Control of electron-transfer and DNA binding properties by the tolyl spacer group in viologen linked acridines

Article abstract of DOI:10.1021/jp027248q

Novel water soluble viologen and pyridinium linked tolylacridines 1a,b and 2a,b were synthesized and their photophysical and DNA binding properties including the photoinduced electron-transfer reactions were investigated. When compared to the cases of the model tolylacridines 3a,b and the pyridinium linked derivatives 2a,b, the singlet excited states of 1a and 1b were efficiently quenched in water and methanol. Intramolecular quenching rate constants (k_ET) calculated in water are found to be 1.2 × 10¹⁰ and 8.8 × 10¹⁰ s^-1 for 1a and 1b and 1.4 × 10⁸ and 0.9 × 10⁸ s^-1 for 2a and 2b, respectively, suggesting thereby that the viologen moiety quenches the fluorescence of the acridine chromophore efficiently when compared to the case of the pyridinium moiety. From the intermolecular electron-transfer studies, it was observed that the singlet and triplet excited states of the acridine chromophore are capable of donating an electron to the viologen moiety. DNA binding studies indicated that the p-tolylachridine derivatives 1a and 2a exhibit strong binding to DNA with binding constants of 1.0 × 10⁵ and 3.3 × 10⁵ M^-1, respectively, whereas the o-tolylacridine derivatives 1b and 2b showed negligible affinity for DNA. The rate constants for the static quenching of 1a and 2a by DNA (k_DNA) are found to be 7 × 10⁹ and 3 × 10⁹ s^-1, respectively, indicating that 1a is an efficient DNA oxidizing agent. Nanosecond laser flash photolysis studies of these systems in aqueous solutions did not show any transients. However, in the presence of DNA, 1a gave transient absorption due to the reduced methyl viologen radical cation. These results demonstrate that the tolyl spacer group in these systems constitutes an interesting variation which controls both the electron transfer and DNA binding properties, and hence, such molecules and derivatives thereof can have potential application as probes for nucleic acids and as DNA cleaving agents.

Full text of DOI:10.1021/jp027248q

4444
J. Phys. Chem. B 2003, 107, 4444-4450
Control of Electron-Transfer and DNA Binding Properties by the Tolyl Spacer Group in
Viologen Linked Acridines
Joshy Joseph, Nadukkudy V. Eldho,† and Danaboyina Ramaiah*
Photosciences and Photonics DiVision, Regional Research Laboratory (CSIR), TriVandrum, India 695 019
ReceiVed: October 18, 2002; In Final Form: February 15, 2003
Novel water soluble viologen and pyridinium linked tolylacridines 1a,b and 2a,b were synthesized and their
photophysical and DNA binding properties including the photoinduced electron-transfer reactions were
investigated. When compared to the cases of the model tolylacridines 3a,b and the pyridinium linked derivatives
2a,b, the singlet excited states of 1a and 1b were efficiently quenched in water and methanol. Intramolecular
quenching rate constants (kET) calculated in water are found to be 1.2 × 1010 and 8.8 × 1010 s-1 for 1a and
1b and 1.4 × 108 and 0.9 × 108 s-1 for 2a and 2b, respectively, suggesting thereby that the viologen moiety
quenches the fluorescence of the acridine chromophore efficiently when compared to the case of the pyridinium
moiety. From the intermolecular electron-transfer studies, it was observed that the singlet and triplet excited
states of the acridine chromophore are capable of donating an electron to the viologen moiety. DNA binding
studies indicated that the p-tolylacridine derivatives 1a and 2a exhibit strong binding to DNA with binding
constants of 1.0 × 105 and 3.3 × 105 M-1, respectively, whereas the o-tolylacridine derivatives 1b and 2b
showed negligible affinity for DNA. The rate constants for the static quenching of 1a and 2a by DNA (kDNA
)
are found to be 7 × 109 and 3 × 109 s-1, respectively, indicating that 1a is an efficient DNA oxidizing agent.
Nanosecond laser flash photolysis studies of these systems in aqueous solutions did not show any transients.
