DNA binding, antioxidant activity, and DNA damage protection of chiral macrocyclic Mn(III) salen complexes

Article abstract of DOI:10.1002/chir.22098

We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral complexes showed ability to bind with DNA, where complex S-1 exhibits the highes

Full text of DOI:10.1002/chir.22098

CHIRALITY (2012)
DNA Binding, Antioxidant Activity, and DNA Damage Protection of
Chiral Macrocyclic Mn(III) Salen Complexes
1
1
1
1
1
2
*
NIRALI PANDYA, NOOR-UL H. KHAN, K. JEYA PRATHAP, RUKHSANA I. KURESHY, SAYED H. R. ABDI, SANDHYA MISHRA, AND
HARI C. BAJAJ1
1Discipline of Inorganic Materials and Catalysis, Central Salt and Marine Chemicals Research Institute (CSMCRI), Council of Scientific & Industrial
Research (CSIR), Bhavnagar, Gujarat, India
2Discipline of Marine Biotechnology and Ecology, Central Salt and Marine Chemicals Research Institute (CSMCRI), Council of Scientific & Industrial
Research (CSIR), Bhavnagar, Gujarat, India
ABSTRACT
We are reporting the synthesis, characterization, and calf thymus DNA binding
studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral
complexes showed ability to bind with DNA, where complex S-1 exhibits the highest DNA binding
constant 1.20ꢀ 106 Mꢁ1. All the compounds were screened for superoxide and hydroxyl radical
scavenging activities; among them, complex S-1 exhibited significant activity with IC50 1.36 and
2.37mM, respectively. Further, comet assay was used to evaluate the DNA damage protection in
white blood cells against the reactive oxygen species wherein complex S-1 was found effective in
protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage. Chirality
00:000–000, 2012. © 2012 Wiley Periodicals, Inc.
KEY WORDS: chiral macrocyclic Mn(III) salen complex; DNA binding; superoxide radical;
hydroxyl radical; plasmid DNA; WBC cell DNA protection
INTRODUCTION
inflammation.16 The low-molecular weight complexes of Mn,
Fe, Ni, Cu, and Zn have been reported to show beneficial effects
for protecting tissues against ROS.17–21 The possible reason
for favoring manganese-containing ligand systems when
compared with copper and iron could be due to its low toxicity
in vivo.17,22–24 Superoxide Dismutase (SOD) mimics of non-
chiral Mn(III) and Mn(II) complexes based on porphyrin,25–28
corrole,29,30 salen,14,31,32 macrocycles,22,32,33 and tripodal
ligands34–36 have been reported in the past. Mn(III) salen and
Mn(II) macrocyclic complexes have been suggested as a
potential class for SOD mimics32,37 due to their high thermody-
namic stability. Besides, Mn(III) salen complexes exert benefi-
cial effects in vitro and in vivo animal models of human
diseases,38–41 which protect from the oxidative stress. Addition-
ally, dinuclear metal complexes have been recognized as
probes for DNA as they have large size, charge, and variety of
molecular shapes for stronger DNA binding affinity.42 The mul-
tinuclearity and stereoselectivity have been one of the success-
ful strategies to increase the efficiency and selectivity of the
DNA interaction as well as therapeutic agents due to the poten-
tial cooperative effects between the metal centers.43 We have
also reported the DNA interaction and antioxidant activity by
chiral mononuclear Mn(III) salen complexes with different sub-
stituent at 5,50 position and observed the influence of chirality
on biological activity.44 In the present article, we report the
synthesis and characterization of chiral dinuclear macrocyclic
Mn(III) salen system viz. S-1, R-1, S-2, R-2 and examined
their activities in calf thymus (CT)-DNA binding by absorption
spectroscopy, circular dichroism spectroscopy (CD), viscosity,
Chirality plays a significant role in pharmaceutical industry
because one enantiomer of a chiral drug molecule is likely to
show desirable therapeutic effects while other enantiomer can
have a different or adverse biological response. A classical
example of this behavior was noticed in the use of chiral drug
ethambutol—a drug in which S enantiomer has shown bacte-
riostatic antimycobacterial activity and has been prescribed
for tuberculosis treatment; however, R enantiomer has resulted
in blindness. Recently, the interaction of transition metal
complexes with DNA evoked great interest due to their impor-
tance in designing of new and promising drugs, probes for
nucleic acids,1,2 DNA-dependent electron transfer reactions,
sequence-specific cleaving agents, and antitumor drugs.3–5 As
DNA is chiral, its interaction with a molecule having a chiral
center(s) is expected to be influenced by diastereomeric
ion-pair formation between the two. Thus, different enantio-
mers of chiral metal complexes are expected to show different
metallo-intercalation capacity with essentially chiral DNA and
protein molecules.6–13 As a consequence of this interaction,
different enantiomers of a metal complex may have variable
effects on various biological reactions, especially those involved
in reactive oxygen species (ROS) like hydrogen peroxide
(H2O2) decomposition, superoxide anion (O2ꢁ) dismutase,
ꢂ
catalase, and ribonuclease reduction.
Reactive oxygen species, such as O2ꢁ, hydroxyl radical
ꢂ
ꢂ
( OH), and H2O2, generate under normal metabolic activities
on regular bases in human body. Human body employed an
array of enzymatic and nonenzymatic antioxidant machinery
to keep their level below toxic limits. However, the ROS level
could exceed the threshold point in the body due to severe
exposure of various environmental pollutants like smoke,
ultraviolet (UV), and infrared radiations. Oxidative stress
condition also arises if endogenous antioxidant defense system
fails to neutralize the ROS.14,15 ROS can induce molecular and
cellular damages to DNA, lipids, and proteins, which eventually
result in various diseases like arthritis, stroke, cancer, and
© 2012 Wiley Periodicals, Inc.
