Synthesis, characterization, antioxidant activities, and DNA-binding studies of (E)-N′-[1-(pyridin-2-yl)ethylidene]isonicotinohydrazide and its Pr(III) and Nd(III) complexes

Article abstract of DOI:10.1248/cpb.58.1306

A new ligand, (E)-N′-[1-(pyridin-2-yl)ethylidene]isonicotinohydrazide (HL), was prepared by condensation of 2-acetylpyridine and isonicotinohydrazide in ethanol. Its two lanthanide(III) complexes, [Nd^III(L) ₂(NO₃)(CH₃OH)₂]?CH ₃CH₂OH (1), and [Pr^III(L)₂(NO ₃)(CH₃OH)₂]?CH₃CH ₂OH (2), have been synthesized and characterized on the basis of element analyses, molar conductivities and IR spectra. The structure of complex 2 has been confirmed by X-ray diffraction. In addition, the DNA-binding properties of the two complexes have been investigated by electronic absorption spectroscopy, fluorescence spectroscopy, circular dichroic (CD) spectroscopy and viscosity measurements. The experimental results suggest that the two complexes bind to DNA via a groove binding mode, and the binding affinity of complex 2 is higher than that of complex 1. Furthermore, the antioxidant activities (superoxide and hydroxyl radical) of the ligand and its metal complexes were determined by spectrophotometry methods in vitro. These complexes were found to possess potent antioxidant activity and be superior to standard antioxidant like mannitol.

Full text of DOI:10.1248/cpb.58.1306

1306
Regular Article
Chem. Pharm. Bull. 58(10) 1306—1312 (2010)
Vol. 58, No. 10
Synthesis, Characterization, Antioxidant Activities, and DNA-Binding
Studies of (E)-N؅-[1-(Pyridin-2-yl)ethylidene]isonicotinohydrazide and Its
Pr(III) and Nd(III) Complexes
Zhong-Yan HAO, Qi-Wan LIU, Jun XU, Lei JIA, and Shao-Bai LI*
College of Chemistry and Chemical Engineering, State Key Laboratory of Applied Organic Chemistry, Lanzhou
University; Lanzhou 730000, P. R. China.
Received December 23, 2009; accepted June 20, 2010; published online July 13, 2010
A new ligand, (E)-N؅-[1-(pyridin-2-yl)ethylidene]isonicotinohydrazide (HL), was prepared by condensa-
tion of 2-acetylpyridine and isonicotinohydrazide in ethanol. Its two lanthanide(III) complexes,
[NdIII(L)2(NO3)(CH3OH)2]·CH3CH2OH (1), and [PrIII(L)2(NO3)(CH3OH)2]·CH3CH2OH (2), have been synthe-
sized and characterized on the basis of element analyses, molar conductivities and IR spectra. The structure of
complex 2 has been confirmed by X-ray diffraction. In addition, the DNA-binding properties of the two com-
plexes have been investigated by electronic absorption spectroscopy, fluorescence spectroscopy, circular dichroic
(CD) spectroscopy and viscosity measurements. The experimental results suggest that the two complexes bind to
DNA via a groove binding mode, and the binding affinity of complex 2 is higher than that of complex 1. Further-
more, the antioxidant activities (superoxide and hydroxyl radical) of the ligand and its metal complexes were
determined by spectrophotometry methods in vitro. These complexes were found to possess potent antioxidant
activity and be superior to standard antioxidant like mannitol.
