DNA binding and antibacterial properties of ternary lanthanide complexes with salicylic acid and phenanthroline

Article abstract of DOI:10.1002/aoc.3104

Twelve ternary lanthanide complexes RE(sal)₃phen (RE ³⁺ = La³⁺, Pr³⁺, Nd³⁺, Sm ³⁺, Eu³⁺, Gd³⁺, Tb³⁺, Dy ³⁺, Ho³⁺, Tm³⁺, Yb<

Full text of DOI:10.1002/aoc.3104

Full Paper
Received: 18 August 2013
Revised: 11 October 2013
Accepted: 29 October 2013
Published online in Wiley Online Library: 21 January 2014
(wileyonlinelibrary.com) DOI 10.1002/aoc.3104
DNA binding and antibacterial properties of
ternary lanthanide complexes with salicylic
acid and phenanthroline
Bin Yue, Hui-Juan Sun, Ying-Nan Chen, Kai Kong, Hai-Bin Chu and
Yong-Liang Zhao*
Twelve ternary lanthanide complexes RE(sal)3phen (RE3+ = La3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Tm3+, Yb3+, Lu3+
sal = salicylic acid, phen = phenanthroline) were prepared. Interactions between the complexes and calf thymus DNA (ct-DNA)
were investigated using UV–visible spectrophotometry, fluorescence quench experiment and viscosity measurement.
Hypochromicity and red shift of the absorption spectra of complexes were observed in the presence of DNA. The enhanced
emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of lanthanide complexes,
which indicated that the lanthanide complexes displaced EB from its binding sites in DNA. Based on the systematic research
of the binding constant (Kb) and the fluorescence quenching constant (Kq) of the 12 complexes, we found that the complexes
with smaller lanthanide ion radius had stronger binding abilities with DNA. Viscosity measurement showed that the relative
viscosity of the DNA solution was enhanced with increasing the amounts of the complexes. All these results suggested that
the complexes could bind to DNA and the major binding mode was intercalative binding. Moreover, all these complexes
exhibited excellent antibacterial abilities against Escherichia coli. Also, the antibacterial activities of complexes with heavy rare
earth were higher than those of complexes with light rare earth. Copyright © 2014 John Wiley & Sons, Ltd.
,
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Keywords: lanthanide complexes; ct-DNA; fluorescence quenching; binding mode; antibacterial activity
and lower toxicity than that of isolated Hsal.[15] The
antibacterial activities of the ternary lanthanide complexes
Introduction
Owing to the potential applications of metal complexes as
anticancer drugs with other biological functions,[1–5] the interac-
tions of metal complexes with nucleic acids have aroused
tremendous interests in recent years.[6,7] Small molecules interact
with DNA primarily by three binding modes: electrostatic
binding, groove binding and intercalative binding (Fig. 1),[8–10]
which can be tested by spectroscopy, electrochemical analysis,
viscosity measurement and gel electrophoresis. Electrostatic
binding is the interaction between cationic species and the
negatively charged DNA phosphate backbone. It occurs along
the external DNA double helix and does not possess selectivity.
In groove binding, interactions with the two grooves of the DNA
double helix generally involve direct hydrogen bonding or van
der Waals interactions with the nucleic acid bases in the deep
major groove or the wide shallow minor groove of the DNA helix.