*Correspondence to: Noor-ul H. Khan, Discipline of Inorganic Materials and
Catalysis, Central Salt and Marine Chemicals Research Institute (CSMCRI),
Council of Scientific & Industrial Research (CSIR), G. B. Marg, Bhavnagar-
364 021, Gujarat, India.
E-mail: khan251293@yahoo.in
Received for publication 04 April 2012; Accepted 13 June 2012
DOI: 10.1002/chir.22098
Published online in Wiley Online Library
(wileyonlinelibrary.com).

PANDYA ET AL.
and thermal denaturation study. We have also evaluated the
(s, 4H), 2.47 (s, 8H), 1.40 (s, 18H). (13C) NMR (CDCl3,
200 MHz): (d (ppm)): 198, 162, 138, 137, 132, 130, 123, 65, 54,
35, 31. FT-IR (KBr, n/cmꢁ1): 3137, 2952, 2811, 2312, 1931,
1806, 1781, 1652, 1597, 1435, 1380, 1321, 1292, 1261, 1232,
1203, 1155, 1008, 966, 927, 888, 801, 754, 709, 619, 551, 528.
Elemental analysis data: calcd (%) for C28H38N2O4: C 72.07, H
8.21, N 6.00. Found (%): C 72.11, H 8.24, N 6.03. Electronspray
ionization (ESI)-MS: m/z: 467 (M + 1).
Synthesis of chiral macrocyclic salen ligands S-10, R-10, S-20,
and R-20. A solution of dialdehyde (C) in tetrahydrofuran
(2 mmol, 2 equivalent, 1.2 ml) was taken in a single neck
50-ml round bottom flask to which the solution of (1S,2S)-(+)-
1,2-diaminocyclohexane/(1R,2R)-(ꢁ)-1,2-diaminocyclohexane
and 1S,2S-(ꢁ)-1,2-diphenylethane-1,2-diamine/1R,2R-(+)-1,2-
diphenylethane-1,2-diamine in tetrahydrofuran (2 mmol,
2 equivalent, 0.8 ml) was added slowly, and the resultant solu-
tion was stirred at room temperature. After completion of the
reaction (3 h), checked on thin layer chromatography (TLC),
the solvent was removed completely under reduced pressure.
The yellow solids were extracted with CH2Cl2 (30 ml), and the or-
ganic layer was washed with water (3 ꢀ 10 ml), brine (3 ꢀ 10 ml)
and finally dried over anhydrous Na2SO4. The removal of
dichloromethane under reduced pressure gave chiral dimeric
macrocyclic salen ligands S-10, R-10, S-20, and R-20 in high
yield, which was used as such for the preparation of the metal
complexes S-1, R-1, S-2, and R-2 without further purification.
Characterization data of macrocyclic salen ligands S-10, R-10,
S-20, and R-20. Ligand S-10. Yellow solid, mp = 237–239 ꢃC.
Yield: 90%. 1H NMR (CDCl3, 200 MHz): (d (ppm)): 13.75
(s, 4H), 8.27 (s, 4H), 7.18 (br s, 4H), 6.96 (s, 4H), 3.38–3.23
(br s, 12H), 2.41 (s, 16H), 1.92–1.62 (m, 16H), 1.36 (s, 36H).
13C NMR (CDCl3, 200 MHz): (d (ppm)): 167.4, 161.5, 138.6,
132.4, 131.9, 128.7, 120.2, 74.4, 64.3, 54.7, 36.7, 35.2, 31.3, 26.4.
FT-IR (KBr, n/cmꢁ1): 3431, 2935, 2864, 2807, 1628, 1444,
1387, 1321, 1266, 1205, 1158, 1097, 1011, 927, 876, 820, 707.
Elemental analysis data: calcd (%) for C68H96N8O4: C 74.96, H
8.88, N 10.28. Found (%): C 74.98, H 8.89, N 10.27. ESI-MS:
role of chirality in antioxidant potential and DNA damage pre-
vention by using chiral macrocyclic Mn(III) salen complexes.
To the best of our knowledge, this is the first report where
chiral macrocyclic Mn(III) salen system was used to study
the influence of chirality on DNA damage protection against
the ROS. Among the chiral complexes S-1, R-1, S-2, R-2
investigated here, the complex S-1 exhibited strong DNA inter-
action with binding constant 1.20 ꢀ 106 Mꢁ1. The complex S-1
showed strong superoxide and ꢂOH scavenging ability with
IC50 1.36 and 2.37mM, respectively.
EXPERIMENTAL SECTION
Materials and Methods
Calf thymus DNA, pUC 19 plasmid DNA, (1S,2S)-(+)-1,2-diaminocyclohexane/
(1R,2R)-(ꢁ)-1,2-diaminocyclohexane and 1S,2S-(ꢁ)-1,2-diphenylethane-
1,2-diamine/1R,2R-(+)-1,2-diphenylethane-1,2-diamine, nitro blue tetrazolium
(NBT), 2-deoxy-D-ribose, low melting point agarose were purchased from
Sigma Aldrich Chemicals pvt. Ltd, Bangalore, India and used as received.
