Organoruthenium(II) compounds with pyridyl benzoxazole/benzthiazole moiety: studies on DNA/protein binding and enzyme mimetic activities

Article abstract of DOI:10.1080/00958972.2017.1309649

We report herein synthesis and characterization of four new organoruthenium(II) complexes of the type [RuH(CO)(PPh₃)₂(L_1,2)]Cl (1, 3) and [Ru(CO)(Cl)₂(AsPh₃)(L_1,2)] (2, 4) derived from the reaction of [RuHCl(CO)(EPh₃)₃] (E?=?P or As) with 2-(pyridine-2yl)benzoxazole (L₁) and 2-(pyridine-2yl)benzthiazole (L₂). Single-crystal X-ray diffraction data of 2 proved octahedral geometry of the complexes with a 1 : 1 ratio between the metal and the coordinated ligands. The binding affinities of 1–4 toward calf-thymus DNA (CT-DNA) and BSA were thoroughly studied by various spectroscopic techniques. Furthermore, the coordination compounds exhibit catecholase-like activities in the aerial oxidation of 3,5-di-tert-butylcatechol to the corresponding o-quinone and phosphatase-like activities in the hydrolysis of 4-nitrophenyl phosphate to 4-nitrophenolate ion. The kinetic parameters have been determined using Michaelis–Menten approach. The highest k_cat values suggested that coordination compounds exhibit higher rates of catalytic efficacy.

Full text of DOI:10.1080/00958972.2017.1309649

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Organoruthenium(II) compounds with pyridyl
benzoxazole/benzthiazole moiety: Studies on
DNA/protein binding and enzyme mimetic
activities
Asaithambi Gomathi, Paranthaman Vijayan, Periasamy Viswanathamurthi,
Shanmugam Suresh, Raju Nandhakumar & Takeshi Hashimoto
To cite this article: Asaithambi Gomathi, Paranthaman Vijayan, Periasamy Viswanathamurthi,
Shanmugam Suresh, Raju Nandhakumar & Takeshi Hashimoto (2017): Organoruthenium(II)
compounds with pyridyl benzoxazole/benzthiazole moiety: Studies on DNA/protein
binding and enzyme mimetic activities, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2017.1309649
To link to this article: http://dx.doi.org/10.1080/00958972.2017.1309649
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Date: 21 March 2017, At: 01:39

Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2017.1309649
Organoruthenium(II) compounds with pyridyl benzoxazole/benzthiazole
moiety: Studies on DNA/protein binding and enzyme mimetic activities
ASAITHAMBI GOMATHI†, PARANTHAMAN VIJAYAN†, PERIASAMY VISWANATHAMURTHI*†,
SHANMUGAM SURESH‡, RAJU NANDHAKUMAR‡ and TAKESHI HASHIMOTO§
†Department of Chemistry, Periyar University, Salem-636 011, India
‡Department of Chemistry, Karunya University, Karunya Nagar, Coimbatore - 641 114, India
§Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University,
7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan
We report herein synthesis and characterization of four new organoruthenium(II) complexes of
the type [RuH(CO)(PPh3)2(L1,2)]Cl (1, 3) and [Ru(CO)(Cl)2(AsPh3)(L1,2)] (2, 4) derived from the
reaction of [RuHCl(CO)(EPh3)3] (E = P or As) with 2-(pyridine-2yl)benzoxazole (L1) and
2-(pyridine-2yl)benzthiazole (L2). Single-crystal X-ray diffraction data of 2 proved octahedral
geometry of the complexes with a 1:1 ratio between the metal and the coordinated ligands. The
binding affinities of 1-4 toward calf-thymus DNA (CT-DNA) and BSA were thoroughly studied
by various spectroscopic techniques. Furthermore, the coordination compounds exhibit
catecholase-like activities in the aerial oxidation of 3,5-di-tert-butylcatechol to the corresponding
o-quinone and phosphatase-like activities in the hydrolysis of 4-nitrophenyl phosphate to
4-nitrophenolate ion. The kinetic parameters have been determined using Michaelis–Menten
approach. The highest kcat values suggested that coordination compounds exhibit higher rates of
catalytic efficacy.
Keywords: Ruthenium(II) compound; X-ray structure; DNA/BSA binding; Enzyme kinetic
studies
1. Introduction
*Corresponding author. Email: viswanathamurthi72@gmail.com
1

Design and synthesis of small synthetic systems that distinguish specific sites of DNA are
essential areas of current research. Such investigations deliver insights for the mechanism of
action of antitumor activity [1-6]. Towards this direction, there is a continuing search for new
metal compounds that strongly interact with DNA [7-9]. Furthermore, investigation of the
interaction of transition metal compounds with DNA helps in the development of new agents that
are potentially useful in molecular biology and also in the design of reputed drugs [10-16].
Moreover, strong evidence has been accumulated, showing that metal-to protein interactions are
also extremely important in promoting the anticancer activity of transition metal compounds and
it has been demonstrated that such interactions could occur with metal ions in either oxidation
states [17-24]. In this regard, ruthenium is the most attractive metal owing to its chemical and air
stability, structural diversity, low toxicity and ability to mimic iron binding in biological systems,
which finally supported ruthenium compounds as highly potent anticancer agents rather than
platinum-based drugs [25-29]. The platinum compounds such as cisplatin and carboplatin are
still hindered by clinical problems, including acquired or intrinsic resistance, limited spectrum of
activity, and high toxicity leading to side-effects. The entrance of two ruthenium-based drugs,
NAMI-A and KP1019, into clinical trials for the treatment of metastatic tumors also increased
the interest in this metal [30, 31].
Subsequently, many effective biological systems based on ruthenium have been
developed because of the fascinating reactivities exhibited by the resultant compounds and the
nature of the ligands that dictate the property of these compounds [32-34]. In this regard,
benzoxazole/benzthiazole ligands have potential biological applications, and hence their metal
compounds exhibit diverse pharmacological applications, due to the structural similarity of
benzoxazole/benzthiazole nucleus with natural compounds [35-37].
On the other hand, catechol oxidase (CO) is a member of the type-III copper proteins,
which catalyzes the oxidation of catechols to quinones, highly reactive intermediates that
undergo auto-polymerization to produce melanin, a brown pigment responsible for protecting
damaged tissues against pathogens and insects of higher plants [38-40]. Researchers have
focused on dinuclear systems to match the original enzyme structurally, where there exists a
dimeric copper active center. Recent investigations have shown that some manganese(II/III),
nickel(II), copper(II), zinc(II) and cobalt(II/III) species can also mediate such catechol oxidation
[41, 42]. Similarly, catalytic cleavage of phosphate esters either by hydrolysis or trans
2