Intercalative binding is stronger than the other two binding modes
because the surface of intercalative molecule is sandwiched
between the aromatic, heterocyclic base pairs of DNA.[11]
Intercalative binding leads to the melting chain and elongation
of the DNA double helix, which is an important feature of
intercalative binding. Also, the hypochromicity and red shift in
the absorption spectra of complexes indicate the intercalative
interaction between complexes and DNA bases.[12–14]
with Hsal and the second ligand (such as 1,10-phenanthroline
(phen), 8-hydroxyquinoline) are expected to be better than the
lanthanide chloride or ligands.[16] Lanthanide ions with unique
biological activity are becoming increasingly attractive within the
chemical and pharmaceutical research field. The DNA binding of
some lanthanides has been explored.[17] Synthesis and characteriza-
tion of lanthanide complexes with Hsal and phen have been studied
for their optical properties,[18] but little work has been done on their
DNA binding properties. Furthermore, the relation between types of
lanthanide complexes and their binding ability with DNA is still
unclear.[19]
Here, 12 lanthanide complexes RE(sal)3phen (RE3+ =La3+, Pr3+,
Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Tm3+, Yb3+, Lu3+) were
synthesized, and we used UV–visible spectra, fluorescence
spectra and viscosity measurements to explore the interaction
between RE(sal)3phen complexes and ct-DNA. Our results
provided evidence that the interaction mode between the
complexes and DNA are intercalation binding.[20] Furthermore,
the binding constant (Kb) and fluorescence quenching constant
*
Correspondence to: Yong-Liang Zhao, College of Chemistry and Chemical Engineering,
Inner Mongolia University, Huhhot, 010021, China. Email: hxzhaoyl@163.com
Salicylic acid (Hsal) can easily form a stable complex with the
metal ion. A large number of studies confirmed that metal
complexes with salicylic acid have higher biological activity
College of Chemistry and Chemical Engineering, Inner Mongolia University,
Huhhot 010021, China
Appl. Organometal. Chem. 2014, 28, 162–168
Copyright © 2014 John Wiley & Sons, Ltd.

DNA binding and antibacterial properties of ternary lanthanide
Figure 1. Illustration of electrostatic groove and intercalative binding of small molecules to the DNA double helix.
(Kq) were calculated, and a negative correlation between Kb or
Kq and the lanthanide ion radius was found. Antibacterial
activities of the 12 complexes were also investigated and we
found that the antibacterial activities of the heavy rare earth
complexes were better than those of light rare earth com-
plexes. These findings may be helpful in understanding the
mechanism of the interactions between DNA and lanthanide
complexes. Also, they should be useful for the development
of potential probes for DNA structure and new therapeutic
reagents for tumours and other diseases.
solution. The preparations of TbCl3 and PrCl3 ethanol solution were
similar to other RECl3 preparations except that H2O2 was added.
Synthesis of RE(sal)3phen
Hsal (1.5 mmol) was dissolved in 5 ml ethanol, and aqueous ammo-
nia was added to adjust the pH level to 7.0. An RECl3 (0.5 mmol)
solution was then added dropwise to the solution under stirring at
60°C. After reaction for 1 h, 0.50 mmol phen dissolved in 5 ml
ethanol was added dropwise to this mixture under stirring for a
further 2 h at 60°C. The precipitate was filtered out, washed twice
with ethanol and dried at 50°C for several hours to obtain the
product of RE(sal)3phen.
Experimental
[La(sal)3phen] Anal. Calcd (%) for LaC33H23O9N2: C 54.26, H 3.17, N
3.84; Found: C 54.48, H 3.01, N 4.21. ESI-MS (DMF), m/z: 632.03,
739.07, 773.21, 845.89.
Materials and Instrumentation
Calf thymus DNA (ct-DNA, Sigma Chemical Co., USA), tris
(hydroxymethyl) aminomethane hydrochloride (Tris–HCl)
and ethidium bromide (EB) were used as received. Purity of
salicylic acid and 1,10-phenanthroline (Sinopharm Chemical
Reagent Co., China) was 99.5%. Purity of rare earth oxides
were 99.99%. Other reactants and solvents were of analytical
grade.
Elemental analysis (C, N and H) were performed with a Vario EL
cube elemental analyser. The contents of rare earth elements
were determined by ethylenediaminetetraacetic acid (EDTA)
titration. Molar conductivity was measured on a DDS-11A con-
ductivity meter with a DJS-I platinum black electrode at 25°C
using N,N-dimethylformamide (DMF) as solvent, and the concen-
trations of complexes were 1.0 × 10-3 mol lꢀ1. IR spectra were
obtained on a Nicolet Nexus 670 FT-IR spectrophotometer in
the range 4000–500 cmꢀ1. Electrospray ionization (ESI) mass
spectra were recorded on an LCQ Advantage MAX mass spectro-
photometer with DMF as the proton source. Absorption spectra
were recorded on a TU1901 spectrophotometer, using DMF as
solvent, and the concentration of the complexes was 1.0 × 10ꢀ5
mol lꢀ1. Fluorescence spectra were carried out using a Shimadzu
RF-5301PC spectrophotometer with excitation and emission slits
of 10.0 nm at room temperature. Viscosity experiments were
conducted on a Ubbelohde Viscomter at 25.0 0.1°C.