Ethidium bromide (EB), riboflavin (vitamin B2) and L-methionine (MET)
were purchased from S.D. Fine Chemicals Ltd., Mumbai, India. A solution
of CT-DNA in phosphate buffer gave a ratio of UV absorbance at 260 and
280 nm of approximately 1.8–1.9, indicating that the DNA was sufficiently
free of protein.45 DNA concentration per nucleotide was determined by absorp-
tion spectroscopy using the molar absorption coefficient (6600 Mꢁ1 cmꢁ1
)
at 260 nm. Commercial grade solvents were distilled before use for the
preparation of ligands and complexes. 3-tert-butyl-salicylaldehyde (A) and
chloromethylsalicylaldehyde (B), dialdehyde (C) and macrocyclic chiral
salen ligands (S-10 and R-10) were synthesized by our earlier reported
method.46 Elemental analysis of complexes was done on a CHNS Analyzer,
Perkin Elmer model 2400 (United State of America). Proton nuclear
magnetic resonance (1H NMR) spectra were obtained with a Bruker
F113V spectrometer (made in Switzerland) (200 MHz) and are referenced
internally with trimethylsilane. Fourier transform infrared spectroscopy
(FT-IR) spectra were recorded on Perkin Elmer Spectrum GX spectropho-
tometer in KBr window. High-resolution mass spectra were obtained
with a liquid chromatography mass spectroscopy (LC-MS) (Q-time of
flight [TOF]) LC (Waters, USA), MS (Micromass, UK), matrix-assisted laser
desorption/ionization (MALDI)-TOF, Model make Ultra flex TOF/TOF,
Bruker Daltonics Germany instruments. The spray voltage, tube lens
offset, capillary voltage, and capillary temperature were set at 4.50kV,
30.00V, 23.00 V, and 200ꢃC, respectively, and the m/z values were quoted
for the major peaks in the isotope distribution. UV–visible (UV–vis) and
CD spectra were recorded on Shimadzu UV 3101 PC NIR spectrophotome-
ter (Japan) and J-815 CD spectrophotometer (Japan), respectively.
m/z: 1089 (M+ 1).
Ligand R-10. Yellow solid, mp = 235–239 ꢃC. Yield: 88%.
1H NMR (CDCl3, 200 MHz): (d (ppm)): 13.78 (s, 4H), 8.23
(s, 4H), 7.15 (br s, 4H), 6.94 (s, 4H), 3.34–3.20 (br s, 12H),
2.42 (s, 16H), 1.91–1.61 (m, 16H), 1.35 (s, 36H). 13C NMR
(CDCl3, 200 MHz): (d (ppm)): 167.1, 161.3, 138.5, 132.3,
131.7, 128.5, 120.2, 74.5, 64.3, 54.6, 36.6, 35.1, 31.3, 26.2. FT-IR
(KBr, n/cmꢁ1): 3430, 2934, 2866, 2808, 1627, 1442, 1386,
1320, 1266, 1208, 1158, 1096, 1010, 927, 878, 820, 706. Elemental
analysis data: calcd (%) for C68H96N8O: C 74.96, H 8.88, N 10.28.
Preparation of ligand precursors
Synthesis of 5,50-(piperazine-1,4-diylbis(methylene))-bis-(3-tert-
butyl-2-hydroxybenzaldehyde). A solution of chloromethylsali-
cylaldehyde (B) in dry toluene (20 mmol, 20 ml) was added
dropwise to a stirring solution of piperazine in dry toluene
(10 mmol, 20 ml), and reaction mixture was stirred at room
temperature. The resulting yellow solution was allowed to heat
at 80 ꢃC with vigorous stirring for 6 h. After completion of the
reaction, dialdehyde dichloride was obtained as a white precipi-
tate, which was washed with ether (2 ꢀ 10 ml) and toluene
(2 ꢀ 10 ml) to remove unreacted starting materials. The resulting
solid was treated with excess of saturated sodium bicarbonate,
and the desired dialdehyde (C) was extracted with CH2Cl2
(3 ꢀ 10 ml). The organic layer was washed with water, brine,
and dried over anhydrous Na2SO4. The removal of the organic
solvent provided dialdehyde (C) in high yield, which was used
for the preparation of chiral macrocyclic salen ligands.
Found (%): C 74.95, H 8.90. N 10.29. ESI-MS: m/z: 1089 (M+11).
Ligand S-20. Yellow solid, mp = 245–248 ꢃC. Yield: 88%. H
NMR (CDCl3, 200 MHz): (d (ppm)): 13.66 (s, 4H), 8.31
(s, 4H), 7.18–6.92 (br m, 28H), 4.72 (s, 4H), 3.31 (br s, 8H),
2.34 (s, 16H), 1.37 (s, 36H). 13C NMR (CDCl3, 200 MHz):
(d (ppm)): 168.7, 161.2, 141.4, 138.7, 132.7, 132.4, 130.1, 129.8,
129.3, 120.0, 81.9, 64.3, 54.5, 36.6, 31.2. FT-IR (KBr, n/cmꢁ1):
3430, 2951, 2872, 2805, 2359, 1626, 1444, 1387, 1321, 1265,
1205, 1156, 1054, 1011, 919, 880, 801, 769, 699. Elemental analysis
data: calcd (%) for C84H100N8O4: C 78.47, H, 7.84, N 8.72. Found
(%): C 78.49, H 7.87, N 8.73. ESI-MS: m/z: 1286 (M + 1).
Ligand R-20. Yellow solid, mp = 246–249 ꢃC. Yield: 87%. 1H
NMR (CDCl3, 200MHz): (d (ppm)): 13.68 (s, 4H), 8.32 (s, 4H),
7.17–7.13 (br m, 28H), 4.73 (s, 4H), 3.33 (br s, 8H), 2.35
(s, 16H), 1.39 (s, 36H). 13C NMR (CDCl3, 200MHz): (d (ppm)):
168.5, 161.1, 141.2, 138.5, 132.6, 132.4,130.2, 129.6, 129.1, 120.0,
Characterization data of dialdehyde (C). White solid, mp =
141–144 ꢃC. Yield: 90%. 1H NMR (CDCl3, 200MHz): (d (ppm)):
11.72 (s, 2H), 9.86 (s, 2H), 7.43 (s, 2H), 7.35 (s, 2H), 3.46
Chirality DOI 10.1002/chir

DNA BINDING, ANTIOXIDANT ACTIVITY
81.5, 64.1, 54.5, 36.5, 31.2. FT-IR (KBr, n/cmꢁ1): 3429, 2952,
S-1, R-1, S-2, and R-2 (Scheme 1). The characterization data
2873, 2807, 2360, 1628, 1442, 1388, 1322, 1267, 1207, 1155,
1053, 1013, 920, 879, 800, 770, 706. Elemental analysis data: calcd
(%) for C84H100N8O4: C 78.47, H 7.84, N 8.72. Found (%): C 78.48,
H 7.88, N 8.75. ESI-MS: m/z: 1286 (M +1).