Key words crystal structure; Pr(III) complex; groove binding; antioxidant activity
It is well-known that Schiff bases play important role in Experimental
Materials Nitroblue tetrazolium (NBT), methionine (MET), vitamin B2
(VitB2), calf thymus DNA (CT-DNA) and ethidium bromide (EB) were ob-
tained from Sigma Chemical Co. All experiments involving interaction of
the complexes with CT-DNA were carried out in doubly distilled water
bioinorganic chemistry and their metal complexes have been
studied extensively for decades. These interesting ligand sys-
tems contain different donor sites in heterocyclic rings, e.g.,
NNO or NNS, which give rise to the architectural beauty of
these coordination complexes.1) Among the ligand systems,
hydrazide and hydrazone occupy important places because
transition metal complexes of these ligands have been devel-
oped due to their chelating capability, structural flexibility,
interesting electrical as well as magnetic properties, and vari-
ous pharmacological activities, such as antibacterial, antitu-
moral, antiviral, antimalarial, and antituberculosis actions.2,3)
Investigation of molecular complexes of lanthanide ions
has attracted significant attention, owing to their broad rang-
ing fluorescent applications in biochemistry, material chem-
istry, medicine and so forth.4—6) However, there have been
only a few DNA-binding investigations of such complexes
have been relatively few. Because the interaction between
metal complexes and DNA is closely related to their potential
buffer containing 5 mM Tris [Tris(hydroxymethyl)-aminomethane] and
50 mM NaCl, and adjusted to pH 7.2 with hydrochloric acid. The DNA con-
centrations of the solutions were determined from their UV absorption at
260 nm using the molar absorption coefficient e260ϭ6600 molϪ1 cmϪ1. DNA
Purity was checked by monitoring the ratio of the absorbance at 260 nm to
that at 280 nm. The solution gave a ratio of A260/A280Ͼ1.80, indicating that
DNA was sufficiently free from protein.11)
Physical Measurements The UV–vis absorption measurements were
conducted by using a Varian Cary 100 spectrophotometer equipped with
quartz cells. All fluorescence emission spectra were measured using a Hi-
tachi F-4500 spectrofluorophotometer equipped with a xenon lamp source
and a quartz cell of 5 cm path length. Viscosity experiments were carried out
on an Ubbelodhe viscometer. The circular dichroic (CD) spectra were
recorded on a Jasco J-810 spectropolarimeter. The 1H-NMR spectra were
recorded with a Bruker ACF300 FT-NMR instrument using tetramethylsi-
lane (TMS) as an internal reference in D2O for the ligand. Electrospray ion-
ization (ESI) mass spectra were recorded on a Finnigan MAT LCQ mass
spectrometer using the syringe pump method. High resolution-mass spectra
biological and pharmaceutical activities, studies on the DNA- (HR-MS) were obtained by the ESI ionization sources. All conductivity
measurements were performed in N,NЈ-dimethyl formamide (DMF) with a
binding of metal complexes are very important in the devel-
DDS-11A conductor at 25 °C. The antioxidant activities were tested on a
opment of new therapeutic reagents and DNA molecular
721E spectrophotometer (Shanghai Analytical Instrument Factory, China).
probes.7,8) Various coordination compounds of aroylhydra-
DNA Binding Experiment Methods Absorption titration experiments
zones have been reported to act as enzyme inhibitors and are
useful due to their pharmacological applications.9,10)
Here, we synthesized (E)-NЈ-[1-(2-pyridinyl)ethylidene]-
isonicotinohydrazide (HL) and prepared its two of its lan-
thanide(III) complexes (Chart 1). The DNA-binding proper-
ties and antioxidant activities of the two lanthanide(III)
complexes with HL are reported herein.
were performed by fixing the concentrations of complexes 1 and 2 as con-
stant at 10 mM while varying the concentration of CT-DNA (10Ϫ4 mol/l). Fit-
ting was completed using an Origin 6.0 spreadsheet, where the values of the
binding constants Kb were calculated. Fluorescence spectra of the competi-
tive binding experiments were obtained by maintaining the EB and CT-DNA
concentrations at 3 mM and 30 mM, respectively, while increasing the concen-
trations of the complexes.
Viscosity experiments were carried out on an Ubbelodhe viscometer, im-
Chart 1. Synthesis Route of the Ligand
∗ To whom correspondence should be addressed. e-mail: bsli@lzu.edu.cn
© 2010 Pharmaceutical Society of Japan

October 2010
1307
mersed in a thermostated water-bath maintained at 25.0Ϯ0.1 °C. Titrations Table 1. Crystal and Experimental Data for Complex 2
were performed for the complexes (1—6 mM), and each complex was intro-
duced into DNA solution (50 mM) present in the viscometer. Flow time was
measured with a digital stopwatch and each sample was measured three
times and an average flow time was calculated.