esterification process has received significant attention as an important biochemical process, e.g.
hydrolysis of amino-acid esters by esterases, peptides by peptidases, and phosphate esters by
phosphoesterases. Earlier investigations showed that dinuclear zinc(II), iron(II), copper(II)
compounds were used as mimic catalysts for hydrolysis reaction [43-48]. Therefore, design and
synthesis of functional models for catechol oxidase/phosphatase hydrolysis containing metal ions
other than copper, say ruthenium, seem to be interesting and at the same time challenging.
Based on the above facts and considering the role and activity of ruthenium and its
coordination compounds in biological systems, along with the significance of
benzoxazole/thiazole in medicine, we report in this work a systematic study on the synthesis, the
structural characterization of organoruthenium(II) complexes containing benzoxazole/thiazole
ligands and their interaction with nucleic acids (DNA) and proteins together with
catecholase/phosphatase-like activities.
2. Experimental
2.1. Materials
All reagents and solvents were of analytical grade except those employed in photophysical
experiments, which were of spectroscopic grade. Doubly distilled water was used to prepare all
buffers. Calf-thymus DNA (CT-DNA), bovine serum albumin (BSA) and 4-nitrophenyl
phosphate disodium salt hexahydrate (4-NPP) were obtained from Himedia. Ethidium bromide
(EB) and 3,5-di-tert-butylcatechol (3,5-DTBC) were purchased from Sigma-Aldrich.
2.2. General methods
Elemental analysis (C, H, N, S) were carried out on a Vario EL III CHNS analyzer. Infrared
spectra were recorded as KBr pellets using a Perkin-Elmer FT-IR spectrophotometer from 4000-
400 cm-1. Electronic spectra were obtained on a JASCO V-570 spectrophotometer. 1H, 13C and
31P NMR spectra were recorded at room temperature with a Bruker AV400 instrument with
chemical shifts relative to tetramethylsilane (1H, 13C) and o-phosphoric acid (31P). Mass spectra
for the coordination compounds were performed on an advanced Q-TOF micro™ mass
spectrometer using electro-spray ionization probe. All MS results are given in the form: m/z,
assignment. Fluorescence spectral data were performed on a JASCO FP-8200 fluorescence
spectrophotometer at room temperature. Single crystal X-ray diffraction data collections were
3

carried out at 93.0(5) K on a Rigaku AFC8-CCD diffractometer. Melting points were checked on
a technico micro heating apparatus and are uncorrected. Stock solutions of compounds
(1.0×10-4 M in DMF) were stored at 4 °C and required concentrations were prepared for all
experiments. All the stock solutions used after no more than four days. Solutions of compounds
were prepared freshly 1 h prior to biochemical evaluation. Data have been expressed as the
mean standard deviation from three independent experiments.
2.3. Synthesis of ligands (L1 and L2)
The ligands 2-(2-pyridyl)benzoxazole (L1) and 2-(2-pyridyl)benzothiazole (L2) were prepared
according to reported methods [49].
2-(2-pyridyl)benzoxazole (L1), Yield: 75% (0.213 mg); Color: Yellowish orange; m.p:
130 °C; Micro analytical data for C12H8N2O: C, 73.46; H, 4.41; N, 14.28%. Found: C, 73.21; H,
4.18; N, 14.12%. IR (KBr, cm-1): 1608, ν(C=N1)oxazole; 1572, ν(C=N2)phenyl; 1085, ν(C-O).
UV-vis [CH2Cl2, λmax, nm]: 248, 305. 1H NMR (300.13 MHz; CDCl3, ppm): 8.72 (d, 1H, J=4.5),
7.56 (t, 1H, J=4.4), 7.75 (t, 1H, J=6.5), 7.80 (d, 1H, J=6.1), 8.26 (d, 1H, J=7.8), 7.72 (t, 1H,
J=5.5), 7.31 (t, 1H, J=5.1), 7.36 (d, 1H, J=5.8).
2-(2-pyridyl)benzothiazole (L2), Yield: 78% (0.245 mg); Color: Yellowish orange; m.p:
123 °C; Micro analytical data for C12H8N2S: C, 67.90; H, 3.80; N, 13.30; S, 15.11%. Found: C,
67.73; H, 3.61; N, 13.12; S, 15.34%. IR (KBr, cm-1): 1623, ν(C=N1)thiazole; 1452, ν(C=N2)phenyl
;
1075, ν(C-O). UV-vis [CH2Cl2, λmax, nm]: 314, 235. 1H NMR (300.13 MHz; CDCl3, ppm): 8.66
(d, 1H, J=4.6), 7.52 (t, 1H, J=7.4), 7.83 (t, 1H, J=7.5), 8.10 (d, 1H, J=8.1), 8.32 (d, 1H, J=7.8),
7.44 (t, 1H, J=7.9), 7.35 (t, 1H, J=6.3), 7.94 (d, 1H, J=7.8).
2.4. Synthesis of ruthenium(II) complexes 1-4
2.4.1. [RuH(CO)(PPh3)2(L1)]Cl (1). A solution of [RuHCl(CO)(PPh3)3] (0.100 g, 0.105 mmol)
in ethanol (20 mL) was treated with L1 (0.0481 g, 0.105 mmol) and the mixture was gently
refluxed for 6 h. The reaction was monitored by thin-layer chromatography (TLC) using silica
gel coated glass plates with 2:8 mixture of ethyl acetate/petroleum ether as the mobile phase.
After the reaction was completed, the resulting solution was cooled to room temperature, the
suspension was filtered and the solid was thoroughly washed with cold ethanol and diethyl ether.
Yield: 80% (0.124 mg); m.p: 220 °C; Micro analytical data for C49H39N2O2P2Ru: C, 69.16; H,
4