[Pr(sal)3phen] Anal. Calcd (%) for PrC33H23O9N2: C 54.11, H 3.17,
N 3.82; Found: C 54.67, H 3.28, N 3.61. ESI-MS (DMF), m/z: 633.87,
740.83, 775.09, 847.61.
[Nd(sal)3phen] Anal. Calcd (%) for NdC33H23O9N2: C 53.87, H
3.15, N 3.81; Found: C 54.21, H 3.36, N 3.78. ESI-MS (DMF), m/z:
636.18, 744.03, 778.32, 848.70.
[Sm(sal)3phen] Anal. Calcd (%) for SmC33H23O9N2: C 53.42, H
3.12, N 3.78; Found: C 52.95, H 3.07, N 4.30. ESI-MS (DMF), m/z:
644.93, 751.82, 786.00, 858.37.
[Eu(sal)3phen] Anal. Calcd (%) for EuC33H23O9N2: C 53.31, H
3.12, N 3.77; Found: C 53.41, H 3.02, N 4.12. ESI-MS (DMF), m/z:
645.94, 752.84, 787.03, 859.60.
[Gd(sal)3phen] Anal. Calcd (%) for GdC33H23O9N2: C 52.93, H
3.10, N 3.74; Found: C 52.58, H 3.39, N 4.27. ESI-MS (DMF), m/z:
650.93, 757.80, 791.98.
[Tb(sal)3phen] Anal. Calcd (%) for TbC33H23O9N2: C 52.81, H
3.09, N 3.73; Found: C 52.81, H 3.09, N 3.73. ESI-MS (DMF), m/z:
651.94, 758.83, 793.03.
[Dy(sal)3phen] Anal. Calcd (%) for DyC33H23O9N2: C 52.56, H
3.07, N 3.72; Found: C 52.14, H 3.22, N 4.12. ESI-MS (DMF), m/z:
656.99, 762.90, 798.08.
[Ho(sal)3phen] Anal. Calcd (%) for HoC33H23O9N2: C 52.40, H
3.06, N 3.70; Found: C 52.66, H 2.94, N 4.16. ESI-MS (DMF), m/z:
657.96, 764.82, 799.08.
[Tm(sal)3phen] Anal. Calcd (%) for TmC33H23O9N2: C 52.12, H
3.05, N 3.68; Found: C 52.26, H 2.94, N 4.09. ESI-MS (DMF), m/z:
661.96, 768.79, 803.09.
[Yb(sal)3phen] Anal. Calcd (%) for YbC33H23O9N2: C 51.84, H
3.03, N 3.66; Found: C 51.69, H 2.81, N 4.03. ESI-MS (DMF), m/z:
773.93, 808.11.
[Lu(sal)3phen] Anal. Calcd (%) for LuC33H23O9N2: C 51.71, H
3.02, N 3.65; Found: C 51.27, H 2.84, N 4.04. ESI-MS (DMF), m/z:
774.81, 809.10.
Synthesis of RE(sal)3phen
Preparation of RECl3
RE2O3 was dissolved in concentrated HCl.[21] The mixture was
heated until a crystallized film appeared above the solution, then
cooled to room temperature. A powdered solid appeared, which
was then dissolved in anhydrous ethanol to obtain 0.1 mol lꢀ1 RECl3
(RE3+ =La3+, Nd3+, Sm3+, Eu3+, Gd3+, Dy3+, Ho3+, Tm3+, Yb3+) ethanol
Appl. Organometal. Chem. 2014, 28, 162–168
Copyright © 2014 John Wiley & Sons, Ltd.