of the complexes are as follows:
Complex S-1. Brown solid, mp=249–252 ꢃC. Yield: 87%. FT-IR
(KBr, n/cmꢁ1): 2930, 2862, 2808, 2359, 1613, 1540, 1429, 1388,
1308, 1263, 1239, 1201, 1165, 1012, 941, 875, 825, 782, 716, 632,
564. Elemental analysis data: calcd (%) for C68H92N8O4:
C 64.50, H 7.32, N 8.85. Found (%): C 64.53, H 7.30, N 8.86.
ESI-MS: m/z: 1267 (M + 1). MS (MALDI, positive): m/z:
1196.29 ([M-2Cl]+). ICP: 8.68/100 mg, Found: 8.65/100 mg.
UV–vis lmax: 315 (13740), 400 (5506) nm.
Synthesis of chiral macrocyclic Mn(III) salen complexes S-1,
R-1, S-2, and R-2. The chiral dimeric macrocyclic salen
ligands S-10, R-10, S-20, and R-20 (1mmol) were dissolved in
25-ml DCM + methanol (1:9) mixture to which Mn(CH3COO)
2ꢂ4H2O (1. mmol) was added in an inert atmosphere, and the
resulting brown solution was refluxed for 7–8 h under nitrogen
atmosphere. After the completion of the reaction (checked on
TLC) and the reaction mixture was cooled to room tempera-
ture, solid lithium chloride (3 mmol) was added. The resulting
mixture was further stirred for 3 h exposed to air. The resulting
mass was filtered, and the solvent was removed from the filtrate
till dryness on a rotary evaporator. The resulting residue was
extracted with dichloromethane, and the organic phase
was washed with water and dried over anhydrous Na2SO4.
The resulting solution was filtered and evaporated to give
dark brown colored Mn(III) macrocyclic salen complexes
Complex R-1. Brown solid, mp = 248–253 ꢃC. Yield: 85%.
FT-IR (KBr, n/cmꢁ1): 2932, 2863, 2809, 2360, 1614, 1542, 1429,
1387, 1310, 1264, 1239, 1202, 1164, 1013, 943, 876, 825, 783, 717,
634, 565. Elemental analysis data: calcd (%) for C68H92N8O4:
C 64.50, H 7.32, N 8.85. Found (%): C 64.52, H 7.34, N 8.86.
ESI-MS: m/z: 1267 (M + 1). MS (MALDI, positive): m/z: 1196.27
([M ꢁ 2Cl]+). ICP: 8.68/100 mg, Found: 8.66/100 mg. UV–vis
lmax: 315 (13144), 400 (6200) nm.
Complex S-2. Brown solid, mp = 254–257 ꢃC. Yield: 84%.
FT-IR (KBr, n/cmꢁ1): 2923, 2805, 2358, 1816, 1608, 1537, 1420,
N
N
N
N
N
N
N
N
N
N
N
N
O
O
Cl
O
t-Bu
t-Bu
t-Bu
t-Bu
O
O
t-Bu
t-Bu
t-Bu
t-Bu
Cl
Cl
Cl
O
O
O
N
N
N
N
R-2
R-1
Mn(OAc)2, LiCl
Mn(OAc)2, LiCl
N
N
N
N
N
N
N
N
N
N
OH
HO
HO
OH
OH
t-Bu
t-Bu
t-Bu
t-Bu
HO
HO
t-Bu
t-Bu
t-Bu
t-Bu
H N
NH
H N
OH
NH
N
N
N
N
N
N
N
N
O
O
t-Bu
R-2'
HO
t-Bu
OH
R-1'
NH
N
N
H N
C
NH
N
N
H2N
H
N
N
N
N
N
N
N
N
OH
HO
HO
t-Bu
t-Bu
t-Bu
t-Bu
OH
OH
HO
HO
t-Bu
t-Bu
t-Bu
t-Bu
N
H
OH
N
O
OH
t-Bu
N
N
N
N
Cl
S-1'
S-2'
B
Mn(OAc)2, LiCl
Mn(OAc)2, LiCl
N
N
N
N
N
N
N
N
N
O
O
t-Bu
t-Bu
t-Bu
t-Bu
O
O
t-Bu
t-Bu
t-Bu
t-Bu
Cl
Cl
Cl
Cl
O
O
O
O
N
N
N
N
N
N
N
S-2
S-1
Scheme 1. General scheme for the synthesis of chiral macrocyclic ligands and complexes.
Chirality DOI 10.1002/chir

PANDYA ET AL.
1389, 1304, 1263, 1198, 1160, 1132, 1008, 935, 824, 778, 699, 616,
(A0) measured under the same condition. All the experiments
were conducted in triplicate, and data were expressed as mean
and standard deviation. The suppression ratio was calculated
by using the following equation.
564. Elemental analysis data: calcd (%) for C84H96N8O4: C 68.98,
H 6.62, N 7.76. Found (%): C 68.94, H 6.64, N 7.77. ESI-MS: m/z:
1463 (M + 1). MS (MALDI, positive): m/z: 1392.27 ([M ꢁ 2Cl]+).
