De novo design, synthesis and spectroscopic characterization of chiral benzimidazole-derived amino acid Zn(II) complexes: Development of tryptophan-derived specific hydrolytic DNA artificial nuclease agent

Article abstract of DOI:10.1016/j.saa.2011.09.006

Novel ternary dizinc(II) complexes 1-3, derived from 1,2-bis(1H- benzimidazol-2-yl)ethane-1,2-diol and l-form of amino acids (viz., tryptophan, leucine and valine) were synthesized and characterized by spectroscopic (IR, ¹H NMR, UV-vis, ESI-MS) and other analytical methods. To evaluate the biological preference of chiral drugs for inherently chiral target DNA, interaction of 1-3 with calf thymus DNA in Tris-HCl buffer was studied by various biophysical techniques which reveal that all these complexes bind to CT DNA non-covalently via electrostatic interaction. The higher K_b value of l-tryptophan complex 1 suggested greater DNA binding propensity. Further, to evaluate the mode of action at the molecular level, interaction studies of complexes 1 and 2 with nucleotides (5′-GMP and 5′-TMP) were carried out by UV-vis titrations, ¹H and ³¹P NMR which implicates the preferential selectivity of these complexes to N3 of thymine rather than N7 of guanine. Furthermore, complex 1 exhibits efficient DNA cleavage with supercoiled pBR322. The complex 1 cleaves DNA efficiently involving hydrolytic cleavage pathway. Such chiral synthetic hydrolytic nucleases with asymmetric centers are gaining considerable attention owing to their importance in biotechnology and drug design, in particular to cleave DNA with sequence selectivity different from that of the natural enzymes.

Full text of DOI:10.1016/j.saa.2011.09.006

Spectrochimica Acta Part A 85 (2012) 53–60
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
De novo design, synthesis and spectroscopic characterization of chiral
benzimidazole-derived amino acid Zn(II) complexes: Development of
tryptophan-derived specific hydrolytic DNA artificial nuclease agent
Shazia Parveen, Farukh Arjmand∗
Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 May 2011
Received in revised form 18 August 2011
Accepted 4 September 2011
Novel ternary dizinc(II) complexes 1–3, derived from 1,2-bis(1H-benzimidazol-2-yl)ethane-1,2-diol and
l-form of amino acids (viz., tryptophan, leucine and valine) were synthesized and characterized by
spectroscopic (IR, 1H NMR, UV–vis, ESI-MS) and other analytical methods. To evaluate the biological
preference of chiral drugs for inherently chiral target DNA, interaction of 1–3 with calf thymus DNA in
Tris–HCl buffer was studied by various biophysical techniques which reveal that all these complexes
bind to CT DNA non-covalently via electrostatic interaction. The higher Kb value of l-tryptophan complex
1 suggested greater DNA binding propensity. Further, to evaluate the mode of action at the molecular
level, interaction studies of complexes 1 and 2 with nucleotides (5ꢀ-GMP and 5ꢀ-TMP) were carried out
by UV–vis titrations, 1H and 31P NMR which implicates the preferential selectivity of these complexes
to N3 of thymine rather than N7 of guanine. Furthermore, complex 1 exhibits efficient DNA cleavage
with supercoiled pBR322. The complex 1 cleaves DNA efficiently involving hydrolytic cleavage pathway.
Such chiral synthetic hydrolytic nucleases with asymmetric centers are gaining considerable attention
owing to their importance in biotechnology and drug design, in particular to cleave DNA with sequence
selectivity different from that of the natural enzymes.
Keywords:
Chiral dizinc(II) complexes
Benzimidazole scaffold
In vitro DNA binding
5ꢀ-GMP and 5ꢀ-TMP
pBR322 hydrolytic cleavage
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Previously, we have been interested in a chiral facile triden-
tate coordinating ligand, 1,2-bis(1H-benzimidazol-2-yl)ethane-
Most important advances of last millennium in the pharma-
cological areas were the proven efficacy of chiral or enantiopure
drugs. Single enantiopure drugs have proven to be better drugs
which exhibit fewer side effects, are safer and more potent than
the racemate drugs [1]. Chiral drugs were assumed to be therapeu-
tically active as most of the biotargets of drugs are chiral in nature.
Therefore, much attention is paid to design and construction of
chiral molecules which are (i) versatile to occupy the active site,
(ii) possess metal-binding domain and (iii) have bioactive organic
functionality (pharmacophore) which preorients the molecule as
drug entity by reducing the toxicity parameters [2]. The introduc-
tion of chirality enhances the pharmacological behavior of metal
complexes by adopting specific conformation and target selective
binding affinity for DNA, as DNA itself exists in nature only in
one chiral form [3]. Complexes bearing amino acid functionality
enhance the enantioselective effect of DNA binding as well for other
therapeutic targets, providing a key recognition element of artificial
nuclease activity to the molecule [4].
1,2-diol and studied its coordinating behavior with different
ancillary ligands [5–7]. Benzimidazole ligand framework con-
stitutes an important class of therapeutic agents in medicinal
chemistry and much of its chemistry was explored for antiviral and
antibacterial studies. Further, crystallographic [8] and NMR stud-
ies [9,10] show ligands incorporating benzimidazoles can interact
selectively with specific nucleotide sequence which binds to minor
groove of A–T tract duplex DNA and benzimidazole motif is confor-
mationally appropriate platform for DNA sequence recognition.