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B. Yue et al.
Preparation of ct-DNA Solution and EB Solution
of complexes predicted by elemental analyses, IR spectra and mass
spectral data.
Ct-DNA was used without further purification, and its stock
solution was prepared by dissolving appropriate solid DNA into
doubly distilled water and storing at 4°C. The concentration of
DNA in stock solution was determined by UV absorption at 260
nm using a molar absorption coefficient ε260 = 6600 l molꢀ1
IR Spectra
The IR spectra of ligands phen, Hsal and all the obtained com-
plexes were determined in the range 4000–500 cmꢀ1, some of
which are given in Fig. S2. The spectra of the 12 lanthanide com-
plexes are similar, which indicates that the complexes have a sim-
ilar structure. As shown in Fig. S2(a), the bands at 3000–3500,
1659 and 1483 cmꢀ1 could be attributed to the ν(O-H)(COOH)
stretching vibration, ν(C=O)(COOH) vibration and δO-H (phenol)
rocking vibration of Hsal.[27,28] After the formation of the com-
plexes, those absorption peaks all disappeared except the peak
of δO-H (phenol). Meanwhile, two new peaks appeared at 1596
and 1389 cmꢀ1, which could be assigned to the antisymmetric
and symmetric stretching vibrations of the carboxylate group,
respectively. These changes indicate that the carboxylate ions
of Hsal coordinate with the lanthanide ions in the complexes.
Evidence of chemical bond formation between Tb3+ ions and
the nitrogen atoms was confirmed as the vibrational band at
1640, 1587, 852 cmꢀ1, assigned to C=C, C=N and CH out-of-plane
deformation[29] in the phenanthroline spectrum, shifted to 1623,
1552 and 841 cmꢀ1 in the RE(sal)3phen complexes, respectively. This
indicates that nitrogen atoms of phenanthroline are coordinating
RE3+ in complexes.[30]
The structures of the lanthanide complexes with salicylic acid
have been discussed by several groups.[31,32] Burns and Baldwin
have reported the crystal structure of samarium salicylate, which
indicates that both the carboxylic and the phenolic oxygen
atoms participate in the coordination with central lanthanide
ions. In our work, δO-H (phenol) of Hsal shows almost no change
after the formation of RE(sal)3phen, which suggests that only
the carboxylic participated in the coordination with RE3+. Gen-
erally, the coordination number of RE ions in organic complexes
is reported to be between 6 and 9. The structure of Tb(sal)3phen
has been predicted by Kaur.[33] On the basis of these previous
reports and our above analysis (elemental analyses, IR spectra
and ESI mass spectral data), the most suitable molecular
structure of the complex is shown in Fig. 2. The coordinate
number of the central metal ion was 8. The metal ion bonded
to a chelating bidentate phen and three bidentate carboxyl
groups from Hsal ligands.
cm
of the absorbance at 260–280 nm. The solution gave a ratio of
260/A280 of 1.8–2.0, indicating that DNA was sufficiently free
.
ꢀ1 [22] Purity of the DNA was checked by monitoring the ratio
A
from protein.[23] Stock solutions of the complexes (1.0 × 10ꢀ3
mol lꢀ1) were prepared by dissolving in DMF and diluting to
1.0 × 10ꢀ5 mol lꢀ1 with double-distilled water. EB stock solution
was prepared by dissolving it in double-distilled water according
to a ratio of CDNA/CEB = 20.
Test of Antibacterial Activity
Antibacterial activity of the ligands and the complexes against
Escherichia coli was studied using the filter paper scraps diffusion
method.[24,25] Small sterile filter papers of 6 mm diameter were
prepared for the purpose of making bacteriostatic slices. The E. coli
was spread on sterile Luria–Bertani (LB) agar plates using a sterile
cotton swab. The 6 mm diameter sterile filter papers were coated
with a 20 μl solution of Hsal, phen and the 12 complexes in DMF,
respectively, and were then carefully placed at the centre of the
lawn without touching the other parts and incubated at 37°C for
16–18 h. The inhibition zone around the film was then photo-
graphed and measured with a vernier caliper to evaluate the
antibacterial performance. All experiments were carried out in paral-
lel three times, and the average diameter values were calculated.