ICP: 7.51/100 mg, Found:7.53/100 mg. UV–vis lmax: 322
(11666), 410 (4934) nm.
O2ꢁ scavenging activityð%Þ ¼ ½ðA0 ꢁ AiÞ=A0 ꢀ 100ꢅ
(1)
ꢂ
Complex R-2. Brown solid, mp= 255–258 ꢃC. Yield: 83%. FT-
IR (KBr, n/cmꢁ1): 2924, 2806, 2359, 1817, 1609, 1538, 1422,
1390, 1306, 1265, 1197, 1162, 1133, 1009, 936, 825, 779, 700,
614, 565. Elemental analysis data: calcd (%) for C84H96N8O4: C
68.98, H 6.62, N 7.76. Found (%): C 68.96, H 6.63, N 7.75. ESI-
MS: m/z: 1463 (M+ 1). MS (MALDI, positive): m/z: 1392.25
([M ꢁ 2Cl]+). ICP: 7.51/100 mg, Found:7.55/100 mg. UV–vis
lmax: 322 (14942), 410 (6310)nm.
Hydroxyl radical scavenging activity. Nonsite-specific
hydroxyl radical-mediated 2-deoxy-D-ribose degradation
method was used to determine the ꢂOH radical scavenging as
described by Halliwell et al.49 The reaction mixture contained
ascorbic acid (50 mM), FeCl3 (20 mM), EDTA (2 mM), H2O2
(1.42 mM), 2-deoxy-D-ribose (2.8 mM), and different concen-
tration of chiral macrocyclic Mn(III) salen complexes S-1,
R-1, S-2, and R-2 (0–7.5 mM) in a final volume of 1 ml in potas-
sium phosphate buffer (10 mM, pH 7.0). The aforementioned
resulting mixture was incubated at 37ꢃC for 1 h, and then,
1 ml of 2.8% trichloroacetic acid (TCA) and 1 ml of 1% thiobarbi-
turic acid (TBA) were added. The mixture was heated in a
boiling water bath for 15 min. It was cooled, and absorbance
was measured at 532 nm. The suppression ratio was calculated
by using the eq. 1.
DNA Binding Study
DNA binding experiments and antioxidant activities of the complexes
were done in a phosphate buffer (10 mM, pH 7.0) using the complex
stock solution which was prepared in Dimethyl Sulphoxide (DMSO),
and all the experiments were conducted by 1% DMSO in phosphate
buffer. In order to evaluate the quantitative DNA binding affinity with
chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2, the
intrinsic binding constants were obtained by absorption spectroscopic
method. The absorption titration experiments were performed by adding
the increasing concentration of DNA solution (0–55 mM) to a fixed con-
centration of chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2,
and R-2 (50 mM), and the spectra were recorded at 300–450 nm. The
intrinsic binding constant was determined by monitoring the changes of
absorbance at ligand to metal chrage transfer (LMCT) position.
Determination of the affinity of chiral enantiomers interaction with DNA
helix was carried out by CD spectroscopy. CD spectra were recorded on a
JASCO J-815 spectrometer at a scanning speed of 50nmminꢁ1 at room
temperature using the fixed concentration of the complexes solution S-1,
R-1, S-2, and R-2 (50 mM) in DMSO and increasing amount of CT-DNA
(0–50mM) solution.
For viscosity measurements, the flow time was measured with digital
stopwatch using Ostwald’s viscometer at 30ꢄ 0.01 ꢃC. The fixed concentration
of CT-DNA solution (50 mM) was mixed with increasing concentration of chiral
macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2 (0–70 mM) in
phosphate buffer (10 mM, pH 7.0). Each sample was measured in triplicate,
and the average flow time was calculated with a digital stopwatch. Data were
presented as (/0)1/3 vs. ([Mn]/ [DNA]), where is the viscosity of DNA in
the presence of the complex and 0 is the viscosity of DNA alone.47
DNA damage protection activity. pUC 19 plasmid
DNA (0.5mg) was treated with chiral macrocyclic Mn(III) salen
complexes S-1, R-1, S-2, and R-2 (0–7.5 mM) and incubated for
15min at room temperature followed by addition of FeSO4
(0.5 mM), H2O2 (20 mM), and phosphate buffer (10 mM, pH
7.4) The total reaction volume was set up to 12 ml, and the
mixture was incubated at 37ꢃC for 20 min. After the incubation,
the extent of DNA damage and the protecting effect of the
chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2,
and R-2 were analyzed on 0.8% agarose gels at 60V at room
temperature.50 The gel was stained with 0.5 mgmlꢁ1 EB, the
DNA bands were visualized using UV light, photographed,
and the intensities of the bands were quantified by using
SYNGENE Gel Document System (United State of America).
The DNA damage protection efficiency of the chiral complexes
was measured in terms of their ability to protect the supercoiled
form (SC DNA). Due corrections were made for the small
amount of open circular (OC) form present in the original SC
DNA and the higher affinity of EB for OC over the SC form.51
To determine the stability of DNA, thermal denaturation experiments
were performed on a TCC 260 temperature controller programmer
UV-3101 PC spectrophotometer by mixing the solutions of S-1, R-1, S-2,
and R-2 (20 mM) in phosphate buffer (10mM, pH 7.0) with a solution of
CT-DNA (0.4mM). The resulting solutions were incubated for 2 min at
different temperatures (25–90ꢃC), and the absorption intensity was
recorded at 260 nm. The melting temperature (Tm) value was determined
from the graph at the midpoint of temperature curve.