5.80; N, 3.29%. Found: C, 69.35; H, 5.58; N, 3.12%. IR (KBr, cm-1): 1632, ν(C=N1)oxazole; 1463,
ν(C=N2)phenyl; 1069, ν(C-O); 1945, ν(C O); 1474, ν(C-CH)in-plane; 1432, ν{Ph(P–Ph)}. UV-vis
[CH2Cl2, λmax, nm]: 235, 275, 438. 1H NMR (300.13 MHz; DMSO-d6, ppm): 7.95−7.73 (m, 4H,
Ar H), 7.69−7.44 (m, 8H, Ar H), 7.33−7.09 (m, 12H, Ar H), 6.87−6.68 (t, 8H, Ar H), 6.55−6.51
(m, 7H, Ar H) -6.82 (s, 1H, Ru-H). 13C NMR (100 MHz, CDCl3, ppm): 205.73 (C≡O), 138.12
(Ar C), 137.33 (Ar C), 137.14 (Ar C), 136.15 (Ar C), 135.80 (Ar C), 135.65 (Ar C), 133.54
(Ar C), 133.46 (Ar C), 132.84 (Ar C), 128.97 (Ar C), 128.43 (Ar C), 128.15 (Ar C), 127.95 (Ar
C), 127.69 (Ar C), 127.15 (Ar C), 126.84 (Ar C).31P NMR (162 MHz, CDCl3, ppm) 30.15. MS
(ESI, m/z): 849.2 [M]+.
2.4.2. Synthesis of [RuCl2(CO)(AsPh3)(L1)] (2). 2 was prepared by adopting the procedure used
for the synthesis of 1 by reacting [RuHCl(CO)(AsPh3)3] with L1 in 20 mL of MeOH/CHCl3 (1:1
v/v) solvent mixture. Yield: 75% (0.086 mg); m.p: 180 °C; Micro analytical data for
C31H23N2O2Cl2AsRu: C, 53.00; H, 3.30; N, 3.98%. Found: C, 53.18; H, 3.11; N, 3.72%. IR
(KBr, cm-1): 1634, ν(C=N1)oxazole; 1448, ν(C=N2)phenyl; 1052, ν(C-O); 1934, ν(C O); 1497,
ν(C-CH)in-plane; 1423, ν{Ph(As–Ph)}. UV-vis [CH2Cl2, λmax, nm]: 239, 272, 347. 1H NMR
(300.13 MHz; DMSO-d6, ppm): 7.86−7.63 (m, 4H, Ar H), 7.57−7.41 (m, 4H, Ar H), 7.38−7.12
(m, 4H, Ar H), 6.84−6.53 (m, 6H, Ar H), 6.37−6.13 (m, 5H, Ar H). 13C NMR (100 MHz, CDCl3,
ppm): 207.24 (C≡O), 137.52 (Ar C), 136.85 (Ar C), 136.72 (Ar C), 134.85 (Ar C), 133.72
(Ar C), 132.63 (Ar C), 131.58 (Ar C), 130.43 (Ar C), 130.12 (Ar C), 129.57 (Ar C), 128.94
(Ar C), 127.45 (Ar C), 125.2 (Ar C). (ESI, m/z): 665.8 [M-Cl]+. Single crystals suitable for X-ray
determination were grown by slow evaporation of methanol/chloroform solution of 2 at room
temperature.
2.4.3. Synthesis of [RuH(CO)(PPh3)2(L2)]Cl (3). 3 was prepared by adopting the procedure
used for the synthesis of 1 by reacting [RuHCl(CO)(PPh3)3] with L2. Yield: 82% (0.093 mg);
m.p: 193 °C; Micro analytical data for C49H39N2OSP2Ru required: C, 67.88; H, 4.53; N, 3.23; S,
3.70%. Found: C, 67.59; H, 4.21; N, 3.02; S, 3.51%. IR (KBr, cm-1): 1638, ν(C=N1)thiazole; 1434,
ν(C=N2)phenyl; 1072, ν(C-O); 1962, ν(C O); 1495, ν(C-CH)in-plane; 1417, ν{Ph(P–Ph)}. UV-vis
[CH2Cl2, λmax, nm]: 237, 281, 418. 1H NMR (300.13 MHz; DMSO-d6, ppm): 7.86−7.80 (m, 4H,
Ar H), 7.78−7.64 (m, 8H, Ar H), 7.55−7.32 (m, 14H, Ar H), 6.84−6.52 (m, 12H, Ar H) -6.04 (s,
5

1H, Ru-H). 13C NMR (100 MHz, CDCl3 ppm): 208.31 (C≡O), 139.20 (Ar C), 138.93 (Ar C),
138.81 (Ar C), 138.67 (Ar C), 136.75 (Ar C), 136.12 (Ar C), 133.74 (Ar C), 132.52 (Ar C),
131.67 (Ar C), 129.72 (Ar C), 128.78 (Ar C), 128.63 (Ar C), 127.82 (Ar C), 127.64 (Ar C),
126.92 (Ar C), 126.44 (Ar C). 31P NMR (162 MHz, CDCl3, ppm) 29.32. MS (ESI, m/z): 865.1
[M]+.
2.4.4. Synthesis of [RuCl2(CO)(AsPh3)(L2)] (4). 4 was prepared by adopting the procedure used
for the synthesis of 2 by reacting [RuHCl(CO)(AsPh3)3] with L2. Yield: 75% (0.856 mg); m.p:
188 °C; Micro analytical data for C31H23N2OSCl2AsRu: C, 51.82; H, 3.20; N, 3.89; S, 4.46%.
Found: C, 51.58; H, 3.06; N, 3.68; S, 4.24%. IR (KBr, cm-1): 1639, ν(C=N1)thiazole; 1442,
ν(C=N2)phenyl; 1063, ν(C-O); 1931, ν(C O); 1482, ν(C-CH)in-plane; 1421, ν{Ph(As–Ph)}. UV-vis
[CH2Cl2, λmax, nm]: 238, 282, 333, 355, 470. 1H NMR (300.13 MHz; DMSO-d6, ppm): 7.94−7.85
(m, 4H, Ar H), 7.67−7.44 (m, 4H, Ar H), 7.39−7.17 (m, 4H, Ar H), 6.95−6.52 (m, 6H, Ar H),
6.48−6.16 (m, 5H, Ar H). 13C NMR (100 MHz, CDCl3, ppm): 207.17 (C≡O), 139.69 (Ar C),
138.84 (Ar C), 138.24 (Ar C), 134.55 (Ar C), 130.92 (Ar C), 129.54 (Ar C), 128.81 (Ar C),
128.31 (Ar C), 127.70 (Ar C), 126.14 (Ar C), 126.54 (Ar C), 125.68 (Ar C), 124.32 (Ar C). (ESI,
m/z): 717.2 [M-Cl]+.
2.5. X-ray structure determination
Crystals of 2 were mounted on a glass fiber and used for data collection. The crystal data were
collected at 93.0(5) K using a Rigaku AFC8-CCD diffractometer. Mo-Kα radiation (0.7170 Å)
was used throughout for studies. The absorption corrections were performed by the multi-scan
method. Corrections were made for Lorentz and polarization effects. Structure solutions and
refinements were performed using the programs SHELXS-2014 [50, 51]. The structure was
solved by direct-methods to locate the heavy atoms, followed by difference maps for the light
non-hydrogen atoms. Details of the data collection and refinement are gathered in table 1 and
important bond lengths and angles are summarized in table 2.
2.6. DNA–binding studies
2.6.1. Emissive titration. Fluorescence spectral titrations were performed at room temperature
in Tris-HCl/NaCl buffer (5 mM Tris-HCl/50 mM NaCl buffer, pH 7.2) to investigate the binding
6