Results and Discussion
Composition Analysis and Molar Conductivity
The results of elemental analyses and rare earth EDTA titration indi-
cated that the compositions of the complexes were RE(sal)3phen
(RE3+ =La3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Tb3+, Dy3+, Ho3+, Tm3+,
Yb3+, Lu3+). All the complexes were stable in air and easily dissolved
in DMF and DMSO but difficult to dissolve in water, ethanol and
acetone. The molar conductance values of lanthanide complexes
in DMF are in the range 2.0–5.7 (S cm2 molꢀ1). These low values
indicate that only a small fraction of lanthanide complexes ionize
in DMF and these complexes are non-electrolytes.[26,27]
Fluorescence Spectral Studies of DNA Binding
Thermogravimetry–Differential Scanning Calorimetry (TG-DSC)
The binding of the complexes to ct-DNA has been studied by the
fluorescence spectral method. EB is one of the most sensitive
fluorescence probes; it can intercalate into DNA and the
Thermal decomposition of the complexes was studied using the
TG technique, which can be regarded as an adjunct for speculat-
ing the composition of complexes. It can be found that the
thermal behaviour of the 12 complexes is quite similar. The
experiment was performed under air atmosphere with a heating
rate of 10°C minꢀ1. The TG-DSC curves of Tm(sal)3phen are
shown (supporting information, Fig. S1) to be in the range
30–700°C. As shown in Fig. S1, the complex begins to
decompose at about 180°C with obvious weight loss. The
weight loss of the complex Tm(sal)3phen around 180–320°C
was 23.05%, which could be assigned to the complex with loss
of one phen (the theoretical value is 23.70%). The weight loss
of 41.48% at 320–640°C is consistent with the complex that
loses three salꢀ with two COOꢀ subtracted (the theoretical
value is 42.51%). This result is in accordance with the composition
OH
O
O
N
OH
O
O
O
O
RE
HO
N
Figure 2. Predicted structure of the RE(sal)3phen complex.
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Appl. Organometal. Chem. 2014, 28, 162–168

DNA binding and antibacterial properties of ternary lanthanide
fluorescence of EB can be effectively increased. The metal
complexes may replace EB in the binding to DNA, thus decreas-
ing the emission intensity of EB. A quantitative solution (1.0 ml
1.0 × 10ꢀ5 mol lꢀ1 DNA and 1.0 ml 5.0 × 10ꢀ7 mol lꢀ1 EB) was
titrated with increasing amounts of lanthanide complex
(1.0 × 10ꢀ3 mol lꢀ1, 10 μl each time). As shown in Fig. 3, the
fluorescence intensity of EB-DNA was quenched steadily with
increasing concentration of Tb(sal)3phen. We selected 537 nm as
the excitation wavelength and recorded the emission spectra from
570 to 630 nm. A remarkable fluorescence decrease of the
EB-DNA system was observed around the fluorescence peak at
592 nm. This indicates that the complex displaces EB from its bind-
ing sites, which leads to a large decrease in the emission intensity of
the EB-DNA system. The fluorescence quenching of EB-DNA by
complexes indicates the strong binding between DNA and the com-
plex. Also, the interaction mode between the complexes and DNA is
probably intercalation binding, which will be further validated by
UV–visible absorption spectra and viscosity studies.
Table 1. Fluorescence quenching constant of
complex interaction with DNA
Complex
Kq (molꢀ1 l)
La(sal)3phen
Pr(sal)3phen
Nd(sal)3phen
Sm(sal)3phen
Eu(sal)3phen
Gd(sal)3phen
Tb(sal)3phen
Dy(sal)3phen
Ho(sal)3phen
Tm(sal)3phen
Yb(sal)3phen
Lu(sal)3phen
(4.69 0.01) × 104
(6.13 0.01) × 104
(6.26 0.01) × 104
(6.46 0.01) × 104
(6.57 0.01) × 104
(6.87 0.01) × 104
(7.00 0.01) × 104
(7.08 0.01) × 104
(7.73 0.02) × 104
(9.01 0.01) × 104
(9.11 0.01) × 104
(1.14 0.01) × 105
The quenching plots illustrate that the fluorescence quenching
of EB-DNA by the complex is in good agreement with the linear
Stern–Volmer equation[34]
:
F0
F
¼ 1 þ Kq½Qꢁ
(1)
where F0 is the emission intensity in the absence of quencher, F is
the emission intensity in the presence of quencher, [Q] is the
quencher concentration, and Kq is the quenching constant.