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions (Å, °)
a
b
C30H34N9O8Pr
789.57
223(2) K
0.71073 Å
Monoclinic
P2/n
The CD spectra of DNA were recorded on a Jasco J-810 spectropolarime-
ter at 25.0Ϯ0.1 °C. Each sample solution was scanned in the range of 200—
350 nm. The CD spectrum, which represented the average of three scans,
was generated from which the buffer background had been subtracted.
X-Ray Crystallography A suitable yellow single crystal of complex 2
with dimensions of 0.40ϫ0.20ϫ0.10 mm3 was mounted in a sealed tube for
data collection performed in a Bruker AXS SMART CCD diffractometer
equipped with a graphite-monochromatic MoKa radiation (lϭ0.71073 Å)
at 223(2) K. Unit cell dimensions were obtained with least-squares refine-
ments. The program SMART was used to collect the intensity data,12)
SAINT for integration of the intensity,12) SADABS for absorption correction
and SHELXTL for structure solution and refinements on F2.13,14) The phenyl
ring was disordered into two positions with occupancy ratio 63 : 37. There
were several residual peaks which were too weak and therefore could not be
characterized. An H atom of N3 was located from different maps and refined
with restraints in bond length and parameters. The H atoms of the solvent
molecules were not located. The whole complex 2 was generated by the two
fold rotation. One half of ethanol was situated at the centre of symmetry.
The H atom of methanol was located from a different map and refined with
restraint in bond length. The C and O atoms of the disordered ethanol were
not refined ansiotropically because the thermal parameters did not behave
well due to the disorder and overlapping. Selected crystallographic data and
structure refinement parameters of complex 2 are summarized in Table 1,
where selected bond distances and angles are summarized in Table 2.
Synthesis of the Ligand and the Complexes The ligand HL was pre-
pared as described previously.15) In short, HL was obtained by the condensa-
tion of 2-acetylpyridine and isonicotinohydrazide in ethanol solution with
the addition of several drops of conc. HCl. IR (KBr) cmϪ1: n(NH) 3189,
n(CϭO) 1671, n(CϭN) 1548. Complex 1 was prepared as follows.
Nd(NO3)3·6H2O (219.1 mg, 0.5 mmol) in ethanol (10 ml) was added to the
solution containing the ligand (240 mg, 1 mmol) in methanol (10 ml). The
mixture was stirred for 12 h to form a precipitate of Nd(III) complex
formed. The precipitate was separated by centrifugation and washed three
times with ethanol and one time with ether, and then dried in vacuo. Anal.
Calcd for C30H36N9O8Nd: C, 45.33; H, 4.56; N, 15.86, Found: C, 45.50; H,
4.67; N, 15.99. IR (KBr) cmϪ1: n(CϭO) 1570, n(CϭN) 1512. The Pr(III)
complex was synthesized in the same way. Slow evaporation of the mixed
methanol/ethanol solution containing Pr(III) complex 2 afforded a yellow
single crystal which is suitable for X-ray diffraction. Anal. Calcd for
C30H36N9O8Pr: C, 45.52; H, 4.58; N, 15.93, Found: C, 45.65; H, 4.69; N,
15.70. IR (KBr) cmϪ1: n(CϭO) 1576, n(CϭN) 1515. HR-MS (ESI) Calcd
for [PrL2]ϩ: 619.0942; [PrL2ϩCH3OH]ϩ: 651.1204; [PrL2ϩCH3COCH3]ϩ:
677.1361; [PrL2ϩ2ϫ(CH3COCH3)]ϩ: 735.1779. Found [PrL2]ϩ: 619.1022;
[PrL2ϩCH3OH]ϩ: 651.1260; [PrL2ϩCH3COCH3]ϩ: 677.1347; [PrL2ϩ2ϫ
(CH3COCH3)]ϩ: 735.1678, LϭC13H11N4OϪ. The complexes are soluble at
room temperature in ethanol, methanol, DMF and dimethyl sulfoxide
(DMSO).