Comet assay. Fresh human lymphocytes were isolated
from whole blood by adding 30-ml blood to 1-ml RPMI 1640
with 10% Fetal calf serum on ice for 30 min and centrifuged
200g (Eppendrof Centrifuge 5804 R, Germany) for 5 min at
4 ꢃC with Histopaque 1077. The isolated lymphocytes were
washed with phosphate buffer saline and centrifuged again.52
The lymphocytes were preincubated with different con-
centration of chiral macrocyclic Mn(III) salen complex
S-1 (0–10 mM) for 30 min at 37 ꢃC. After pretreatment,
cells were washed with phosphate buffer saline and treated
with H2O2 for 15 min in ice. The treated samples were then
centrifuged at 200g for 3 min at 4 ꢃC. Positive control sam-
ples were treated with phosphate buffer saline with H2O2,
and negative control samples were treated with phosphate
buffer saline only. For the comet assay, the control and
treated cells were suspended in 1% low-melting tempera-
ture agarose dissolved in phosphate-buffer saline. 100-ml
aliquot of the solution was layered onto a base slide, which
was precoated with 1% agarose and then covered with a cover-
slip. When the agarose gel solidified, the coverslip was gently
Antioxidant Activity
Superoxide dismutase assay. The superoxide radicals
were generated in the test system by using NBT/VitB2/MET
and determined spectrometrically by NBT photoreduction
method with a minor modification.44,48 Accordingly, increasing
concentration of chiral complexes S-1, R-1, S-2, and R-2
(0–7.5 mM) were added to a mixture containing NBT (65 mM),
L-MET (13 mM), VitB2 (1.5 mM), ethylenediaminetetraacetic
acid (EDTA) (0.1mM), and phosphate buffer (10 mM, pH
7.0). The aforementioned mixture was illuminated with a white
fluorescence lamp (15 W) for 15min, and its absorbance (Ai)
was measured at 560 nm. The aforementioned mixture without
the test compounds was used as control, and its absorbance
Chirality DOI 10.1002/chir

DNA BINDING, ANTIOXIDANT ACTIVITY
8.32 for its ꢂOH and H-CN proton, respectively. The IR spectra
slid off, and another top agarose layer (80 ml, 0.5% low-melting
temperature agarose) was added into slide while covering it
with a new cover slip and then left for 10 min on a chilled
metal plate for solidification of the agarose layer. After
solidification, the cover slip was removed carefully, and the
slides were submerged in alkali lysis solution (2.5 M NaCl,
10 mM Trizma, and 100 mM EDTA) at pH > 13 kept for over-
night at 4 ꢃC. Thereafter, the glass slide was incubated in cold
electrophoresis buffer (0.3M NaOH and 1 mM EDTA, pH 13)
in horizontal electrophoresis tank (model PowerPac 200, Bio-
Rad Co., USA) for 30 min at room temperature to allow for
DNA unwinding. Electrophoresis was performed for 10 min
at 25V and 300 mA in a chamber cooled in an ice bath. After
electrophoresis, the glass slides were neutralized in 0.4 M
Tris–HCl (pH 7.5) buffer, washed twice in distilled water,
and left overnight for drying at room temperature. The slides
were stained following the silver staining method.53 Stained
slides were examined using an Olympus Microscope model-
BX60 (Japan) fitted with an Olympus-DP72 camera. The classi-
fication of comet category and their tail measurements was
carried out according to Garcia et al.54
of chiral ligands showed two characteristic peak near
1628cmꢁ1 for u(H-CN) and 3430cmꢁ1
assigned for u(ꢂOH),
respectively; however, after coordination with metal ions, the
characteristic u(H-CN) peak shifted to a lower wave number
1613cmꢁ1
while the intensity of phenolic ꢂOH peak diminished
significantly, confirming its involvement in complex formation.
The UV–vis spectra of chiral macrocyclic Mn(III) salen
complexes were recorded in aqueous DMSO (1%). The chiral
macrocyclic Mn(III) salen complexes S-1 and R-1 showed
shoulder at 315 and 400 nm; however, the complexes S-2 and
R-2 showed shoulder at 322 and 410 nm.
DNA Binding Properties
Absorption titration spectroscopy was used to measure the
interaction of chiral macrocyclic Mn(III) salen complexes S-1,
R-1, S-2, and R-2 with CT-DNA (Figs. 1 and 2). The
complexes binding with DNA through intercalation generally
results in hypochromism and bathochromism of the absorption
spectral bands due to the strong stacking interaction between
the aromatic chromophore of the complexes with the base pairs
of DNA.55 The changes in the absorption spectra of chiral
complexes in the presence of DNA were monitored in the wave-
length range 300–450 nm. Because of the addition of increasing
concentration of DNA solution into the solution of the
complexes, peak showed hyperchromism at LMCT position,
and the binding affinity was observed for the present
complexes in the order S-1 > R-1 > S-2> R-2 (Figs. 1
and 2). These spectroscopic characteristics suggested that all
the complexes have some interaction with DNA. The intrinsic
binding constant of chiral metal complexes with DNA was
obtained by monitoring the changes in absorbance at LMCT
position and calculated by following functional equation.