Taking Tb(sal)3phen for example, Kq is obtained from the slope
of the F0/F versus [DNA] linear plot (Fig. 3, inset) and is found to
be 7.00 × 104 molꢀ1 l, which indicates a strong interaction of
lanthanide complex with DNA. Plots of F0/F versus [Q] should
appear to be linear. The illustration shows the Stern–Volmer
quenching curve and data of the fluorescence quenching
constant are presented in Table 1.
The fluorescence quenching of EB-DNA by complexes indi-
cates that the complexes have a strong interaction with DNA.
From the sequence of quenching constants of the complexes
binding to EB-DNA, we can see that Kq is related to lanthanide
ion radius, and the sequence of Kq is Lu(sal)3phen > Yb(sal)3phen
Tm(sal)3phen > Ho(sal)3phen > Dy(sal)3phen > Tb(sal)3phen
Figure 4. Absorption spectra of Tb(sal)3phen (2.00 ml 1.0 × 10ꢀ5 mol Lꢀ1
in the presence of increasing amounts of ct-DNA ([DNA] = 0–0.476 μM,
0.01 mol lꢀ1 Tris–HCl, pH 7.2) , 20 μl each time. Arrow a → f shows
the absorbance changes upon increasing ct-DNA concentration.
)
Figure 3. Effect of Tb(sal)3phen (1.0 × 10ꢀ3 mol Lꢀ1, 10 μl each time) on
the fluorescence spectra of EB-DNA system (1.0 ml 1.0 × 10ꢀ5 mol Lꢀ1
DNA and 1.0 ml 5.0 × 10ꢀ7 mol Lꢀ1 EB). λex = 537 nm, λmax–em = 592 nm.
Arrow a → f shows the intensity changes upon increasing concentration
of the complex. The inset plot shows the relative fluorescent intensity
curve of Tb(sal)3phen interaction with EB-DNA.
Figure 5. Pattern of Tb(sal)3phen of [DNA]/(εf ꢀ εa) linear relation to [DNA].
Appl. Organometal. Chem. 2014, 28, 162–168
Copyright © 2014 John Wiley & Sons, Ltd.
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B. Yue et al.
Table 2. Binding constant of complex interaction with DNA
Complex
Pr(sal)3phen
Nd(sal)3phen
Gd(sal)3phen
Tb(sal)3phen
Yb(sal)3phen
(2.63 0.17) × 104
Kb(molꢀ1 l)
(6.86 0.09) × 103
(1.72 0.03) × 104
(1.79 0.01) × 104
(2.19 0.08) × 104
Gd(sal)3phen > Eu(sal)3phen > Sm(sal)3phen > Pr(sal)3phen > Nd
(sal)3phen > La(sal)3phen. The data suggest that the binding
ability of Lu(sal)3phen is strongest whose lanthanide ion radius
is the smallest, and the binding ability of La(sal)3phen is weakest
whose lanthanide ion radius is the largest. This may be the result
of the effect of the trivalent lanthanide ion radius. The Lu3+ has
the smallest radius, largest charge density and strongest electro-
static interaction with DNA in these complexes, so the capacity of
phen in Lu(sal)3phen inserting into the base pairs of DNA is the
strongest and the fluorescence quenching constant is the
largest.[35] Conversely, the capacity of La(sal)3phen inserting into
the base pairs is weakest and the fluorescence quenching
constant is smallest. These data indicate that the Kq of the 12
complexes is inversely related to the lanthanide ion radius.