However, in the presence of DNA, 1a gave transient absorption due to the reduced methyl viologen radical
cation. These results demonstrate that the tolyl spacer group in these systems constitutes an interesting variation
which controls both the electron transfer and DNA binding properties, and hence, such molecules and derivatives
thereof can have potential application as probes for nucleic acids and as DNA cleaving agents.
Introduction
Design of molecules targeting nucleic acids is an active area
of research. Several classes of molecules, which induce DNA
modifications by various mechanisms, have been reported in
the literature.1-13 Among these, the photoactivated DNA cleav-
ing agents show some advantages, such as (i) better control of
the reaction trigger, (ii) selectivity of the reaction center by
adjusting the wavelength of irradiation, and (iii) the ability to
control light, which helps to minimize the side reactions and
thus leads to a better understanding of the reaction mechanism.
By absorption of light, these reagents are known to modify DNA
through different mechanisms, including the electron-transfer
reaction, generation of diffusible intermediates, and H-atom
abstraction.14 In the latter two processes, selectivity of the DNA
cleavage is rather difficult to attain, as these reactions are
generally nonspecific, while the former mechanism is shown
to have base selectivity. The major challenges associated with
the DNA cleavage through the electron-transfer mechanism
include (i) efficiency of the DNA oxidation and (ii) selectivity
of the DNA cleavage. The inefficiency associated with such
reactions is the fast recombination of the charge separated
species, such as the oxidized base and the reduced agent. To
improve the efficiency of the DNA cleavage through the
electron-transfer mechanism, a cosensitization approach has been
Figure 1. Schematic representation of the strategy adopted for the
design of photoactivated DNA cleaving agents.
introduced recently.15 In this approach, the photoactivated DNA
cleaving agent comprises a sensitizer, which is either free or
covalently linked to a cosensitizer moiety and wherein both these
moieties are capable of binding to DNA through noncovalent
interactions.15-17
With the objective of developing efficient photoactivated
DNA cleaving agents, which function through the electron-
transfer mechanism,18 we have designed bifunctional derivatives
(Figure 1), where an intercalator (acridine) is linked to a groove
binder (viologen) through a spacer group. The intercalator is
so chosen that it can act as a sensitizer, which upon excitation
can transfer an electron to the viologen moiety, leading to the
radical cation of acridine and the radical cation of viologen.
The radical cation of acridine, once formed, can oxidize DNA
bases with a preference to guanine and thus can initiate oxidative
modification of DNA. The challenging difficulties involved are
* To whom correspondence should be addressed. Telephone: +91
471 2515362. Fax: +91 471 2490186 or +91 471 2491712. E-mail:
d_ramaiah@rediffmail.com or rama@csrrltrd.ren.nic.in.
† Current address: Laboratory of Membrane Biochemistry and Biophys-
ics, NIH, Baltimore, MD 20852, USA.
10.1021/jp027248q CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/12/2003

Properties of Viologen and Pyridinium Linked Acridines
J. Phys. Chem. B, Vol. 107, No. 18, 2003 4445
Synthesis of 9-(4-Bromomethylphenyl)acridine and 9-(2-
Bromomethylphenyl)acridine. A solution of 9-(4-methylphenyl)-
acridine (3a, 1 mmol), N-bromosuccinimide (NBS, 1 mmol),
and benzoyl peroxide (20 mg) in dry CCl4 (20 mL) was refluxed
for 8 h. The reaction mixture was cooled and filtered. The filtrate
was concentrated to give a residue, which was chromatographed
over a silica gel column. Elution of the column with a mixture
(1:4) of ethyl acetate and petroleum ether gave 9-(4-bromo-
1
methylphenyl)acridine in 60% yield, mp 203-204 °C: H NMR
(CDCl3, 300 MHz) δ 4.7 (2H, s), 7.3-8.05 (8H, m), 8.25-
8.50 (4H, m); 13C NMR (CDCl3, 75 MHz) δ 148.60, 146.28,
137.95, 136.04, 130.85, 129.96, 129.54, 129.06, 126.52, 125.69,
125.69, 124.92, 32.88. Exact mol wt calcd for C20H15NBr (M
+ H)+: 348.0388. Found: 348.0384 (high-resolution mass
spectrometry).