affinity between CT-DNA and complexes. Mixing of such solutions with the aqueous buffer
DNA solutions used in the studies never exceeded 5% DMSO (v/v) in the final solution, which
was needed due to low aqueous solubility of most of the compounds. The CT-DNA
concentration per nucleotide was determined using fluorescence spectrometry by the complexes
excitation wavelength. During titration, an equal quantity of CT-DNA was added to both the
complexes solution and reference solution to eliminate the intensity of CT-DNA itself and the
Tris-HCl/NaCl buffer was subtracted through baseline correction. Emissive titration experiments
were performed with a fixed concentration of 1-4 (25 µM). While gradually increasing the
concentration (0-50 µM) of DNA, the emission intensities were recorded for coordination
compounds in the range of 350-650 nm. Titrations were manually done by a micropipette for the
addition of CT-DNA.
2.6.2. EB-displacement assay. DNA binding tendency of the ruthenium complexes also were
measured by the fluorescence based EB displacement studies. EB-displacement experiments
were achieved by observing changes in the fluorescence intensity on the emission wavelength at
λemis = 602 nm at the excitation wavelength at λex = 500 nm, after aliquot addition of
coordination compounds to an aqueous solution of the EB-DNA. EB alone showed minimal
fluorescence and the fluorescence was enhanced significantly with gradual addition of CT-DNA
until maximum fluorescence was attained due to the formation of an intercalative DNA-EB
adduct. Before the emission spectra were recorded, CT-DNA was pretreated with EB in the ratio
[DNA]/[EB] = 1 and kept for 30 min at room temperature in order to fully react. Then, the
complexes were added to this mixture of EB-DNA by manual titration and the changes in the
fluorescence intensity were noted down.
2.6.3. Viscosity experiment. Viscosity measurement was carried out using an Ubbelodhe
viscometer immersed in a thermostatic water bath maintained at 25 0.1 °C. DNA samples with
approximately 200 base pairs in length were prepared by sonication in order to minimize the
complexities arising from DNA flexibility. Flow times were measured with a digital stopwatch;
each sample was measured three times at 5 min interval, and an average flow time was
calculated. Relative viscosities for CT-DNA in the presence and absence of the complexes were
calculated from the relation η = (t–t0)/t0, where t is the observed flow time of DNA-containing
7

solution and t0 is the flow time of Tris–HCl/NaCl buffer alone. Data are presented as (η/η0)1/3
versus binding ratio, where η is the viscosity of CT-DNA in the presence of the complex and η0
is the viscosity of CT-DNA alone.
2.7. Protein binding study
The binding mode of 1-4 with BSA was performed using the fluorescence spectra at room
temperature with an excitation wavelength of BSA at 280 nm and monitoring the emission at
344 nm by keeping the concentration of BSA constant (2.5 µM) while increasing the complexes
concentration (0-50 µM) regularly. The excitation and emission slit widths (each 5 nm) remained
constant for all the experiments. A scan rate of 200 nm min-1 was used. In addition, absorption
titration experiments were carried out by keeping the concentration of the coordination
compounds (20 µM) and the BSA concentration (2.5 µM) as constant. Furthermore, the type of
quenching mechanism of coordination compounds was determined from the UV-visible
absorption spectra in the range of 200-600 nm.
2.8. Catalytic oxidation of 3,5-DTBC
The kinetic experiments were carried out using fluorescence quenching spectra under pseudo
first-order conditions. Quenching of the emission intensity of 3,5-DTBC at λemis = 440 nm (λex =
401 nm) was monitored by addition of the complexes. The compounds (1×10-4 M) in DMF were
added to 100 equivalents of 1×10-3 M solutions of 3,5-DTBC in DMF at aerobic conditions.
Emissive intensity of the resultant reaction mixture was plotted with respect to wavelength at a
regular interval of 15 min in a fluorescence spectrophotometer in the range of 400-700 nm. The
dependence of the rate on substrate concentration and different kinetic parameters were obtained
by treatment of coordination compounds with 3,5-DTBC and monitoring the increase in
emission intensity at 440 nm (the peak corresponding to the quinone band maxima) as a function
of time.
2.9. Phosphate ester hydrolysis
The hydrolysis of 4-NPP catalyzed by ruthenium compounds at room temperature was studied
using fluorescence quenching spectroscopy. The hydrolytic tendency was detected
spectrophotometrically by monitoring the time evolution of p-nitrophenolate in DMF (λmax
=
8

465 nm) through a wavelength scan from 410–700 nm over 2 h. Quenching of the emission
intensity of 4-NPP at 465 nm (excitation wavelength at 401 nm) was monitored using 1-4. The
hydrolase activity involves the preparation of stock solutions of compounds (0.05×10-3 M) and
the substrate 4-NPP (1×10-3 M), at higher concentrations in pure DMF. The dependence of the
rate on substrate concentration and different kinetic parameters were obtained by treatment of
0.05×10-3 M solution of coordination compounds with 40 equivalents of substrate (the peak
corresponding to the phenolate band maxima) as a function of time.
3. Results and discussion
3.1. Synthesis
L1 and L2 were synthesized through the condensation reaction of picolinic acid and
2-aminophenol or 2-aminothiophenol in polyphosphoric acid according to the previous reported
procedures. The ligands react with 1:1 ratio amount of ruthenium precursor complexes in ethanol
or methanol/chloroform mixture to form the corresponding ruthenium complexes 1-4. The
unprecedented formation of 1-4 is shown in scheme 1. The ligands and their complexes were
characterized by elemental analysis, FT-IR, UV-vis, NMR and ESI-mass spectral techniques. In
addition molecular structure of 2 was confirmed by single-crystal X-ray crystallography.
9