DNA, and εa, εf and εb correspond to the apparent extinction coeffi-
cient Aobsd/[complex], the extinction coefficient for free complex
and for the complex in the fully bound form, respectively. The bind-
ing constant Kb for Tb(sal)3phen has been determined from the plot
of [DNA]/(εa ꢀ εf ) versus [DNA] (Fig. 5). The binding constants of
some complexes are obtained in a similar way and shown in Table 2.
As shown in Table 2, the Kb values were calculated to be from
6.86 × 103 to 2.63 × 104 ꢀ1, suggesting a relatively strong bind-
M
ing of RE(sal)3phen complexes with DNA. The Kb values obtained
for the complexes are close to those of the so-called DNA-
intercalative Ru(II) complexes (1.1 × 104–4.8 × 104 ꢀ1),[44,45] but
M
they are much lower than that of the classical intercalator,
ethidium bromide(107 ꢀ1). Also, the sequence of binding ability
M
of the complexes to DNA is Yb(sal)3phen > Tb(sal)3phen > Gd(sal)
3phen > Nd(sal)3phen > Pr(sal)3phen. This suggests that Kb is also
related to the lanthanide ion radius, which is consistent with the
fluorescence quenching constant Kq. It may be concluded that
the complexes bind to DNA mainly in intercalation mode.
UV–Visible Absorption Spectral Studies of DNA Binding
The UV–visible absorption spectra of ligands phen, Hsal as well as all
the complexes were determined in the range 200–400 nm. The UV–
visible absorption spectra of the 12 lanthanide complexes are simi-
lar, and only that of Tb(sal)3phen is given. As shown in Fig. S3, the
peak of phen is 265 nm in the lanthanide complex, which is similar
to that of isolated phen (264 nm). The peak of salicylic acid at 297
nm shifted to 289 nm in the complex, which indicates that the lan-
thanide ion coordinated with salicylic acid.
The spectra of Tb(sal)3phen with increasing concentration of DNA
are shown in Fig. 4. It can be seen that the absorption spectra
showed obvious hypochromicity at an absorbance peak of 265 nm,
together with a red shift of about 2.5 nm with increasing DNA
concentration. The hypochromicity and red shift in the absorption
spectra of complexes indicated the intercalative binding of com-
plexes to DNA bases. This can be explained as follows. There is a
strong interaction in the molecular stack between the DNA base
pairs and the planar aromatic chromophore of the complexes.[36–38]
An orbit coupling occurs between the empty π*-orbital of the phen
couples and the π*-orbital of the DNA base pairs, which causes an
energy decrease and a decrease of the π–π* transition energy of
the phen. Therefore, the absorption peak of the complexes around
265 nm exhibits a red shift. At the same time, the empty π*-orbital
is partially filled with electrons, reducing the π–π* transition probabil-
ity, which leads to hypochromism of the complexes.
Figure 6. Influence on DNA viscosity at different concentrations of Gd(sal)
3phen. η0 and η are the relative viscosity of DNA solution before and after
the addition of complexes, respectively. CDNA = 1.00 × 10ꢀ4 mol lꢀ1
.
Table 3. Diameters of antibacterial rings of complexes with different
concentrations on E. coli mmꢀ1
In order to further illustrate the binding strength of the complexes,
the intrinsic binding constant Kb was determined. Kb is a tool for
investigating the magnitude of the binding strength of a compound
bound to DNA and represents the binding constant per DNA base
pair. It can be obtained by monitoring the changes in absorbance
at the corresponding λmax with increasing concentration of DNA
and given by the ratio of slope to the y intercept in plots of [DNA]/(εa
Compound
Concentration (mol lꢀ1
)
0.004
<10
0.008
0.012
DMF
<10
<10
<10
17.8
18.5
18.7
19.1
19.7
20.0
<10
<10
13.8
Hsal
<10
<10
Phen
εf) versus [DNA], according to the following equation[39–43]
:
Pr(sal)3phen
Nd(sal)3phen
Gd(sal)3phen
Tb(sal)3phen
Tm(sal)3phen
Yb(sal)3phen
16.8
23.5
23.2
23.7
24.5
24.7
24.8
17.5
17.3
17.8
18.4
18.5
½DNAꢁ ½DNAꢁ
1
¼
þ
(2)
εa ꢀ εf εb ꢀ εf Kbðεb ꢀ εfÞ
The binding constant Kb of the complex to ct-DNA is determined
from equation (2), where [DNA] represents the concentration of
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Appl. Organometal. Chem. 2014, 28, 162–168

DNA binding and antibacterial properties of ternary lanthanide
Figure 7. Antibacterial rings of (a) 0.012 mol lꢀ1 DMF, (b) 0.012 mol lꢀ1 Hsal, (c) 0.012 mol lꢀ1 phen and Yb(sal)3phen at different concentrations on E.