11.6516(8)
11.2406(8)
12.9195(9)
100.034(2)
1666.2(2)
2
1.574
1.525
800
Ϫ15/14, Ϫ14/14, Ϫ12/16
12846
3823/4/222
0.8625, 0.5807
1.088
R1ϭ0.0347, wR2ϭ0.0845
1.395, Ϫ0.776
c
b
Volume (Å3)
Z
Dcalcd (g/cm3)
Absorption coefficient (mmϪ1
F(000)
)
Range of h k l
Reflections collected
Data/restraints/parameters
Max. and min. transmission
Goodness-of-fit on F2
Final R indices [IϾ2s(I)]
Largest diff. peak and hole (ÅϪ3
)
Table 2. Selected Bond Distances and Angles (Å, °) for Complex 2
Pr(1)–O(1)
2.414(2) Pr(1)–O(2)
2.415(2) Pr(1)–N(2)a)
2.558(2) Pr(1)–N(2)
2.558(2) Pr(1)–N(1)a)
2.624(2) Pr(1)–N(1)
2.624(2)
2.687(3)
2.687(3)
2.707(2)
2.707(2)
83.20(7)
70.51(7)
73.3(2)
83.16(8)
134.13(8)
129.18(8)
139.92(8)
123.62(8)
120.07(8)
65.66(7)
59.99(8)
69.58(8)
103.86(7)
169.1(2)
118.89(7)
74.23(8)
69.35(8)
116.84(8)
72.99(8)
144.57(8)
74.72(8)
58.95(8)
Pr(1)–O(1)a)
Pr(1)–O(4)
Pr(1)–O(4)a)
Pr(1)–O(2)a)
O(1)–Pr(1)–O(1)a)
O(1)a)–Pr(1)–O(4)
O(1)a)–Pr(1)–O(4)a)
O(1)–Pr(1)–O(2)a)
O(4)–Pr(1)–O(2)a)
O(1)–Pr(1)–O(2)
O(4)–Pr(1)–O(2)
O(2)a)–Pr(1)–O(2)
O(1)a)–Pr(1)–N(2)a)
O(4)a)–Pr(1)–N(2)a)
O(2)–Pr(1)–N(2)a)
O(1)a)–Pr(1)–N(2)
O(4)a)–Pr(1)–N(2)
O(2)–Pr(1)–N(2)
O(1)–Pr(1)–N(1)a)
O(4)–Pr(1)–N(1)a)
O(2)a)–Pr(1)–N(1)a)
N(2)a)–Pr(1)–N(1)a)
O(1)–Pr(1)–N(1)
O(4)–Pr(1)–N(1)
O(2)a)–Pr(1)–N(1)
N(2)a)–Pr(1)–N(1)
N(1)a)–Pr(1)–N(1)
147.3(2)
O(1)–Pr(1)–O(4)
70.51(7)
83.20(7)
129.19(8)
139.92(8)
83.17(8)
134.13(8)
48.9(2)
59.99(8)
69.58(8)
103.86(7)
123.62(8)
120.07(8)
65.66(7)
72.99(8)
144.57(8)
74.72(8)
58.95(8)
118.89(7)
74.23(8)
69.35(8)
116.84(8)
140.5(2)
O(1)–Pr(1)–O(4)a)
O(4)–Pr(1)–O(4)a)
O(1)a)–Pr(1)–O(2)a)
O(4)a)–Pr(1)–O(2)a)
O(1)a)–Pr(1)–O(2)
O(4)a)–Pr(1)–O(2)
O(1)–Pr(1)–N(2)a)
O(4)–Pr(1)–N(2)a)
O(2)a)–Pr(1)–N(2)a)
O(1)–Pr(1)–N(2)
O(4)–Pr(1)–N(2)
O(2)a)–Pr(1)–N(2)
N(2)a)–Pr(1)–N(2)
O(1)a)–Pr(1)–N(1)a)
O(4)a)–Pr(1)–N(1)a)
O(2)–Pr(1)–N(1)a)
N(2)–Pr(1)–N(1)a)
O(1)a)–Pr(1)–N(1)
O(4)a)–Pr(1)–N(1)
O(2)–Pr(1)–N(1)
N(2)–Pr(1)–N(1)
Superoxide Radical Scavenging Assay Superoxide radicals (OϪ2 ˙) were
generated in vitro using a non-enzymatic system and determined spec-
trophotometrically by a NBT photoreduction method whicn wae a slight
modification of a method described previously.16—19) The amount of O2Ϫ˙ and
suppression ratio for OϪ2 ˙ can be calculated by measuring the absorbance at
560 nm. Light was avoided while preparing solutions of MET, VitB2 and
NBT were prepared avoiding light. The tested compounds were dissolved in
DMF. The assay mixture, in a total volume of 5 ml, contained MET (10 mM),
NBT (46 mM), VitB2 (3.3 mM), the tested compound (5—20 mM) and a phos-
phate buffer (67 mM, pH 7.8). After illuminating with a fluorescent lamp at
30 °C for 10 min, the absorbance of the samples (Ai) was measured at