Thus, in our current strategy, we aim to develop new
chiral artificial nucleases derived from 1,2-bis(1H-benzimidazol-
2-yl)ethane-1,2-diol and amino acid framework which is a source
of chiral carbon center and provides a suitable heteroatom NONO-
donor two compartment coordination platform for dizinc(II) ions.
Synthetic hydrolytic nucleases have attracted much attention
owing to their importance in biotechnology and drug design, in
particular to cleave DNA with sequence selectivity different from
that of the natural enzymes [11]. Among the transition metal com-
plexes, most prominent synthetic hydrolases known are copper(II)
and zinc(II) complexes [12]. Zn(II) based artificial nucleases are
more important in the hydrolytic cleavage of phosphatediesters
[13–16]. Hydrolysis of phosphodiester linkage leads to cleavage
∗
Corresponding author. Tel.: +91 5712703893.
E-mail address: farukh arjmand@yahoo.co.in (F. Arjmand).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.09.006

54
S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
OH
H
N
H
N
O
OH
NH2
4N HCl
-4H2O
OH
+
HO
N
N
NH2
HO
HO
O
1,2-diaminobenzene
L(+) Tartaric acid
L
Scheme 1. The synthesis of ligand, L.
of DNA forming fragments that could be religated enzymatically
[17]. Our interest lies in Zn(II) metal ion, as it is a good Lewis acid,
non-toxic and redox inert, exchanges ligands rapidly, and does
not show ligand field stabilization energy and, as a consequence
it can easily adopt its coordination geometry to best fit structural
requirement of a reaction [18]. Besides this, Zn complexes form
the most propitious forms of Zn-metalloelement for the delivery to
required cellular sites enabling Zn-dependent enzyme syntheses
and facilitation of Zn-dependent biochemical processes [19]. The
bimetallic complexes exhibit enhanced acceleration of phosphate
diester cleavage as compared to the monometallic complexes.
Herein, we report the synthesis, spectroscopic characterization and
enantioselective DNA binding and cleavage studies of dizinc(II)
complexes.
been performed by the standard protocol as described in Refs.
[24,25].
2.3. Syntheses
2.3.1. Synthesis of ligand
1,2-Bis(1H-benzimidazol-2-yl)ethane-1,2-diol (L): The ligand
was synthesized from l(+)-tartaric acid (3.0 g, 20 mmol) and
1,2-diaminobenzene (4.32 g, 40 mmol) in ethane–water mixture
according to the procedure reported earlier (Scheme 1) [2].
2.4. Synthesis of zinc complexes 1–3
The
complexes
[Zn2(l-trp)L(H2O)](NO3)2,
and [Zn2(l-val)L(H2O)](NO3)2,
1,
[Zn2(l-
3 were
leu)L(H2O)](NO3)2,
2
prepared from a general synthetic procedure in which l-trp
(0.204 g, 1 mmol)/l-leu (0.131 g, 1 mmol)/l-val (0.117 g, 1 mmol),
pretreated with aqueous NaOH (0.04 g, 1 mmol) was added to a
10 ml aqueous solution of Zn(NO3)2·6H2O (0.594 g, 2 mmol) and
stirred for 30 min. A 10 ml methanolic solution of ligand L (0.294 g,
1 mmol) was added to the resulting solution and refluxed at 60 ◦C
for 4 h. A white precipitate formed was filtered off. The volume
of the resulting colorless solution was reduced under reduced
pressure which on slow evaporation at room temperature yielded
white product isolated by filtration, washed with hexane and dried
in vacuo (Scheme 2).
2. Experimental
2.1. Materials and physical measurements
1,2-Diaminobenzene, l-(+)-tartaric acid (Merck), zinc nitrate
hexahydrate, sodium hydroxide (Merck) were used as received.
All amino acids were purchased from Sigma and used as such. 6×
loading dye (Fermental Life Science) and supercoiled plasmid DNA
pBR322 (Genei), T4 ligase enzyme (CalBiochem) were utilized as
received. Calf thymus DNA (CT DNA), guanosine-5ꢀ monophosphate
disodium salt (5ꢀ-GMP), thymine-5ꢀ monophosphate disodium salt
(5ꢀ-TMP) were purchased from Sigma Chemical Co. and Fluka,
respectively. All reagents were of best commercial grade and were
used without further purification. Carbon, hydrogen and nitrogen
contents were determined on Carlo Erba Analyser Model 1106.