Figure 2. Structures of the tolylacridine derivatives 1a,b, 2a,b, and
3a,b under investigation.
to get a control over the DNA binding and photoinduced
electron-transfer reaction to produce long-lived charge-separated
species, which regulate the efficiency of the DNA cleavage
through this mechanism.
Similar reaction of 9-(2-methylphenyl)acridine (3b) with NBS
in dry CCl4 in the presence of benzoyl peroxide yielded 9-(2-
bromomethylphenyl)acridine in 56% yield, mp 136-137 °C:
1H NMR (CDCl3, 300 MHz) δ 4.05 (2H, s), 7.10-7.90 (10H,
m), 8.15-8.45 (2H, m); 13C NMR (CDCl3, 75 MHz) δ 148.46,
144.04, 136.49, 135.30, 130.62, 129.75, 129.54, 129.07, 128.26,
126.23, 125.63, 124.96, 30.34. Exact mol wt calcd for C20H15-
NBr (M + H)+: 348.0388. Found: 348.0394 (high-resolution
mass spectrometry).
Another objective of our investigation is to examine the
effect of the spacer group on the DNA intercalating property
of the acridine moiety, which in turn can control the efficiency
of the DNA photocleavage. Here we describe our results on
the photophysical and DNA binding properties, including intra-
and intermolecular electron-transfer reactions of a few viologen
and pyridinium linked tolylacridine derivatives (Figure 2). These
results demonstrate that the tolyl spacer group constitutes an
interesting variation and plays a major role in controlling the
photophysical and DNA binding properties of these novel
molecules.
Synthesis of the Viologen Linked Tolylacridines 1a and 1b.
A solution of 9-(4-bromomethylphenyl)acridine (1 mmol) and
1-butyl-4,4′-bipyridinium bromide (2 mmol) in dry acetonitrile
(30 mL) was stirred at 30 °C for 12 h. The precipitated solid
was filtered and washed with dry acetonitrile and dichloro-
methane to remove the unreacted starting materials. The solid
was further purified by Soxhlet extraction with dichloromethane
and recrystallization from a mixture (4:1) of ethyl acetate and
acetonitrile to give 77% of 1a, mp 268-269 °C: 1H NMR
(DMSO-d6, 300 MHz) δ 0.85-1.05 (3H, t, J ) 2.89 Hz), 1.15-
1.55 (2H, m), 1.80-2.20 (2H, m), 4.70-4.90 (2H, t, J ) 2.80
Hz), 6.3 (2H, s), 7.50-8.40 (12H, m), 8.75-9.05 (4H, m),
9.42-9.60 (2H, m), 9.70-9.85 (2H, m); 13C NMR (DMSO-d6,
75 MHz) δ 149.23, 1483.61, 147.74, 146.19, 146.00, 145.76,
136.21, 134.42, 131.09, 130.64, 129.36, 128.94, 127.26, 126.79,
126.38, 126.21, 124.23, 63.87, 60.64, 32.70, 18.77, 13.33; MS
m/z 561 (M+Br-, 5), 481 (M+, 100), 424 (10). Anal. Calcd for
C34H31Br2N3: C, 63.66; H, 4.87; N, 6.55. Found: C, 63.81; H,
5.09; N, 6.42.
Experimental Section
General Techniques. The equipment and procedure for
melting point determination and spectral recordings are de-
scribed in earlier publications.19,20 The fluorescence quantum
yields were determined by using optically matching solutions.
9-Aminoacridine in methanol (Φf ) 0.99) was used as the
standard.21 The fluorescence lifetimes were measured on an
Edinburgh FL900CD single photon counting system. Fluores-
cence lifetimes were determined by convoluting the instrumental
function with a mono- or biexponential decay and minimizing
the ø2 values of the fit to 1 ( 0.1. Laser flash photolysis
experiments were carried out in an Applied Photophysics model
LKS-20 laser kinetic spectrometer using the third harmonic (355
nm) of a Quanta Ray GCR-12 series pulsed Nd:YAG laser. The
DNA binding studies were carried out in 10 mM phosphate
buffer containing 2, 50, and 100 mM NaCl. The DNA binding
affinities were calculated according to the method of McGhee
and von Hippel by using the data points of the Scatchard
plot.22-24 The spectroscopic measurements in water were carried
out at pH 9 to avoid the protonation of the excited state of
acridine. Petroleum ether used was the fraction with bp ) 60-
80 °C.