560 nm. The sample without the tested compound was used as control and
its absorbance was A0. All experimental results were expressed as the
meanϮstandard deviation (S.D.) of triplicate determinations. The suppres-
sion ratio for OϪ2 ˙ was calculated from the following expression:
Symmetry code: a) Ϫxϩ1/2, y, Ϫzϩ3/2.
Hydroxyl Radical Scavenging Assay The hydroxyl radicals (OH˙) in
aqueous media were generated through the Fenton system.20) The solution of
the tested compound was prepared with DMF. The 5 ml assay mixture con-
tained the following reagents: safranin (11.4 mM), ethylenediaminetetraacetic
acid (EDTA)-Fe(II) (40 mM), H2O2 (1.76 mM), the tested compound (2—
15 mM) and a phosphate buffer (67 mM, pH 7.4). The assay mixtures were in-
cubated at 37 °C for 30 min in a water bath and then the absorbance was
measured at 520 nm. All tests were run in triplicate and are expressed as the
meanϮstandard deviation (S.D.).
suppression ratio (%)ϭA0ϪAi/A0ϫ100
(1)
Drug activity was expressed as the 50% inhibitory concentration (IC50).
IC50 values were calculated from regression lines where: x was the tested
compound concentration in mM and y was percent inhibition of the tested
compounds.
The suppression ratio for OH˙ was calculated from the following expres-
sion:
suppression ratio (%)ϭ[(AiϪA0)/(AcϪA0)]ϫ100
(2)

1308
(Where Aiϭthe absorbance in the presence of the tested compound;
Vol. 58, No. 10
A0ϭthe absorbance in the absence of the tested compound; Acϭthe ab-
sorbance in the absence of the tested compound, EDTA-Fe (II), H2O2.)
Results and Discussion
Synthesis and General Properties Complexes 1 and 2
were prepared in high yield from reactions of the Schiff
base ligand HL in the presence of Pr(NO3)3·6H2O or
Nd(NO3)3·6H2O. The stability of the two complexes in an
aqueous solution had been studied by observing the UV–vis
spectrums and estimating the molar conductivities at differ-
ent time intervals for any possible change. The tested com-
plexes were prepared in methanol and for experiments
freshly diluted in phosphate buffer system (at pH 7.4, 7.8).
Next, the UV–vis spectra and molar conductivities were ex-
amined at different time intervals. These investigations re-
vealed that the UV–vis spectra had remained unaltered for
the solutions and there were no obvious changes in the molar
conductivity values of either freshly prepared solutions or
those used throughout the entire experiment (24 h). This indi-
cated that the complexes are quite stable in solution. The
molar conductivity data were in accordance with the non-
electrolyte.
Fig. 1. Molecular Structure of the Pr(III) Complex Showing 30% Proba-
bility Displacement Ellipsoids and the Atom-Numbering Chart
Hydrogen atoms attached to carbon atoms have been omitted for clarity.