Scheme 1. Synthesis of organoruthenium(II) complexes 1-4.
3.2. Structural characterization
The IR spectra provide valuable information regarding the nature of the functional group
attached to the metal atom. The coordinated stretching vibration bands were assigned by
comparing the IR spectra of the free ligands with the spectra of their metal complexes. A strong
band was observed at 1623-1608 cm-1 in the ligands corresponding to oxazole/thiazole ring C=N
which was shifted to 1639-1632 cm-1 in the complexes indicated the participation of
oxazole/thiazole ring nitrogen in bonding. The pyridine ring (C=N) appeared as sharp peak at
1572-1452 cm-1 in free ligands, shifted to lower frequency 1463-1412 cm-1 in all complexes
indicated that pyridine ring nitrogen is coordinated to central metal. The band in the range 1962-
1931 cm-1 was attributed to the stretching mode of the C O group present in the complexes
[52, 53]. In addition, bands appearing in the region 1497-1474 cm-1 in the spectra of complexes
were assigned to -C-CH in the plane stretching vibration. The bonding between ligand and metal
was also confirmed by the presence of new bands at 548-425 cm-1 in complexes resulting in the
M-N (metal–nitrogen) vibration [54].
The UV-vis spectra were recorded in DMSO solution. The free ligand showed two
intense bands around 235-314 nm which were assigned to intra-ligand transitions of the type
π→π* and n→π*. The spectra of the complexes showed bands around 238-353 nm which might
be assigned to ligand-to-metal charge-transfer transition (LMCT) [55]. The low intensity band in
the region 418-470 nm in the complexes is assigned to metal-to-ligand charge-transfer transition
(MLCT) (figure S1). The pattern of the electronic spectra of the complexes indicated the
presence of an octahedral environment around ruthenium(II) ion similar to other ruthenium
complexes [56, 57].
The 1H NMR spectra of the ligands and their complexes were recorded to confirm the
binding of ligand to the metal ion. The spectra of free ligands and their corresponding
coordination compounds showed peaks for aromatic protons in the region 6.13-8.72 ppm
(figures S2 and S3). The hydride ligand signal has been observed in the high field region at -6.82
and -6.04 ppm for 1 and 3 which indicated the complexes 1 and 3 having Ru-H bond, while in
complexes 2 and 4, there is no Ru-H bond [58] (figures S4-S7). 13C NMR spectra of the
coordination compounds showed a peak at 208.3-205.7 ppm due to carbonyl carbon (C O)
10

present in all complexes. The presence of peak at 139.69-137.52 ppm was assigned to
oxazole/thiazole ring C=N group. The aromatic carbon showed their peaks in the region 138.84-
136.85 ppm [59] (figures S8-S11). 31P NMR spectra of 1 and 3 were recorded to confirm the
presence of triphenylphosphine group and their geometry in the complexes. A sharp singlet was
observed around 29.32–30.15 ppm, due to the presence of two magnetically equivalent
triphenylphosphine ligands coordinated in trans position [60] (figure S12).
The ESI-MS spectra of coordination compounds were recorded to confirm their
molecular ions. Complexes 1 and 3 exhibited the molecular ion peaks at m/z 849.2 and 865.1,
respectively, which were assigned to [M]+ ions and 2 and 4 showed peaks at m/z 665.8 and
717.2, respectively, which were assigned to [M-Cl]+ (figures S13-S16). The obtained molecular
masses are in agreement with the calculated molecular masses.
The crystal structure of 2 along with the numbering scheme is given in figure 1. The
crystallographic data and structure refinement parameters for 2 are summarized in table 1 and
selected bond lengths and angles are depicted in table 2. The unit cell packing diagram for 2 is
given as figure S17. Single-crystal X-ray studies revealed that 2 crystallized in triclinic crystal
system with space group P-1. Complex 1 was formed as a cationic complex in which the
chloride ion presents as counter ion in the outside of the coordination spheres (see Supporting
Information, figures S18 and S19).
Complex 2 was expected as a cationic complex similar to 1. Nevertheless, 2 was formed
as a neutral complex due to the solvent (CHCl3) assisted formation according to the Riemer-
Tiemann reaction mechanism. In 2, the basal plane consists of pyridine nitrogen, oxazole
nitrogen of the ligand in its neutral bidentate fashion, one triphenylarsine group and carbonyl
group. The other coordination sites were filled up by two chloride atoms which are present in
trans position. The bidentate pyridyl chelate ligand coordinated equatorially to the metal ion with
the formation of five-membered ring with the bite angle N(5)-Ru(1)-N(2) is 76.13(12)°. The only
one triphenyl arsine group occupies the cis position of the chlorine ligand, the bond angle Cl(1)-
Ru(1)-As(1) is 95.45(7)°. The equatorial bond lengths are [Ru(1)-N(2)] 2.120(4) Å, [Ru(1)-N(5)]
2.234(3) Å, [Ru(1)-C(1)] 1.829(4) Å and [Ru(1)-As(1)] 2.3974(19) Å, and the axial bond lengths
are [Ru(1)-Cl(1)] 2.4630(18) Å and [Ru(1)-Cl(2)] 2.397(2) Å and are comparable with the
distances found in previously reported ruthenium complexes [61-65]. Carbonyl group (CO)
occupies the trans site to the pyridine ring. This may be a consequence of Ru→CO back
11