Molar conductances were measured at room temperature on con-
ductivity meter CM 180. IR spectra were recorded on Interspec
[Zn2(l-trp)L(H2O)](NO3)2, 1: Yield: 62%, M.p. 190–200 ◦C (d),
Anal. Calc. for C27H26N8O11Zn2: C, 42.30; H, 3.42; N, 14.62%, Found:
C, 43.43; H, 3.85; N, 14.79%. IR (KBr, cm−1) 3403 ꢁ(O–H), 3187
ꢁ(N–H), 1625 ꢁas(COO), 1494 ꢁ(C N), 1414 ꢁs(COO), 1387 vs(NO3),
1284 (imidazole NH), 824 (rocking OH)water, 745 ꢁ(Ar). 1H NMR
(400 MHz, DMSO-d6, ppm): 12.3 (s, 2H, benzimidazole NH); 10.8
(s, 1H, tryptophan indole NH); 7.17–7.46 (m, 8H, benzimidazole
aromatic H); 7.26 (d, 2H, tryptophan aromatic H); 7.33 (d, 2H,
tryptophan aromatic H); 6.3 (s, 1H, benzimidazole OH); 5.47 (d,
1H benzimidazole chiral CH); 3.36 (s, chiral trp H + trp NH2); 2.56
(t, 2H trp CH2). [␣]D (MeOH) +122.70; Molar conductance, ꢂM
(1 × 10−3 M, MeOH): 190 ꢃ−1 cm2 mol−1 (2:1 electrolyte). UV–vis
(10−4 M, MeOH, nm, ε): 244 (12,630), 274 (18,230), 280 (22,030).
ESI (m/z+), 647.8 [C27H26N8O11Zn2−2NO3+2H+].
2020 FTIR spectrometer in KBr pellets from 400 to 4000 cm−1
.
Electronic spectra were recorded on UV-1700 PharmaSpec UV–vis
spectrophotometer (Shimadzu) in methanol using cuvettes of 1 cm
path length. Data were reported in ꢀmax/nm. Fluorescence mea-
surements were determined on a Hitachi F-2500 fluorescence
spectrophotometer. The 1H NMR spectra were obtained on a Bruker
DRX-400 spectrometer operating at room temperature. Electro-
spray mass spectra were recorded on Micromass Quattro II triple
quadrupole mass spectrometer. The viscosity measurements were
carried out using Oswald capillary viscometer maintained at 25 ◦C.
Circular dichroism was performed on Applied Photophysics Chi-
rascan Circular Dichroism Spectrometer with Stop Flow. While
measuring the absorption spectra an equal amount of DNA was
added to both the compound solution and the reference solution to
eliminate the absorbance of the CT DNA itself, and the CD contri-
bution by the CT DNA and Tris buffer was subtracted through base
line correction.
[Zn2(l-leu)L(H2O)](NO3)2, 2: Yield; 52%, M.p. 185 ◦C (d), Anal.
Calc. for C22H27N7O11Zn2: C, 37.90; H, 3.88; N, 14.08%. Found:
C, 38.35; H, 3.98; N, 14.78%. IR (KBr, cm−1) 3386 ꢁ(O–H), 3193
ꢁ(N–H), 1626 ꢁas(COO), 1491 ꢁ(C N), 1418 ꢁs(COO), 1383 vs(NO3),
1291 (imidazole NH), 838 (rocking OH)water, 747 ꢁ(Ar). 1H NMR
(400 MHz, DMSO-d6, ppm): 12.43 (s, 2H, benzimidazole NH);
7.12–7.49 (m, 8H, benzimidazole aromatic H); 6.3 (s, 1H benzim-
idazole OH); 5.38 (d, 1H, benzimidazole chiral CH); 3.35 (t, leu
chiral CH + leu NH2); 2.51 (t, 2H leu CH2), 2.23 (m, 1H leu CH);
0.85 (d, 6H val CH3). [␣]D (MeOH), +101.83, Molar conductance, ꢂM
(1 × 10−3 M, MeOH): 185 ꢃ−1 cm2 mol−1 (2:1 electrolyte). UV–vis
(10−4 M, MeOH, nm, ε): 243 (14,600), 274 (15,820), 281 (26,620).
ESI (m/z+), 697.7 [C22H27N7O11Zn2+2H+].
2.2. DNA binding and cleavage experiments
DNA binding experiments include absorption spectral traces,
emission spectroscopy, viscosity measurements, circular dichro-
ism conformed to the standard methods and practices previously
adopted by our laboratory [20–23]. DNA cleavage experiment has
[Zn2(l-val)L(H2O)](NO3)2, 3: Yield; 44%, M.p. 180 ◦C (d), Anal.
Calc. for C21H25N7O11Zn2: C, 36.97; H, 3.69; N, 14.37%. Found:
C, 37.75; H, 3.86; N, 14.71%. IR (KBr, cm−1) 3396 ꢁ(O–H), 3183
ꢁ(N–H), 1626 ꢁas(COO), 1510 ꢁ(C N), 1410 ꢁs(COO), 1383 vs(NO3),

S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
55
O
R
Zn(NO3)2.6H2O
MeOH, H2O
NH2
H2O
O
O
+
60oC reflux
3-4h
Zn
Zn
L
(NO3)2
N
+
N
L- amino acid +NaOH
N
H
HN
OH
CH2
R =
1
N
H
CH3
CH3
2
3
CH2 CH
CH3
CH
CH3
Scheme 2. Schematic representation of the formation of complexes 1–3.
1281 (imidazole NH), 850 (rocking OH)water, 743 ꢁ(Ar). 1H NMR
(400 MHz, DMSO-d6, ppm): 12.9 (s, 2H, benzimidazole NH);
7.12–7.54 (m, 8H, benzimidazole aromatic H); 6.3 (s, 1H benzimida-
zole OH); 5.38 (d, 1H, benzimidazole chiral CH); 3.63 (d, val chiral
CH + val NH2); 2.21 (m, 1H valine CH); 0.81 (d, 3H val CH3). [␣]D
(MeOH), +187.41, Molar conductance, ꢂM (1 × 10−3 M, MeOH):
218 ꢃ−1 cm2 mol−1 (2:1 electrolyte). UV–vis (10−4 M, MeOH, nm,
ε): 243 (13,200), 274 (15,600), 281 (16,620). ESI (m/z+), 679
[C21H25N7O11Zn2−2H+].