IR Spectra The IR spectra of the two complexes are
very similar. The n(CϭO) of the free ligand are at
1671 cmϪ1, for complexes 1 and 2 this peak shifted to 1570
and 1576 cmϪ1, and the n(ligand–complexes) is equal to 101
and 95 cmϪ1, thus indicating that the O atom of CϭO is in-
volved in coordination to the metal ion. The band at
1548 cmϪ1 for the free ligand is assigned to the n(CϭN)
stretch, which shifts to 1512 and 1515 cmϪ1 for complex 1
and 2, indicating that the N atom of CϭN is involved in co-
ordination to the metal ion. The vibrations n(N–H) had dis-
appeared in the spectra of the two complexes indicating that
the ligand is deprotonated. The new bands at 561 and
555 cmϪ1 for complexes 1 and 2 are assigned to n(M–O), and
the weak peaks at 470 and 476 cmϪ1 for complexes 1 and 2
are assigned to n(M–N), respectively.21,22)
Fig. 2. Coordination Polyhedron of Complex 2
Crystal Structure Complex 2 crystallized in the mono-
clinic space group P2/n. The X-ray crystal structure of the 147.3(2)°). The coordination polyhedron around Pr3ϩ is a
[PrIII(L)2(NO3)(CH3OH)2]·CH3CH2OH with atom number- distorted bicapped square antiprism, as shown in Fig. 2.
ing chart is presented as an ORTEP figure in Fig. 1. The
DNA-Binding Studies. Electronic Absorption Titration
crystal structure revealed that the discrete molecules of Electronic absorption spectroscopy is universally employed
[Pr(C5H4N–C(CH3)ϭN–NϭC(O)–C5H4N)2] are held to- to examine the binding mode of DNA with small molecules.
gether by very weak van der Waal’s forces. Under the reac- The ligand has no effect on the absorption of DNA solutions.
tion condition, the ligand molecule tautomerises from the ke- The absorption spectra of complexes 1 and 2 in the absence
tonic [C5H4N–C(CH3)ϭN–NH–C(O)–C5H4N] to enolic form and presence of CT DNA are given in Figs. 3a and b. In the
[C5H4N–C(CH3)ϭN–NϭC(OH)–C5H4N]. These two ligand absence of CT DNA, the UV–Vis absorption spectra of com-
molecules are coordinated to the Pr(III) centre in the plex 1 has three well-solved bands at lmaxϭ206, 263 and
monoanionic form after deprotonation of the enolic oxygen 344 nm, while complex 2 has three bands at lmaxϭ205, 265
atom, being coordinated to Pr(III) via the pyridine and hydra- and 342 nm. With increasing DNA concentration, the absorp-
zone nitrogen atoms as well as via the a-oxyazine oxygen. tion bands around 205 and 265 nm of the two complexes
Their coordination in the a-oxyazine form results in the for- show obvious decreases in molar absorptivity (hypo-
mation of four 5-membered chelate rings around the Pr(III) chromism) as well as marked bathochromism. In order to
centre. The experimental parameters of the single-crystal X- study the binding ability of the compounds with DNA quan-
ray analysis of the Pr complex 2 indicate that the mononu- titatively, the binding constant Kb was determined using the
clear Pr atom is surrounded by ten coordinated donor atoms. following Eq. 3,23)
Six of them belong to two deprotonated ligands, two to the
bidentate nitrate group and two to coordinated methanol mol-
[DNA]/(eaϪef)ϭ[DNA]/(ebϪef)ϩ1/Kb(ebϪef)
(3)
ecules (with all the Pr–O bond lengths rang from 2.414(2) Å where [DNA] is the concentration of DNA in base pairs, ea,
to 2.624(2) Å and the O–Pr–O angles ranging from 48.9(2) to ef, and eb are the apparent extinction coefficient correspon-

October 2010
1309
Fig. 3. Electronic Spectra of the Complex 1 (a) and Complex 2 (b) in the
Presence of 0—20 mM CT-DNA
Arrow shows the absorbance intensity changes upon increasing DNA concentration.