coli: (d) 0.004 mol lꢀ1, (e) 0.008 mol lꢀ1, (f) 0.012 mol lꢀ1
.
Viscosity Measurements
than those of the light rare earth complexes. The enhanced
antibacterial activities of the complexes presumably result from
the increase in liposolubility of the lanthanide complexes by
the chelate effect.[23,50–52]
Viscosity studies were carried out in order to further investigate
whether the interactions between DNA and lanthanide complex
involve intercalation. The viscosity measurements of DNA are
regarded as the least ambiguous and the most critical test of a
binding model in solution in the absence of crystallographic
structural data.[46,47] A classical intercalation model is known to
cause a significant increase in the viscosity of a DNA solution
due to an increase in lengthening in the DNA helix. Furthermore,
non-classical intercalation (partial intercalation) of the complex
would reduce the DNA viscosity, and the non-intercalation bind-
ing (such as electrostatic binding and groove binding) causes no
obvious change of DNA viscosity.[48,49] As shown in Fig. 6, the
relative viscosity of the DNA solution was enhanced with increase
in Gd(sal)3phen. Such behaviour further suggests that the major in-
teraction mode between Gd(sal)3phen and DNA should be
intercalation binding. This may be caused by complexes that
exhibit a strong tendency to interact with DNA. The increasing vis-
cosity profile indicates that the complexes are effective intercalators.
Conclusions
Twelve ternary lanthanide complexes RE(sal)3phen were pre-
pared. Fluorescence quench, hypochromism studies and viscosity
measurements suggest that the complexes exhibit a strong inter-
action towards DNA via intercalative binding. In particular, both
the quenching constant Kq and the binding constant Kb have a
negative correlation with the lanthanide ion radius. In addition,
all these complexes exhibit excellent antibacterial ability against
E. coli, and the antibacterial activities of the heavy rare earth com-
plexes are stronger than those of the light rare earth complexes.
The systematic study of the binding mode of the 12 RE(sal)3phen
complexes may provide useful information about the mechanism
of anticancer drugs binding to DNA, and thus will be beneficial to
the design of new lanthanide DNA binding agents.
Antibacterial Activities of Ligands and Complexes
Acknowledgements
The ligands and complexes were evaluated by the filter paper
scraps diffusion method against E. coli. A large diameter of the
inhibition zone indicates stronger antibacterial activity of a
complex. As shown in Table 3 and Fig. 7, all the complexes
exhibited stronger inhibitory effect on E. coli than the ligands.
The antibacterial activities of the lanthanide complexes increased
with increasing concentration of the complexes in the range
0.004–0.012 mol lꢀ1. A complex is usually considered to have a
strong inhibitory effect when the inhibition zone diameter is
more than 20 mm and, as we can see from Table 3, all these
complexes exhibit excellent antibacterial ability against E. coli
when the concentration was 0.012 mol lꢀ1. Furthermore, the
antibacterial activities of heavy rare earth complexes are stronger
The research work is supported by the National Natural Science
Foundation of China (21161013), Natural Science Foundation of
Inner Mongolia (2011MS0202), the Opening Foundation for
Significant Fundamental Research of Inner Mongolia (2010KF03)
and a fund from the Inner Mongolia University ‘211 project’
innovative training project.
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