donation as indicated by the short Ru(1)-C(16) [1.848(4) Å] (1) and Ru(2)-C(32) [1.834(2) Å]
(2) bonds and low stretching frequency, which preferred weak σ- or π-donors occupying the
opposite site to CO favor the back donation. The bonding parameters around the ruthenium
center confirm a slightly distorted octahedral geometry and are in a comparable range to those of
the closely related ruthenium complexes in the literature [66-68]. Unfortunately, the high-quality
crystals of 3 and 4 suitable for X-ray single-crystal diffraction have not been obtained,
suggesting that refined structural factors are critical to stabilize these species. But the similarities
in their spectroscopic characteristics suggest that 1 is a good structural model for 3 and 2 is
structural model for 4.
3.3. DNA binding studies
3.3.1. Stability of compounds. Before carrying out DNA-binding studies, the stability of 1-4
was checked by UV-vis absorption spectra. The spectra for the complexes in the presence of
Tris-HCl/NaCl buffer at four different time points (0 min, 1 h, 6 h and 12 h) were recorded.
The spectral results did not reveal any noticeable change in either the intensity or the
position of the absorption bands in Tris-HCl/NaCl buffer solution. These results established
that the complexes retain a stable structure under physiological conditions (figure S1).
3.3.2. Fluorescence emission technique. In general the interaction of metal compounds with
DNA takes place via both covalent and non-covalent interaction. In the case of covalent binding,
the labile ligand of the complexes is replaced by a nitrogen of DNA-base, whereas in non-
covalent interactions, the metal complexes interact with the outside of the DNA-helix. Emissive
spectroscopy is an effective method to examine the binding mode of metal compounds with
DNA and its magnitude. The emission spectra of 1-4 at fixed concentration (25 μM) is titrated
with varying amounts of DNA (0-50 μM) were recorded. Then, aliquot addition of CT-DNA to
metal complexes leads to continual hypochromism (19.1% for 1, 12.8% for 2, 19.27% for 3,
19.3% for 4) with red-shift of 2-3 nm (figures 2 and S20). The observed hypochromism is due to
an intercalative mode of binding involving a strong stacking interaction between extended
aromaticity of the ligands and the DNA-base pairs. The binding of complexes to DNA leads to
hypochromism, which provides a measure of the strength of the intercalation. In order to
determine quantitatively the binding strength of the complexes with CT-DNA, intrinsic binding
12

constants (Kbin) were obtained by monitoring the changes in the wavelength and the
corresponding intensity of emission of 1-4. The binding constant was obtained by the following
Scatchard equation [69],
CF = CT[I/I0-P]/[1-P]
(1)
where CT is the concentration of the complex added, CF is the concentration of the free complex,
I0, I were its emission intensities in the absence and presence of DNA, respectively, and P is the
ratio of the observed emission quantum yield of the bound complex to the free complex. The
value of P was obtained from a plot of I/I0 vs 1/[DNA] such that the limiting emission yield is
given by their intercept. The amount of bound complex CB at any concentration was equal to
CT −CF. The obtained Scatchard plots of r/CF versus r for tested compounds with increasing
concentration of CT-DNA are depicted in figure S21. A plot of r/CF versus r (= CB/[DNA]) gives
the intrinsic binding constant Kbin and the values are listed in table 3. From the results, it has
been concluded that the complexes bind to DNA via intercalative mode [70-75]. The Kbin values
for 1, 2, 3 and 4 were 4.16×104, 4.83×104, 4.50×104 and 4.99×104 M-1, respectively, which were
lower than the Kbin values reported for [Ru(dmb)2(ipad)](ClO4)2 (2.2×106 M-1),
[Ru(dmp)2(ipad)](ClO4)2 (1.0×106 M-1), [Ru(dip)2(ipad)](ClO4)2 (0.5×106 M-1) [71], but
comparable with [Ru(bpy)2(L1)](PF6)4·CH3OH (5.7×104 M-1), [Ru(bpy)2(L2)](PF6)4·2H2O
(4.1×104 M-1) [76], and larger than those for cis-[Ru(bpy)2(OFX)]Cl·2H2O (1.12×104 M-1), cis-
[Ru(dmbpy)2(OFX)]Cl·2H2O (1.78×104 M-1), cis-[Ru(phen)2(OFX)]Cl·2H2O (2.36×104 M-1)
[77]. The stronger binding affinity of 4 and 2 might be due to slightly longer Ru–As bond
distance when compared to Ru–P bond distance in 3 and 1, which affects how far the aromatic
chromophore would be able to intercalate into the DNA. The Ru–P distance of 2.3738(6) Å is
about 0.09 Å, shorter than the Ru–As bond of 2.4630(8) Å, which places the phenyl rings of the
PPh3 or AsPh3 units closer to the aromatic chromophore by this distance value for the
compounds containing PPh3, thus hindering further intercalation of the chromophore into the
DNA due to steric interaction with the phenyl groups. For compounds containing AsPh3, the
phenyl groups are further removed from the chromophore, thus allowing for a slightly deeper
intercalation accompanied by more pronounced π-stacking interactions between the
chromophore and the DNA-bases resulting in ultimately a slightly higher intrinsic binding
constant [78]. Moreover, the complexes containing benzthiazole ligand showed slightly higher
13

binding ability than the complexes containing benzoxazole ligand [79].
3.3.3. EB-displacement study. Usually, competitive binding experiments were carried out using
metal complexes as a quencher, which provide strong information about the binding of the
complexes to DNA. EB is widely used as sensitive fluorescence probe for native DNA. It emits
intense fluorescent light in the presence of DNA due to strong intercalative interaction between
the adjacent DNA base pair [80]. Interaction of 1-4 with CT-DNA in EB solution leads to
substantial quenching in fluorescence intensity due to the displacement of EB from EB-DNA
adduct. This displacement technique is based on the decrease of fluorescence intensity resulting
from the displacement of bound EB from a DNA sequence by a quencher and the quenching is
due to the reduction of the number of binding sites on the DNA that is available for EB. Hence,
this method serves as indirect evidence to identify intercalative binding modes. While adding
increasing concentration of 1-4 (0-50 μM) to fixed concentration of EB-DNA solution (7.5 μM)
significant decrease in fluorescence intensity with the hypochromism (39.6% for 1, 32.6% for 2,
36% for 3, 52.2% for 4) and noticeable red-shift 2-4 nm was observed (figures 3 and S22). From
this evidence, it is concluded that EB is being released from EB-DNA compound because of its
exchange by corresponding ruthenium complexes. The quenching parameter of 1-4 have been
calculated using the Stern-Volmer equation,
I0/I = 1 + Ksv [Q] (2)
where I0 and I are the emission intensities of EB bound CT-DNA in the absence and presence of
the quencher (complexes) concentration [Q], respectively, which gave the Stern-Volmer
quenching constant (Ksv). The Ksv value is obtained with a slope from the plot of I0/I versus [Q]
which are shown in figure S23. The quenching constant (Ksv) values are listed in table 4.
Further, the apparent DNA binding constant (Kapp) values were also calculated using the
following equation:
KEB [EB] = Kapp [M50%
]
(3)
where KEB = 1.0×10-7 M-1 is the DNA-binding constant of EB, [EB] is the concentration of EB
(7.5 μM) and [M50%] is the concentration of the compound used to obtain a 50% reduction in
14