Inset: plots of [DNA]/(eaϪef) vs. [DNA] for the titration of compound.
Fig. 4. Emission Enhancement Spectra of Complexes 1 (a) and 2 (b) in the
Presence of 0—50 mM CT-DNA
Arrow shows the emission intensities changes upon increasing the DNA concentra-
tion.
ding to Aobsd/[M], the extinction coefficient for the free com-
pound and the extinction coefficient for the compound in the ligand is added, it could compete with EB for DNA binding
fully bound form, respectively. In plots of [DNA]/(eaϪef) sites and then a decrease in the fluorescence intensity is ob-
versus [DNA], Kb is given by the ratio of the slope to the in- served. Therefore EB can be used as a common fluorescent
tercept, as shown in Fig. 3.
probe for DNA structure and has been employed in investiga-
The binding constants Kb for complexes 1 and 2 were tions of the mode and the process of metal complex binding
Ϫ1
found to be 4.31ϫ104 M and 1.24ϫ105 MϪ1, respectively. to DNA.28)
The results indicate that the binding strength of complex 2 is
The emission spectra of the DNA–EB system in the pres-
stronger than that of complex 1. The Kb value obtained here ence of increasing amounts of complexes 1 and 2 are shown
is lower than that reported for classical intercalator (for in Fig. 4. The fluorescence intensity of EB at 584 nm showed
ethidium bromide and [Ru(phen)DPPZ] whose binding con- a remarkable decreasing trend with increasing concentrations
stants have been found to be in the order of 106—107 M).24,25) of the complexes, indicating that some EB molecules are re-
Such a change in lmax and the observed binding constant is leased from EB–DNA after an exchange with the complexes
more in keeping with the groove binding with DNA, as ob- which results in the fluorescence quenching of EB. This may
served in the literature.26,27)
be due either to the metal complex competing with EB for
Fluorescence Spectra Further support for the com- the DNA binding sites and thus displacing the EB (whose
plexes binding to DNA was given through the competitive fluorescence is enhanced upon DNA binding) or it could be a
binding experiment. EB is a conjugate planar molecule. Its more direct quenching interaction on the DNA itself. We as-
fluorescence intensity is very weak, but the intensity greatly sume it is the former which implies that complexes 1 and 2
increases when EB is intercalated into DNA. When a second can bind more strongly to DNA than EB.

1310
Vol. 58, No. 10
According to the classical Stern–Volmer Eq. 4,29)
F0/Fϭ1ϩKq[Q]
(4)
where F0 and F represent the emission intensity in the ab-
sence and presence of quencher, respectively, Kq is a linear
Stern–Volmer quenching constant and [Q] is the quencher
concentration. The quenching plots illustrate that the quench-
ing of EB bound to DNA by the complexes is in good agree-
ment with the linear Stern–Volmer equation (Fig. 4, inset). In
the plots of F0/F versus [Q], Kq is given by the ratio of
the slope to the intercept. The Kq values of the two com-
Ϫ1
Ϫ1
plexes are 1.2ϫ104 M and 2.3ϫ104 M for complex 1
and complex 2, respectively. These results suggest that com-
plex 2 is more capable than complex 1 at replacing the strong
DNA intercalator EB, which are in accordance with the
above absorption titration results.
CD Spectroscopy Circular dichroic spectral techniques
may provide useful information on how the conformation of
the CT-DNA chain is influenced by the bound complex. The
CD spectrum of CT-DNA consists of a positive band at
274 nm that can be due to base stacking and a negative band
at 243 nm that can be due to helicity and is also characteristic
of DNA in a right-handed B form.30) The changes in CD sig-
nals of CT-DNA observed on interaction with drugs may
often be assigned to the corresponding changes in CT-DNA
structure.31) Thus simple groove binding and electrostatic in-
teraction of small molecules show less or no perturbation on
the base-stacking and helicity bands, whereas intercalation
enhances the intensities of both the bands stabilizing the
right-handed B conformation of CT-DNA as observed for the
classical intercalator methylene blue.32)
Fig. 5. CD Spectra of CT-DNA (120 mM) in the Absence or Presence of
Complexes 1 and 2 (60 mM)
The CD spectra of DNA taken after incubation of the com-
plexes with CT DNA are shown in Fig. 5. In all cases, the in-
tensities of both the negative and positive bands decrease sig-
nificantly. This suggests that the DNA binding of the com-
plexes induces certain conformational changes, such as the
conversion from a more B-like to a more C-like structure
within the DNA molecule.33—35) These changes are indicative
of a non-intercalative mode of binding of these complexes
and offer support to their groove binding nature.36) The
changing intensity follows the order of 2Ͼ1.