RESULT AND DISCUSSION
Synthesis and General Properties
The chiral macrocyclic Mn(III) salen complexes S-1, R-1,
S-2, and R-2 were synthesized in high yield via the reaction
of respective chiral macrocyclic salen ligands S-10, R-10,
S-20, and R-20 with Mn(CH3COO)2ꢂ4H2O under refluxing con-
dition followed by the addition of LiCl with aerobic oxidation
(Scheme 1). The complexes were characterized by various
spectroscopic techniques, and data is given in experimental
section. Chiral salen ligands displayed the molecular ion peak
(M+ 1) corresponding to their ESI-MS spectra. In the mass
spectra, the molecular ion peak was observed for complexes
S-1 and R-1 at m/z = 1267 [M+ 1] and complexes S-2 and
R-2 at m/z = 1463 [M+ 1] in ESI mode, and the reasonable
fragment peak was detected at m/z= 1,196 ([Mꢁ2(Cl)]+) for
complexes S-1 and R-1 and 1,392 ([M-2(Cl)]+) for complexes
(2)
½DNAꢅ=ðea ꢁ efÞ ¼ ½DNAꢅ=ðeb ꢁ efÞ þ 1=Kbðeb ꢁ efÞ
where [DNA] is the concentration of DNA in base pairs, ea
corresponds to Aobs/[Mn], ef and eb correspond to the extinc-
tion coefficients of the free chiral macrocyclic Mn(III) salen
complex and of the chiral macrocyclic Mn(III) salen complex
in fully bound form, respectively. Binding constant (Kb) was
obtained from the ratio of the slope to intercept by using the
plot of [DNA]/(eaꢁef) vs. [DNA] (Figs. 1 and 2). The Kb values
for complexes S-1, R-1, S-2, and R-2 are 1.20ꢀ 106, 1.51ꢀ 105,
1
S-2 and R-2 in MALDI mode. The H NMR spectra of chiral
salen ligands S-10 and R-10 showed singlet at d 13.75 and
d 13.78 for phenolic ꢂOH proton and d 8.27 and d 8.23 for its
1
H-CN proton, respectively. However, the H NMR spectrum
of S-20 and R-20 showed singlet at d 13.66, 13.68 and d 8.31,
Fig. 1. Absorption spectra of chiral macrocyclic Mn(III) salen complexes (50 mM). (A) S-1 and (B) R-1 in phosphate buffer (10 mM, pH 7.0) in the presence of
increasing concentration of DNA (0–55 mM). Inset plot is [DNA]/(ea ꢁ ef) vs. DNA.
Chirality DOI 10.1002/chir

PANDYA ET AL.
Fig. 2. Absorption spectra of chiral macrocyclic Mn(III) salen complexes (50 mM). (A) S-2 and (B) R-2 in phosphate buffer (10 mM, pH 7.0) in the presence of
increasing concentration of DNA (0–55 mM). Inset plot is [DNA]/(ea ꢁ ef) vs. DNA.
Fig. 3. Circular dichorism spectral changes of chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2 in the presence of increasing concentration of
DNA (0–50 mM). Complex concentration = 50 mM.
2.57 ꢀ 105, and 9.03 ꢀ 104 Mꢁ1, respectively. These Kb values
clearly indicate that the enantioselective approach of the com-
plexes accentuate the stronger binding affinity of S-complexes
with DNA as compared with R-complexes. The Kb values of
complex S-1 were quite lower than the classical intercalator
such as EB (1.43 ꢀ 106 Mꢁ1) indicating the intercalative mode
of binding;56 however, complexes R-1, S-2, and R-2 bind with
DNA through groove binding or external binding nature.
The CD spectroscopic technique was utilized to study
the interaction of chiral macrocyclic Mn(III) salen complexes
S-1, R-1, S-2, and R-2 with DNA which provides direct
information on how the DNA helix and chiral Mn(III) salen
complexes interact with each other.
The CD spectra of S-1, S-2 metal complexes and their
respective enantiomers R-1, R-2 showed bands of opposite
configuration at 320 and 420 nm (Fig. 3). Because of the
addition of increasing concentration of DNA solution into
the complexes S-1 and R-1, the 320-nm peak showed a blue
shift, and spectral strength decreased by 31.26% and 9.51%,
respectively; however, the complexes S-2 and R-2 showed
only decreased spectral strength by 19.17% and 10.41%,
respectively. These results could be attributed to distinct
interaction behavior of both the enantiomers with DNA.
Among all the complexes studied here, complex S-1 showed
strong interaction with DNA.
To further explore the DNA binding ability, viscosity study
was carried out to determine the relative viscosity of CT-DNA
in the presence and absence of chiral metal complexes. An
intercalative binding mode of complexes with DNA is known
to increase the base pair separation which in turn increases
the relative viscosity of DNA. However, groove binding or
surface binding with DNA generally results to either minor
or no change in the relative viscosity of DNA. 57,58 As shown
in Figure 4, complex S-1 induced the maximum increase in
the relative viscosity due to the strong interaction with
DNA. A comparison of the relative viscosity changes with
EB (DNA intercalator) and Hoechst dye (DNA groove
binder) indicated that complex S-1 bind intercalative with
DNA. The complexes R-1, S-2, and R-2 showed only minor
changes in the relative viscosity, demonstrated the groove
or surface binding nature of the complexes with DNA.
Thermal denaturation experiments were carried out to
investigate the effect of DNA duplex stability due to the binding
with metal complexes. Duplex DNA becomes unwind to give
single-stranded DNA and thus increase the absorbance at
260 nm. The DNA intercalator EB stabilized the duplex DNA
to a significant extent and thus results DNA to melt at consider-
ably higher temperature.59,60 The Tm of CT-DNA alone was
found to be 70 ꢃC; however, in the presence of complex S-1, it
was increased up to 80 ꢃC. The complex S-2 showed the
Chirality DOI 10.1002/chir

DNA BINDING, ANTIOXIDANT ACTIVITY
TABLE 1. IC50 values of chiral macrocyclic Mn(III) salen
complexes against the free radicals.
IC50 value (mM)
Free radicals
scavenging
S-1
R-1
S-2
R-2
O2ꢁscavenging
1.36
2.37
4.20
5.30
3.43
4.09
4.85
5.15
ꢂ
ꢂOH scavenging
S-2, and R-2 (Table 1) strongly suggest that chiral complexes
possess excellent antioxidant activities, much better than
standard antioxidants like butylated hydroxyltoluene and bu-
tylated hydroxyanisole.44,51,61 The chiral macrocyclic Mn(III)
salen complexes showed antioxidant activity in the order
of (S)-1 > (S)-2 > (R)-1 > (R)-2 and indicates that the S
enantiomers of the complexes are more active than their
respective R enantiomers.