ꢁ(C N) of the imidazole moiety and a characteristic ꢁ(N–H) stretch-
ing band at 3180 cm−1, ꢁ(C–N) and ꢁ(C–O) frequency at 1317 and
1267 cm−1, respectively [27,28]. In the complexes 1–3, the ꢁ(O–H)
stretching band shifted to a lower wavelength around 3400 cm−1
indicating the coordination of one O atom of one alcoholic group
to the metal center while other remains uncoordinated [29]. The
band due to ꢁ(N–H) remains unaltered in all the complexes indi-
cating non-participation of NH group in complex formation. The
ꢁ(C N) undergoes a negative shift at 1494 cm−1 (in the case of
complex 1), 1491 cm−1 (in complex 2) and 1510 cm−1 (in com-
plex 3) which is indicative of the involvement of N atoms in the
formation of dinuclear zinc complexes. The IR spectra of these
complexes displayed bands in the region 1280–1281 cm−1 due to
the presence of imidazole ring NH [30]. Coordinated water was
confirmed by the presence of band at ∼824–850 cm−1, the rock-
ing mode of water [31]. The presence of the vibrational bands in
the region of 1621–1629 cm−1 and 1410–1414 cm−1 is character-
istic of antisymmetric and symmetric COO stretching vibrations,
respectively. The ꢄꢁ value which represents the separation of
the ꢁas(COO) and ꢁs(COO) reflects the coordination modes of the
carboxyl groups [32] was greater than 200 cm−1 indicating uniden-
tate carboxylate ligation. Further the bands around 1660 cm−1 and
1430 cm−1 observed in free amino acids corresponding to ꢁas(COO)
and ꢁs(COO), respectively shift to a lower frequency indicating that
the terminal coordination mode of carboxylate group of the amino
acid to metal ion is via deprotonation [33]. The IR spectra of the
complexes exhibited a very broad band at ∼3387 cm−1 correspond-
ing to the coordinated amino group of tryptophan, leucine and
valine, which shifted from 3400 cm−1 suggesting that the coordi-
nation of –NH2 group amino acid to the metal ions [29].
3. Results and discussion
The complexes 1–3 due to the presence of chiral auxiliary, l-
amino acids (viz., tryptophan, leucine and valine), exhibited optical
rotation, [␣]D values of +122.70, +101.83 and +187.41, respec-
tively, which indicate that all the complexes are optically active.
l-Enantiomers of Zn(II) were chosen for this work as it has been
previously demonstrated in literature that l-enantiomer binds to
DNA more avidly than d- and dl-enantiomers [3,25,26]. All the
complexes were stable towards air and moisture and were sol-
uble in methanol and DMSO. Molar conductance values of these
complexes in MeOH (1 × 10−3 M) at 25 ◦C suggest their 2:1 elec-
trolyte nature (185–218 ꢃ−1 cm2 mol−1). In all the complexes both
the Zn(II) metal ions were four coordinate preferably in favor-
able tetrahedral coordination geometry. DNA binding profile of the
complexes 1–3 was studied employing UV–vis, fluorescence spec-
troscopy, circular dichroic and viscometric studies. DNA binding
modes were evaluated by the interaction of complexes 1 and 2 to
5ꢀ-GMP and 5ꢀ-TMP by absorption titrations and further validation
of interaction was done by 1H and 31P NMR techniques. The DNA
cleavage activity of complex 1 was also carried out.
3.2. NMR spectral studies
3.1. IR spectra
The 1H NMR spectra of zinc complexes 1–3 were recorded in
DMSO-d6 solution and displayed signals for aliphatic and aromatic
protons with chemical shift values in accordance with their pro-
posed structure. In complex 1 there was a signal at 10.8 ppm which
The IR spectra of complexes 1–3 were recorded in the region
of 4000–400 cm−1. The free ligand, L displayed ꢁ(O–H) bands at
3497 cm−1, a medium intensity band at 1538 cm−1 assigned to

56
S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
was attributed to the indole NH proton of the tryptophan moiety.
The peaks at 6.8 ppm, 3.36 ppm were assigned to aromatic protons
of tryptophan moiety and the chiral CH, respectively, which further
confirm the presence of the tryptophan moiety in the complexes
[34]. In complex 2, the presence of NMR signals at 2.51, 2.23 and
0.85 ppm confirms the presence of leucine moiety [35]. Similarly
in complex 3, the valine moiety was confirmed by the appearance
of signals at 2.09 and 0.81 ppm [36]. The ligand revealed OH pro-
ton signal at 5.5 ppm [37] which has disappeared in the complexes
suggesting coordination of the –OH group to the metal center.
However, a new signal at 6.3 ppm suggests that one –OH group
is uncoordinated and involved in H-bonding with the anion and
solvent [2].
