methylene protons of the free glycine.
2
4

Z.D. Petrovi ´c et al. / Bioorganic Chemistry 37 (2009) 162–166
165
inhibition of soybean lipooxygenase (LOX), as a histidine-contain-
ing enzyme. Availability and stability of mammalian lipoxygenases
is limited, and therefore research on lipoxygenases was done with
readily obtainable enzyme from soybean seeds. The active site in
soybean LOX is non-heme Fe(III) atom coordinated by three histi-
dines, isoleucine, asparagine and a hydroxide group [47], Fig. 2.
Inhibition of this enzyme was investigated by using complexes 1
and 2, and DEA.
Considering the radical mechanism of inhibition of LOX and the
fact that palladium(II) ion is ‘‘soft” Lewis acid, we assumed that
complexes 1 and 2 can act as free radical scavengers in LOX-cata-
lyzed reaction of dioxigenation of fatty acids.
The generally accepted mechanism of action of LOX is radical
mechanism which involves hydrogen abstraction from the fatty
acid by the iron(III)-hydroxide group, accompanied with the reduc-
tion of LOX to Fe(II) form (Scheme 1) The obtained pentadienyl
radical is then trapped by dioxigen to yield a peroxyl radical of
fatty acid as a catalytic intermediate. Reduction of the peroxyl rad-
ical by the Fe(II) ion yields the hydroperoxide product and regen-
erates the Fe(III) ion (active LOX form) [48].
site of the enzyme molecule [49], by preventing the formation of
the activated Fe(III) form of LOX [50] or by trapping the free radi-
cals formed during the lipoxygenase-catalyzed oxygenation of
polyunsaturated fatty acids [51]. In our cases it is reasonable to ex-
pect that palladium(II)–chlorido complexes, as electrophiles, react
as free radical scavengers by trapping radical intermediate(s) and
blocking the catalytic cycle.
UV absorbance-based enzyme assays with diethanolamine pal-
ladium(II) complexes 1 and 2, and DEA were done in order to eval-
uate their inhibitory activity of soybean LOX. Perusal of% inhibition
values, or IC50 values, shows that DEA as a complex precursor has
lower IC50 than complexes. Significant higher inhibitor activity of
complex 1, and especially 2, relative to DEA (Table 1) clearly shows
that palladium(II)-chlorido moiety of these complexes is meritori-
ous for higher inhibitor activity. Complex 2 is very active and more
potent than the reference compound caffeic acid. These results are
supported by our DFT calculations and structure–activity relation-
ship (SAR) investigation. In Fig. 1 the LUMO map of complex 1 is
depicted. It is shown that the most electron-deficient area of
complex 1 is delocalizated over palladium (II) ion and four chlorido
2
ꢀ
2ꢀ
4
Studies on inhibitors of soybean LOX show several possible
mechanisms, e.g., by binding inhibitor to sites around the active
ligands, namely over the [PdCl
4
]
ion. Therefore, the [PdCl ]
moiety of the complex acts as weak Lewis acid capable for
accepting electrons from other species, such as radicals. In case
of complex 2, LUMO orbital is delocalizated over palladium (II)
ion, two chlorido ligands and two nitrogen atoms of diethanola-
mine ligands, Fig. 3. For this reason, one can suppose that com-
plexes 1 and 2 can be used as successful radical scavengers.
In order to prove free radical scavenger properties of complexes
N
O
1
and 2, and DEA, assays in vitro with superoxide anion radical
N
were done. The superoxide-producing system was set up by mix-
ing phenazine methosulfate (PMS), nicotinamide-adenine-dinukle-
Ile
O
OH
N
Table 1
Fe
O
ꢀ
Å
%
Superoxide radical scavenging activity ðO2 Þ; in vitro inhibition of soybean
lipoxygenase (LOX) (IC50).
N
N
N
ꢀÅ
ꢀÅ
Compound
ðO Þ (%) 0.01 mM
ðO Þ (%) 0.1 Mm
LOX IC50 (lM)
2
2
Complex 1
Complex 2
DEA
78
64
83
100
100
92
210
76
500
600
CA
45
NH3
Asn
CA caffeic acid; DEA diethanolamine.
Each value represents the mean of two independent experiments.
Fig. 2. The active site in soybean LOX.
Lox-Fe3+
active)
(
H
H
H
,
,
R
R
R
R
O2
Lox-1
H+
pH 9.0
HOO
Lox-Fe2+
Lox-Fe3+
2+
Lox-Fe
,
.
R
R
O2
.
OO
OO
radical
peroxyl radical
R= -(CH ) CH
3
2
n
,
R= -(CH ) COOH
2
n
Scheme 1. Radical mechanism for lipoxygenase-catalyzed oxidation of polyunsaturated fatty acids under aerobic conditions.

1
66
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2
The reaction between PdCl and diethanolammonium chloride
in a molar ratio of 1:2, leads to the easy formation of the
HDEA] [PdCl ] complex (1), whose structure was optimized by
DFT methods. In order to prove hydrolytic activity, the reaction be-
tween the [HDEA] [PdCl ] complex with MeCOHis-Gly dipeptide at
[
2
4
[
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pH = 2.0 and 60 °C was done. Selective cleavage of peptide bond
was observed under these experimental conditions during the
course of 43 h. Also, complexes 1 and 2, and DEA, as their precur-
sor, were tested for their in vitro soybean LOX inhibitory and free
radical scavenging activities. UV absorbance-based enzyme assay
and assay with superoxide anion radical were done. The scaveng-
ing activities of the complexes were measured and compared with
those of their precursor and vitamin C. Complex 2 with high anti-
oxidant ability and low IC50 value is considered as agent with po-
tential antioxidant activity, and can therefore be candidate for
further stages of screening in vitro and/or in vivo.
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