Fig. 4. The effect of increasing concentration of ethidium bromide (EB),
Hoechst dye, and complexes S-1, R-1, S-2, and R-2 on the relative viscosity
of calf thymus (CT) DNA. [CT-DNA] = 50 mM.
Hydroxyl radical scavenging activity. Hydroxyl radical
scavenging activities of chiral complexes S-1, R-1, S-2, and
R-2 were determined at the same concentration as mentioned
earlier (0–7.5 mM). The present chiral complexes showed sig-
nificantly higher ꢂOH scavenging activities as compared with
butylated hydroxyanisole and butylated hydroxyltoluene.51,61
It is evident from Figure 7 and Table 1 that complex S-1 pos-
sesses strong antioxidant activities as compared with other chiral
metal complexes, and it indicates that the chiral complexes may
have driven differential and selective nature for scavenging
ꢁ
ꢂ
and ꢂOH radicals. We have also conducted the negative
O2
control experiments parallel, while incubating the reaction
mixture with 1% DMSO solvent only, but there is no ꢂOH
scavenging ability.
Fig. 5. Thermal denaturation graph of calf thymus DNA (0.4 mM) in the
presence of S and R enantiomers of chiral macrocyclic Mn(III) salen
complexes S-1, R-1, S-2, and R-2 (20 mM).
DNA Damage Preventive Activity by Chiral Macrocyclic Mn
(III) Salen Complexes
increase in Tm value (75 C) of CT-DNA; however, R-1 and
R-2 does not alter the Tm value of DNA (Fig. 5). Complex S-1
showed the high Tm value which indicates the stronger DNA
binding through intercalation.
Hydroxyl radicals generated by fenton reactants and attack
on DNA may result in strand breakage and transformation
from SC DNA to OC DNA or linear DNA.62 The protective
effect of complexes S-1, R-1, S-2, and R-2 was confirmed
through pUC 19 plasmid DNA damage induced by ꢂOH radi-
cals. Control plasmid DNA showed two bands, one of OC
DNA that was slightly visible and the other of SC DNA form.
Treatment with FeSO4 and H2O2 in the absence of any chiral
Antioxidant Activity
Superoxide radical scavenging activities. The SOD
activities of the chiral macrocyclic Mn(III) salen complexes
were determined by NBT photoreduction method. All the
metal complexes inhibited the reduction of NBT chloride,
with the increasing concentration of complexes ranging from
0 to 7.5 mM (Fig. 6). The IC50 values of complexes S-1, R-1,
Fig. 6. Scavenging effect of the chiral macrocyclic Mn(III) salen com-
Fig. 7. Hydroxyl radical scavenging activities by chiral macrocyclic Mn
plexes on O2ꢁ by MET/VitB2/NBT system.
(III) salen complexes.
ꢂ
Chirality DOI 10.1002/chir

PANDYA ET AL.
Fig. 8. (A) Electrophoretic pattern of pUC 19 plasmid DNA breaks by ꢂOH generated from the fenton reaction and prevented by chiral complexes. (B) Percent-
age of DNA damage and protection by ꢂOH shown in bar diagram.
Fig. 9. (A) Electrophoretic pattern of pUC 19 plasmid DNA breaks by ꢂOH generated from the fenton reaction and prevented by increasing concentration of
complex S-1. (B) Percentage of DNA damage and increasing amount of protection by complex S-1 shown in bar diagram.
Fig. 10. Comets showing tails of different length induced by H2O2 and protected by different concentrations of S-1 that pretreated the white blood cells.
complexes led to the formation of OC DNA by the strand
scission of SC DNA, while the presence of 7.5 mM of chiral
complexes protected the strand scission of plasmid DNA to
the considerable extent, and results are given in Figure 8A.
Densitometry analysis and percent of DNA protection are
shown in Figure 8B. Bar diagram represents that the control
plasmid DNA contains 92% SC and 8% OC forms; however, in
the presence of FeSO4 and H2O2, the 92% SC DNA have been
completely converted to OC DNA form. The presence of
of SC DNA form increased with parallel reduction of the OC
DNA with increasing concentration of complex S-1. We have
also conducted the negative control experiments parallel,
while incubating the plasmid DNA with chiral complex S-1
alone, where complex alone is not capable to cleave DNA
(data not shown).
DNA Damage Protection by Comet Assay
chiral complexes protected the DNA damage against the
White blood cells (WBC) were extracted from the healthy
human volunteers, were used to study the extent of DNA
damage protection by chiral complex S-1, and were quanti-
fied by comet assay (Fig. 10). The DNA damage protection
was quantified by measuring the displacement of genetic
material between the cell nucleus (comet “head”) and
ꢂOH radical, being complex S-1 protected significant 89%
DNA damage over the other complexes. Further, we have
evaluated the protection of DNA damage against ꢂOH by
using increasing concentration of complex S-1 (0–7.5 mM),
and results are shown in Figure 9A and 9B. The percentage
Chirality DOI 10.1002/chir

DNA BINDING, ANTIOXIDANT ACTIVITY
Fig. 11. White blood cell incubated with DMSO and complex S-1 solution alone and the tail of the DNA damage observed by comet assay.
resulting “tail”, and migration of tail length was calculated by
computerized image analysis.54 The human WBC was
incubated in the presence of H2O2, resulted in greater DNA
damage, and indicated by high tail moment values with major-
ity of comet category 4. However, the WBC cells were
preincubated with complex S-1 and showed a decrease in
the tail moment values with the comet of categories 1 and 2,
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