3.3. Mass spectra
The formation of metal–amino acid dizinc(II) complexes
and the speciation of various ionic forms in methanol solution
were studied with ESI-MS. ESI-MS spectra of the complexes
1–3 did not display the molecular ion peaks but displayed
other fragmentation peaks. Major peaks at 647.8, 421 and
357 were observed for complex 1 corresponding to the frag-
ments [C27H26N8O11Zn2−2NO3+2H+], after the removal of
both the nitrate ions, [C27H26N8O11Zn2−2NO3−H2O−Trp],
after the removal of water molecule and the tryptophan
moiety,
exhibited fragmentation peaks at 697.7, 421 and
360 corresponding to the fragments [C22
27N7O11Zn2+2H+],
[C22H27N7O11Zn2−2NO3−H2O−Leu] and [C22H27N7O11Zn2−
2NO3−H2O−Leu−Zn]. Similarly, complex exhibited peak
[C27
H26N8O11Zn2−2NO3−H2O−Trp−Zn.
Complex
2
H
3
at 679, 537 and 360 corresponding to the fragments
[C21H25N7O11Zn2−2H+],
[C21H25N7O11Zn2−2NO3−H2O]
and
Fig. 1. Absorption spectral traces of (a) complex 1, (b) complex
2 in 0.1 M
[C21H25N7O11Zn2−2NO3−H2O−Val−Zn]. All three complexes
exhibited one more peak at 295 corresponding to the benzimida-
zole ligand moiety [L+2H+] after the removal of second zinc metal
ion.
Tris–HCl/10 M NaCl buffer at pH 7.2 upon the addition of CT DNA. Inset: plots of
[DNA]/εa − εf (m2 cm) vs. [DNA] for the titration of CT DNA with complexes ꢀ, exper-
imental data points; full lines, linear fitting of the data. [Complex] 6.66 × 10−6 M,
[DNA] 0–4.0 × 10−5 M.
spectral changes typical of a metal complex’s association with the
DNA helix. Hyperchromic effect reflects the corresponding changes
of the DNA in its conformation and structure after the complexes
are bound to DNA. Hypochromism results from the contraction of
the DNA helix while hyperchromism results from DNA helix struc-
tural damage [41]. The intrinsic DNA binding constants (Kb) of the
complexes to CT DNA have been calculated to quantify the extent of
DNA binding (Supplementary Information Table 1). The Kb follows
the order 1 > 2 > 3, indicating that the tryptophan complex binds
to the CT DNA with greater propensity than other two complexes.
The higher binding propensity is due to the presence of extended
planar aromatic ring of the tryptophan moiety, facilitating non-
covalent interactions between the ␲-system of the ligand and DNA
base pairs [42]. This is well supported by literature reports which
describe that system which lacks planar aromatic moiety displays
poor DNA binding ability [43]. Further, the higher the ring polariz-
ability, more stable is the complex [44]. This clearly validated the
order of the DNA binding ability. Recent studies on some ternary
complexes of amino acids indicated that size, shape and polarity
of side chains of different amino acids may influence their bind-
ing mode to DNA [45]. The 1,2-benzimidazolyl ligand, L as well as
the tryptophan moiety in complex 1 possess an aromatic moiety,
therefore, the probability of binding of these complexes to CT DNA
via partial intercalation cannot be ruled out, nonetheless hyper-
chromism supports electrostatic interactions [46].
3.4. Electronic spectra
The UV–vis spectra of complexes 1–3 were carried out in the
region of 190–1100 nm in methanol at room temperature. The
complexes showed three absorption bands in the UV region. All
the complexes showed ligand centered n–␲* and ␲–␲* transitions
at ∼273 nm and ∼281 nm [6]. All the complexes revealed a band
around 244 nm corresponding to intraligand transitions [38]. The
similarity of the position of these bands in the UV–vis spectra of
these complexes indicates similar chromophore with different N,O
coordinated ligand [40]. The electronic spectra of complexes 1–3
displayed transitions at 281 nm characteristic of the tetrahedral
environment around the zinc metal center [26].
3.5. DNA binding studies
3.5.1. Absorption titrations
The binding mode of the metal complexes to DNA helix is often
examined by the electronic absorption spectroscopy, followed by
the changes in the absorbance and shift in wavelength [39]. The
complexes 1–3 displayed intense absorption bands around 273 nm
assigned to intraligand transition. Upon addition of increasing
amounts of CT DNA, these complexes showed an increase in the
absorption spectral intensity of the intraligand absorption band
at 273 nm with a blue shift of 1–2 nm (Fig. 1 and Supplementary
Information, S1). These changes are characteristic of the com-
plexes bound to DNA through non-covalent interactions or the
complex could uncoil the helix and made more bases embedding
in DNA exposed [40]. Hyperchromism and hypochromism are the
3.5.2. Interaction with 5ꢀ-GMP and 5ꢀ-TMP
The complex 1 showed highest binding propensity towards CT
DNA, hence to determine the coordination of zinc metal ion to

S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
57
Fig. 2. Absorption spectral traces of (a) complex 1, (b) complex 2 in Tris–HCl upon
the addition of 5ꢀ-GMP. [Complex] 6.66 × 10−6 M, [DNA] 0–2.0 × 10−5 M.