fluorescence intensity of DNA pretreated with EB. The Kapp value for 1-4 are given in table 4.
3.3.4. Viscosity measurement. Further exploration of the interaction mode between the
ruthenium(II) complexes and CT-DNA was carried out by viscosity measurements with a view
to discover the DNA-binding properties assessed from the above spectral studies. In general, the
viscosity of double-stranded DNA increases when complexes bind DNA in an intercalating mode
but remains unchanged when complexes bind in an electrostatic mode; the groove-binders have
little effect on DNA-viscosity [81]. From this information, these measurements could provide
strong support in favor of intercalative binding. When ruthenium(II) complexes 1-4 were treated
with CT-DNA (200 µM), the concentration of complexes (0-120 µM) increased from a ratio of R
= 0-0.1 (1/R = [compound]/[DNA]) (figure 4), the relative viscosity of DNA solution exhibited
an increase upon addition of the complexes. The observed behavior of DNA-viscosity upon
addition of the compounds may be considered as evidence of the existence of an intercalative
binding mode to DNA, a conclusion that elucidates the preliminary indications derived from
fluorescence spectroscopy studies.
3.4. BSA binding
3.4.1. Fluorescence spectroscopy. Serum albumin is a major transport protein found in the
blood plasma. Studies on binding of metal complexes with protein are becoming increasingly
important for interpreting the metabolism and transporting process. In the present work, BSA
was chosen as model protein due to its structural similarity with human serum albumin. The
binding activity of the complexes with serum albumin has been studied from the concentration
dependence upon the change in the fluorescence intensity of protein after the addition of
complexes [82]. To investigate the interaction mechanism and binding, fluorescence titration
experiments have been performed by using BSA (2.5 μM) and varying concentration of
ruthenium complexes 1-4 (0-50 μM) at room temperature; the results are illustrated in figures 5
and S24. As seen in the figures, the effect of complexes on the fluorescence intensity of protein
at 347 nm show significant trends, indicating that interaction of complexes with BSA could
cause the conformational change in the protein structure. To obtain deep insight into the
quenching progression, the quenching constant (Kq) was analyzed by the Stern-Volmer equation
[69],
15

I0/I = 1+ Ksv [Q] = 1+ Kq τ [Q] (4)
o
where I0 and I are the fluorescence intensity in the absence and presence of the quencher,
respectively, τo is the fluorescence life time of BSA (τ = 10-8 s), kq is the quenching rate constant
o
and Ksv and [Q] are the Stern-Volmer quenching constant and the concentration of the quencher,
respectively. Quenching constants (Kq) have been obtained from the plot of log (I0-I) / I versus
log [Q] (figure S25). The quenching parameters of BSA for all the complexes are shown in
table 4. The quenching mechanism of the complexes-BSA systems have been proved through
following UV-visible spectral study.
3.4.2. UV-vis spectroscopic study. The fluorescence quenching mechanisms are usually
classified as either static or dynamic quenching. Static quenching usually results from the
formation of a complex between the quencher and the fluorophore in the ground state, whereas in
dynamic quenching, the fluorophore and the quencher come into contact with each other during
the transient existence of the excited state. The type of quenching can be determined by using
UV-vis spectral study. The UV-vis absorption spectra of BSA in the presence of complexes are
shown in figure 6. The absorption intensity of BSA was enhanced as the complexes were added,
and there was a little blue/red-shift, indicating that the complexes can interact with BSA by static
quenching mechanism [83].
3.4.3. Binding constant and binding site number. When static quenching interaction occurs, it
is assumed that the complex binds independently to a set of equivalent binding sites in BSA, the
binding parameters can be determined according to the Scatchard equation,
log [I0-I / I] = log Kbin + n log [Q] (5)
where Kbin is the binding constant of the compound with BSA and “n” is the number of binding
sites. The number of binding sites “n” and the binding constant (Kbin) have been found from the
plot of log (I0-I) / I versus log [Q] (figure S26). The calculated values of Kq, Kbin and “n” values
are gathered in table 4. The value of “n”, which is approximately equal to 1, indicates that the
binding site in BSA is unique. The higher values of Kq and Kbin indicated a strong interaction
16

between BSA and the complexes.
3.5. Catecholase activity
The catecholase-like activities of the four organoruthenium(II) complexes were determined by
the catalytic oxidation of 3,5-DTBC. 3,5-DTBC is chosen as model substrate for this reaction,
because its low reduction potential makes it easy to oxidize and bulky tertiary groups present in
the compound prevent further oxidation reaction such as ring opening [84]. However, the
oxidation product 3,5-DTBQ (3,5-di-tert-butyl quinone) is highly stable and exhibited
characteristic emission at λemis at 440 nm in DMF solvent. Based on the above, the bio-catalytic
activity of ruthenium(II) complexes 1-4 for the oxidation of 3,5-DTBC to the corresponding
quinone in DMF were carried out at room temperature by fluorescence emission spectroscopy.
Catecholase like activities of 1-4 (1×10-4 M) were confirmed by adding the complexes to 100
equivalents DMF solution of 3,5-DTBC (1×10-2 M) at room temperature under aerobic
conditions and the progress of the reaction was studied based on the fluorescence spectra of the
mixture at 15 min intervals up to 2 h. Upon addition of 3,5-DTBC, a new band gradually
appeared about 400 nm due to the formation of 3,5-DTBQ (figures 7 and S27). Thus, the
experiment clearly proves that the oxidation of 3,5-DTBC to 3,5-DTBQ which is catalyzed by
the new complexes, as it is well established that 3,5-DTBQ shows a maximum at λemis = 440 nm
in pure DMF.
3.5.1. Kinetics of catecholase activity. The kinetics of the oxidation of 3,5-DTBC to 3,5-DTBQ
was determined by the method of initial rates [85], following the oxidation product 3,5-DTBQ
emission increase at 440 nm. The oxidation rates and various kinetic parameter of the substrate
concentration were determined using 10-4 M solution of complexes with the different
concentration of 3,5-DTBC (10-100 equivalents), under the aerobic condition. In the complexes,
a first-order kinetics was observed at low concentration of 3,5-DTBC. However, in higher
concentration, saturation kinetics was observed. The catalytic behavior treatment was based on
the Michaelis–Menten model seemed to be appropriate under excess substrate conditions [86].
17