Fig. 6. Effect of Increasing Amounts of Complexes 1 and 2 on the Rela-
tive Viscosity of CT-DNA at 25 °C
[DNA]ϭ50 mM, [complex]ϭ1, 2, 3, 4, 5, 6 mM.
tions, typically cause less pronounced (positive or negative)
Viscosity Studies Hydrodynamic measurements that are or no change in DNA solution viscosity.38) As a validation of
sensitive to the length change (i.e., viscosity and sedimenta- the above verdict, viscosity measurements were carried out.
tion) are regarded as the least ambiguous and the most criti- The effects of the compounds together with EB on the vis-
cal tests of a binding model in solution in the absence of cosity of DNA at 25.0Ϯ0.1 °C are shown in Fig. 6. The re-
crystallographic structural data. Viscosity studies were car- sults reveal that complexes 1 and 2 effect relatively inappar-
ried out as a means for further clarifying the binding of com- ent increase in DNA viscosity, consistent with the DNA
plex 1 with DNA, viscosity studies are carried out. Data are groove binding suggested above, which is also known to en-
presented as (h/h0)1/3 versus 1/R, where Rϭ[DNA]/[com- hance DNA viscosity.39) The increased degree of viscosity,
pound]; and h and h0 are the relative viscosities of DNA in which may depend on its affinity to DNA follows the order of
the presence and absence of compound, respectively. The rel- 2Ͼ1, which is consistent with our foregoing hypothesis.
ative viscosity values are calculated from the flow time of
Scavenger Measurements of O؊2 ˙ Figure 7a depicts the
DNA-containing solution (t) and the flow time of buffer inhibitory effect of the compounds on OϪ2 ˙. The average sup-
alone (t0), using the expression (5),37)
pression ratio for OϪ2 ˙ increases with the increase of the com-
plex concentration in the range of tested concentrations. The
average suppression ratio of the ligand metal salts can not be
hϭ(tϪt0)/t0
(5)
Intercalating agents are expected to elongate the double read. The Pr(III) complex 2 (IC50ϭ4.71Ϯ0.22 mM) is the
helix to accommodate the compounds in between the base most effective inhibitor in these complexes, whereas the
leading to an increase in the viscosity of DNA. In contrast, Nd(III) complex 1 is the poorest one. The improvement of
complexes that bind exclusively in the DNA grooves by par- the complexes of the antioxidative properties of the Schiff
tial and/or non-classical intercalation, under the same condi- base ligand may be explained by the redox behavior of the

October 2010
1311
plexes we studied in this paper are potential drugs for some
diseases and therefore should be further researched.
Conclusion
In this paper, we have synthesized and characterized HL
and its Pr(III) and Nd(III) complexes. In addition, the DNA-
binding properties were investigated by electronic absorp-
tion, fluorescence, CD spectroscopy, and viscosity measure-
ment. The results support the fact that complexes 1 and 2 can
bind to CT-DNA by the mode of groove binding and the
binding affinity of complex 2 is higher than that of complex
1. Furthermore, the two complexes have active scavenging
effects on OϪ2 ˙ and OH˙. According to the IC50 values ob-
tained, they are found to exert superior activity in comparison
to the ligand HL. These findings clearly indicate that lan-
thanide-based complexes have many potential practical appli-
cations, such as the development of nucleic acid molecular
probes and new therapeutic reagents for diseases.
Acknowledgements This work was supported by the State Key Labora-
tory of Applied Organic Chemistry, Lanzhou University.
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