Fig. 3. Absorption spectral traces of (a) complex 1, (b) complex 2 in Tris–HCl upon
the addition of 5ꢀ-TMP. [Complex] 6.66 × 10−6 M, [DNA] 0–2.0 × 10−5 M.
the specific site of CT DNA, interaction with low molecular build-
ing blocks of DNA molecule viz., guanosine-5ꢀ monophosphate and
thymine-5ꢀ monophosphate was done by UV–vis absorption titra-
tions. To evaluate the enantioselective binding to nucleotides, the
studies of complexes 1 and 2 were carried out. On addition of 5ꢀ-
GMP and 5ꢀ-TMP, there is an increase in the absorption intensity
of the band at 273 nm in 1 and 2 with no shift in the case of 5ꢀ-
GMP and a blue shift of 1–2 nm in the case of 5ꢀ-TMP (Figs. 2 and 3a
and b) indicating the binding of these complexes to 5ꢀ-GMP and
5ꢀ-TMP through electrostatic interaction. The Kb values calculated
suggested greater binding with 5ꢀ-TMP than 5ꢀ-GMP which clearly
indicates that zinc specifically binds to TMP through its N3 nitrogen
(Supplementary Information Table 2) [47]. The Kb values calcu-
lated clearly indicated that the l-tryptophan complex binds to CT
DNA with greater propensity than l-leucine complex. Literature
survey reveals that Zn complexes in contrast to Cu(II) break the
hydrogen bonds of A–T base pairs of the DNA helix, binding selec-
tively to thymine, altering the local structure of the A–T regions
of the DNA. The complex show preference for thymine due to two
effects—when thymine binds, strong hydrogen bonds are formed
between NH2 groups of the ligand and C O groups of thymine,
which stabilizes the complex and secondly, a significant stronger
MO interaction was identified in thymine in comparison to gua-
nine. The presence of two electron withdrawing oxo groups at C2
and C3 position of thymine ring lower the energy of the lone pair
orbital at N3 of thymine base [25,47].
exhibited H8 signal at 8.07 ppm and the ribose peak signals at
3.85–5.8 ppm, respectively. On addition of complexes 1 and 2, there
was no significant shift in H8 signal of free 5ꢀ-GMP attributing
to non-participation of guanine on binding with these complexes
through coordination mode. Similarly, the 31P NMR signal appeared
at 3.73 ppm in free 5ꢀ-GMP did not show any shift on the addi-
tion of these complexes. On interaction of complexes 1 and 2 with
5ꢀ-TMP, the N3-H signal of free 5ꢀ-TMP (7.64 ppm) showed a sig-
nificant shift (7.93 ppm in 1 and 8.13 ppm in 2). The C6-H atom of
free 5ꢀ-TMP showed a signal at 6.2 ppm was shifted to 6.18 ppm
and 6.19 ppm with increased intensity upon addition of the com-
plexes 1 and 2, respectively. Further the T-CH3 peaks at 1.95 ppm
shifted to a slightly higher frequency to that of free 5ꢀ-TMP. The
31P NMR signal at 3.67 ppm in free 5ꢀ-TMP was shifted upfield to
2.15 ppm and 2.08 ppm upon the addition of complexes 1 and 2,
respectively (Supplementary Information Figs. S2 and S3a, b). These
findings indicate that the Zn(II) complexes preferentially bind to N3
of 5ꢀ-TMP via coordination rather than 5ꢀ-GMP.
3.5.3. Fluorescence spectroscopic studies
No luminescence was observed for complexes 1–3 either
in MeOH or in the presence of DNA. Hence, steady state
competitive binding studies of these complexes were moni-
tored in order to investigate if the complexes could displace
EB from its EB–DNA complex. EB, 3,8-diamino-5-ethyl-
To validate our hypothesis, the binding of complexes 1 and 2,
to 5ꢀ-GMP and 5ꢀ-TMP, 1H and 31P NMR techniques at physiologi-
cal pH were carried out. The 1H NMR of free 5ꢀ-GMP in DMSO-d6
6-phenylphenanthridinium bromide, is
a
planar aromatic

58
S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
Fig. 5. Effect of viscosity of complex (a) 1 and (b) 2 on the viscosity of CT DNA. Rel-
ative viscosity ꢅ/ꢅ0 vs. [complex]/[DNA]. [Complex] 0.2–1 × 10−6 M, [DNA] 10−6 M.
3.5.4. Viscosity measurements
In the absence of any crystallographic data, viscosity mea-
surements are regarded as the most critical and least ambiguous
method to determine the binding mode of the DNA in the solu-
tion [51]. While intercalation results in substantial increase in the
viscosity of DNA due to stiffening and lengthening of the helix,
the other types of interactions produce only substantial changes
in the structure of the DNA and DNA remains essentially in unper-
turbed form [52]. For the complexes 1–3, the plot of relative specific
viscosity ꢅ/ꢅ0 vs. [complex]/[DNA] shows irregular changes in the
viscosity of the DNA (Fig. 5). A low complex concentration induces a
very large decrease in the viscosity which reverses on further addi-
tion of the complex. The initial decrease in the viscosity is due to the
substrate induced DNA kinking, since changes in the viscosity of a
rigid rod like DNA solutions are directly proportional to changes in
its hydrodynamic length. While at higher binding ratios much more
kinks produce rod like DNA structure with contour lengths compa-
rable to DNA molecules. The consequent increase in the length of
DNA may also be due to the formation of supercoiled helical struc-
ture [53]. These observations are in accordance with the absorption
and fluorescence spectral titrations and account for an external
DNA binding mode—electrostatic and/or groove binding for the
complex [54].