The Michaelis–Menten constant (KM) and maximum initial rate (Vmax) were determined
by linearization using Lineweaver-Burk plots (figure S29). The observed rate constants ki were
obtained by initial rate method. Plots of ki versus [3,5-DTBC] gave non-linear curve of
decreasing slope. The turnover number (kcat) values can be calculated by dividing the Vmax values
by the concentration of the corresponding complexes as described in table 5. A probable
mechanistics for 3,5-DTBC oxidation which was promoted by ruthenium(II) complexes is
schematically depicted in scheme 2. Unfortunately, we were unable to characterize the
intermediates. After proper workup, when a mixture of starch-potassium iodide solution was
added to a mixture of complex and 3,5-DTBC, blue coloration developed, which indicates that
hydrogen peroxide was produced during the course of reaction. It is interesting to note that no
blue coloration was observed in the absence of 3,5-DTBC. A believable mechanistic path of the
formation of H2O2 as by product during the oxidation of 3,5-DTBC to 3,5-DTBQ catalyzed
complexes was suggested by Chyn and Urbach [87]. It is concluded that 1-4 belong to the highly
efficient catalyst group.
18

Scheme 2. Probable mechanism for oxidation of 3,5-DTBC by complexes.
3.6. Phosphatase activity
The substrate 4-NPP was chosen to study the phosphatase mimic activity of synthesized
complexes. Its hydrolytic tendency was distinguished using emissive intensities by monitoring
the time evolution of p-nitrophenolate ion (4-NP) in DMF at λemis = 465 nm through a
wavelength scan from 400-800 nm over 15 min intervals up to 2 h. The emission performances
of the complexes are shown in figures 8 and S30.
3.6.1. Kinetic studies. Kinetic studies of phosphatase hydrolysis were performed by the initial-
slope method [85], by monitoring the rate of increase in the emissive intensity band at 475 nm,
which corresponds to increase in 4-NP concentration. The dependence of the initial rate on the
concentration of the substrate was monitored at the respective wavelength by fluorescence
emissive spectroscopy. The hydrolytic cleavage (phosphate ester hydrolysis) rate initially
increases linearly as the 4-NPP concentration increases but deviates progressively from linearity
and finally tends toward saturation curve, and a treatment based on the Michaelis–Menten model
seemed to be appropriate under excess substrate conditions. The kinetic parameters such as Vmax
,
19

KM and kcat for the catalyzed reaction were determined from the plots 1/v versus 1/[NPP]
(Lineweaver Burk plot) as per Michaelis-Menten approach for enzymatic kinetics (figure S32).
Kinetic parameters of all the complexes are given in table 6. The results indicated first-order rate
constant values, which are comparable to the reported values for phosphate bond cleavage [88].
4. Conclusion
A set of four new organoruthenium(II) complexes containing pyridyl benzoxazole/benzthiazole
ligands were synthesized. Characterization of the synthesized complexes was performed by
elemental analysis and various spectroscopic techniques like IR, UV-vis, NMR (1H, 13C, 31P) and
ESI-mass spectrometry. The molecular structure of 1 and 2 was confirmed by single-crystal
X-ray crystallography which revealed an octahedral geometry around ruthenium ion. Stability of
the complexes in Tris-HCl/NaCl buffer was investigated using absorption spectroscopy which
confirmed that they retain their molecular composition in the chosen buffer solution. Binding
behavior of the new organoruthenium(II) complexes with DNA revealed the existence of
intercalative mode of interaction. The binding ability of the new compounds is comparable to
previously reported ruthenium complexes [70, 75, 76]. However, the new complexes are simple
and their syntheses are relatively easy when compared to reported compounds. Moreover, 4 and
2 bind with DNA stronger than 3 and 1, which could be a result of a deeper intercalation of the
chromophore into the DNA due to the longer Ru–As (4, 2) versus the Ru–P (1, 3) bond
distances. From the protein binding studies, the mechanism of quenching of BSA was found to
be a static one, which indicates that the complexes bind to BSA via hydrophobic interaction.
Apart from the above mentioned interaction with biomolecules, 1-4 exhibited promising
catecholase-like activity for the oxidation of 3,5-DTBC to 3,5-DTBQ and phosphatase-like
activity in the hydrolysis of 4-NPP to 4-NP.
Supplementary material
CCDC 1437107 (1) and 1437108 (2) contain the supplementary crystallographic data for this
paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; [Fax: +44–1223/150 336033; E-mail:
deposit@ccdc.cam.ac.uk]. Representative NMR (1H, 13C, 31P) and ESI-MS spectra of the
20

complexes. Unit cell packing diagram for 1 and 2. Fluorescence titrations of 1-3 (25 μM) with
CT-DNA (0-50 μM). Fluorescence titrations of 1-3 (0-50 µM) with EB bound CT-DNA
(7.5 µM). Fluorescence titrations of 1-3 (0-50 μM) with BSA (1 μM). Scatchard and Stern-
Volmer plots of 1-4. Oxidation of 3,5-DTBC by 2-4 monitored by fluorescence spectroscopy.
Hydrolysis of 4-NPP by 2-4 monitored by fluorescence spectroscopy. Non-linear plots of
catecholase and phosphatase activity of 1-4.
Acknowledgement
One of the authors (A.G.) thanks the Department of Science and Technology, Ministry of
Science and Technology, Government of India, for a fellowship under the INSPIRE scheme
(No. IF140098). The authors also thank UGC, India for financial assistance to Department of
Chemistry, Periyar University, Salem under SAP.
21

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26

Graphical abstract
27

Figure 1. Perspective view (30% probability ellipsoids) of 2 with the atom numbering scheme.
28

Figure 2. Fluorescence titrations of 4 (25 μM) with CT-DNA (0-50 μM).
29