Fig. 4. Emission spectra of EB bound to DNA in the presence of (a) complex 1, (b)
complex 2 in Tris–HCl buffer at pH 7.2. Arrows indicate the intensity changes upon
increasing concentration of the complexes. Inset: fluorescence quenching curve of
DNA bound EB with complexes ꢀ, experimental data points; full lines, linear fitting
of the data. [Complex] 6.66–4 × 10−6 M, [EB–DNA] 6.66 × 10−7 M.
heterocyclic indicator of intercalation. In the presence of DNA, it
emits intense fluorescence due to intercalation of planar phenan-
thridinium ring between adjacent base pairs on the DNA double
helix [40]. The fluorescence intensities of EB–DNA at 560 nm
(270 nm excitation) with an increasing amount of complex con-
centration were recorded. EB was non-emissive in Tris–HCl buffer
medium due to fluorescence quenching of free EB by solvent
molecules [48]. Addition of second DNA binding molecule results
in a decrease in emission intensity of EB–DNA system. Two mech-
anisms, replacement of EB by the second molecule or electron
transfer, have been proposed to account for this reduction in
emission intensity [49]. The complexes 1–3 show a decrease in the
emission intensity of EB–DNA system (Fig. 4). As these complexes
bind to DNA via electrostatic interactions, i.e. surface binding
hence observed quenching is due to the photoelectron transfer
mechanism [50]. The quenching efficiency for each complex is
evaluated by the Stern–Volmer quenching constant, Ksv, obtained
as the slope of I0/I vs. r (=[complex]/[DNA]) for complexes 1–3
are found to be 0.48, 0.33 and 0.22, respectively. The maximum
value obtained for l-tryptophan complex, 1 is in agreement with
its highest Kb value, i.e. it binds to CT DNA very strongly, hence
maximum quenching. High Ksv value of the complex reveals that
they can bind to CT DNA avidly [46].
3.5.5. Circular dichroic studies
Circular dichroic spectral technique is useful in monitoring the
conformational variations of DNA during complex–DNA interac-
tions [55]. The observed CD spectrum of CT DNA consists of a
positive band at 275 nm (UV, ꢀmax, 260 nm) due to base stacking
and a negative band at 245 nm caused by helicity, which are char-
acteristic of DNA in the right-handed B-form [56]. Simple groove
binding and electrostatic interaction of the complexes with DNA
shows less or no perturbation on the base stacking while interca-
lator enhances the intensities of both the bands. Upon the addition
of complexes 1–3 to CT DNA, the CD spectrum of DNA undergoes

S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
59
Fig. 8. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by 1 in the presence of reactive oxygen species at 310 K after incu-
bation for 45 min. Lane 1: DNA control; Lane 2: 50 ␮M of complex 1 + sodium azide
(0.4 M) + DNA; Lane 3: 50 ␮M of complex 1 + SOD (15 units) + DNA; Lane 4: 50 ␮M of
complex 1 + DMSO (0.4 M) + DNA; Lane 5: 50 ␮M of complex 1 + tert-butyl alcohol
(0.4 M) + DNA.
Fig. 6. CD spectra of CT DNA alone (- - - - dashed line) (a) in the presence of complex
1, (b) in the presence of complex 2 and (c) in the presence of complex 3. [Complex]
1 × 10−4 M, [DNA] 1 × 10−4 M.
Fig. 9. DNA religation by T4 ligase enzyme of pBR322 plasmid DNA cleaved by com-
plex 1. Lane 1: DNA control; Lane 2: DNA cleaved by complex 1; Lane 3: ligation of
linearised DNA by T4 DNA ligase.
changes in both positive and negative bands (Fig. 6). The inten-
sities of both the bands decrease (negative band tending to zero
levels). The decrease in ellipticity is due to destabilization of DNA
form attributed to the opening of DNA structure by the formation
of intra-strand DNA cross-linkings [57]. The decrease in the posi-
tive band suggests that the complex can unwind the DNA helix and
lead to loss of helicity [58].
3.5.6.2. DNA cleavage in the presence of minor and major groove
binders. DNA recognition elements (groove binding) DAPI (minor
groove binding) [62] and methyl green (major groove binding) [63]
were used to probe the potential interacting site of complex 1 with
supercoiled pBR322 DNA. As shown in Fig. 7, upon the addition of
DAPI (Lane 2), there is apparent inhibition of the cleavage activity
of 1 suggesting that the complex is a minor groove binder which is
in conformity with many literature reports revealing that benzim-
idazole ligands play important roles in several DNA minor groove
binding agents such as Hoechst 33258 and Hoechst 33342 [64,65].
3.5.6. DNA cleavage studies
3.5.6.1. DNA cleavage without added reductant. Since complex 1
showed maximum binding propensity with CT DNA, the DNA
cleavage abilities of this complex was studied by agarose gel elec-
trophoresis using supercoiled pBR322 plasmid DNA as a substrate.
Change in the electrophoretic mobility of plasmid DNA on agarose
gel is commonly taken as evidence for direct DNA–metal inter-
actions. The DNA cleavage ability of complex 1 was assessed by
the conversion of DNA from Form I (supercoiled form) to Form II
(nicked circular form) or Form III (linear form) [25]. A concentra-
tion dependent DNA cleavage by complex 1 was first performed
in which pBR322 DNA (300 ng) was incubated at 310 K for 45 min.
During electrophoresis, while scission occurs on one strand (nick-
ing), the supercoiled form relaxes to generate nicked form (Form II)
[59]. When cleavage occurs on both the strands, a linear form (Form
III) is generated which migrates between Forms I and II [60]. With
the increase in concentration of complex 1, DNA was converted
from Form I to Form II without the formation of Form III, suggesting
single strand DNA cleavage (Fig. S4). Complex 1 shows discern-
able cleavage with increase in concentration and intensified nicked
(Form II) was observed. Since the nuclease efficiency of complexes
is usually dependent on activators [61]. Therefore, DNA cleavage
activity of 1 in the presence of ascorbic acid, glutathione, H2O2
and 3-mercaptopropionic acid (MPA) were evaluated (Fig. S5). The
cleavage activity was significantly enhanced by these activators and
followed the order H2O2 > MPA ≈ Asc ꢁ GSH.
3.5.6.3. Reactive oxygen species (ROS) responsible for DNA cleavage.
To postulate a mechanism responsible for DNA cleavage mediated
by complex 1, the cleaving ability in the presence of various rad-
ical scavengers was carried out such as DMSO, tert-butyl alcohol
as OH radical scavenger [66], NaN3 as 1O2 scavenger [67] and SOD
as superoxide anion radical scavenger [68]. Upon the addition of
NaN3 and SOD (Lanes 2 and 3, respectively), the cleavage ability
is enhanced suggesting the non-involvement of the singlet oxygen
radical and superoxide anion radical in the mechanistic pathway
of DNA cleavage. While on the other hand, the hydroxyl radi-
cals, DMSO and TBA (Lanes 4 and 5, respectively) inhibit the DNA
cleavage which is suggestive of the involvement of diffusible OH
radicals as one of the ROS responsible for DNA breakage (Fig. 8)
[69]. These OH free radicals participate in the oxidation of the
deoxyribose moiety followed by the hydrolytic cleavage of sugar
phosphate backbone [70]. Therefore, cleavage of the DNA occurs via
hydrolytic pathway. This hypothesis is supported by previous liter-
ature which reveals that Zn(II) complexes generally cleave the DNA
by hydrolytic pathway [71]. To ascertain the discernible hydrolytic
DNA cleavage pathway in the presence of 1, DNA religation experi-
ment was further performed in which supercoiled pBR322 DNA was
treated with T4 ligase enzyme and subjected to gel electrophoresis
[72]. In our case, the nicked form (Form II) was relegated to a large
extent in the presence of T4 ligase enzyme in comparison to con-
trol DNA alone in supercoiled form (Fig. 9) providing a supportive
evidence in favor of hydrolytic mechanism.
4. Conclusion
In this work, the synthesis of complexes 1–3, their structural
characterization and in vitro DNA binding studies of dizinc(II)
ternary amino acid complexes towards CT DNA have been carried
out. The DNA binding studies of the complexes 1–3 using various
biophysical and spectroscopic techniques reveal that l-trp complex
Fig. 7. Agarose gel electrophoresis pattern for the cleavage of pBR322 plasmid DNA
(300 ng) by 1 (0.25 mmol) in the presence of DNA minor groove binding agent DAPI
and major binding agent methyl green at 310 K after incubation for 45 min. Lane 1,
DNA + 1 + methyl green; Lane 2, DNA + 1 + DAPI (8 mM); Lane 3; DNA control (2.5 mL
of 1 0.01 mg/ml solution).

60
S. Parveen, F. Arjmand / Spectrochimica Acta Part A 85 (2012) 53–60
exhibits highest propensity for DNA binding and DNA binding mode
was essentially non-covalent via electrostatic interactions. Never-
theless, the DNA binding propensity of complex 1 was much higher
than 2 and 3 which clearly attenuate the effect of side chain on
specific binding affinity. Specific interaction with nucleotides viz.,
guanosine-5ꢀ monophosphate and thymine-5ꢀ monophosphate by
UV–vis and 1H and 31P NMR reveal that these complexes bind to N3
of thymine showing lower affinity for N7 of guanine residue. Fur-
thermore, the cleavage activity of 1 was performed by agarose gel
electrophoretic assay and noticeably, the complex exhibited effec-
tive DNA cleavage via hydrolytic mechanistic pathway. Therefore,
l-tryptophan derived dizinc(II) complex, 1 was developed as DNA
hydrolytic cleavage agent.
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The authors are highly indebted to the Regional Sophis-
ticated Instrumentation Center (RSIC), Central Drug Research
Institute, Lucknow, India for providing CHN analysis data, ESI-Mass
and polarimetry, Sophisticated Analytical Instrumentation Facility
(SAIF), Panjab University, Chandigarh for providing the NMR spec-
tra and Advanced Instrumentation Research Facility (AIRF), JNU,
New Delhi for providing CD facility.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.09.006.
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