Materials. 4,4′-Bipyridine and methyl viologen dichloride
hydrate (98%) were obtained from Aldrich and used as received.
A solution of calf thymus DNA (Pharmacia Biotech, USA) was
sonicated for 1 h and filtered through a 0.45 µM Millipore filter.
The concentrations of DNA solutions were determined by using
the average value of 6600 M-1 cm-1 for the extinction
coefficient of a single nucleotide at 260 nm.25 1-Butyl-4,4′-
bipyridinium bromide was obtained in 95% yield by the reaction
of 4,4′-bipyridine with 1-bromobutane in the molar ratio 3:1 in
dry acetonitrile.26 The synthesis of 9-(4-methylphenyl)acridine
(3a), mp 188-189 °C (lit. mp 189 °C),27 and 9-(2-methylphenyl)-
acridine (3b), mp 212-213 °C (lit. mp 214 °C),27,28 was
achieved as per the reported procedures.
Similar reaction of 9-(2-bromomethylphenyl)acridine (1
mmol) with 1-butyl-4,4′-bipyridinium bromide (2 mmol) in dry
acetonitrile and purification as in the earlier case gave 1b in
75% yield, mp 224-225 °C: 1H NMR (DMSO-d6, 300 MHz)
δ 0.80-1.00 (3H, t, J ) 2.89 Hz), 1.05-1.50 (2H, m), 1.70-
2.10 (2H, m), 4.65-4.90 (2H, t, J ) 2.85 Hz), 5.60 (2H, s),
7.15-8.70 (16H, m), 9.30-9.55 (4H, m); 13C NMR (DMSO-
d6, 75 MHz) δ 148.50, 147.80, 145.82, 145.27, 142.77, 135.64,
132.08, 131.90, 131.36, 130.55, 130.44, 129.98, 129.47, 127.08,
126.68, 126.49, 125.95, 125.34, 124.24, 62.02, 60.64, 32.68,
18.78, 13.35; MS m/z 561 (M+Br-, 7), 481 (M+, 100), 424 (7).
Anal. Calcd for C34H31Br2N3: C, 63.66; H, 4.87; N, 6.55.
Found: C, 63.69; H, 5.06; N, 6.46.
Synthesis of the Pyridinium Linked Tolylacridines 2a and 2b.
A solution of 9-(4-bromomethylphenyl)acridine (1 mmol) and
dry pyridine (2 mmol) in dry acetonitrile (30 mL) was stirred
at 30 °C for 12 h. The precipitated solid was filtered and washed
with dry acetonitrile and dichloromethane to remove the
unreacted starting materials. The solid was further purified by
Soxhlet extraction with dichloromethane and recrystallization
from a mixture of ethyl acetate and acetonitrile (4:1) to give 2a

4446 J. Phys. Chem. B, Vol. 107, No. 18, 2003
Joseph et al.
1
in 87% yield, mp 270-272 °C: H NMR (DMSO-d6, 300 MHz)
δ 6.05 (2H, s), 7.55-7.60 (6H, m), 7.78-7.86 (4H, m), 8.20-
8.25 (4H, m), 8.67-8.72 (H, t, J ) 5.7 Hz), 9.31-9.33 (2H, d,
J ) 5.6 Hz); 13C NMR (DMSO-d6, 75 MHz) δ 148.63, 146.67,
145.56, 135.21, 134.90, 131.12, 131.623, 130.90, 129.85,
129.55, 129.13, 126.83, 126.65, 124.72, 63.58. Exact mol wt
calcd for C25H19N2 (M+): 347.1548. Found: 347.1546 (high-
resolution mass spectrometry). Anal. Calcd for C25H19BrN2: C,
70.26; H, 4.48; N, 6.56. Found: C, 70.06; H, 4.55; N, 6.61.
Similar reaction of 9-(2-bromomethylphenyl)acridine (1
mmol) with dry pyridine (2 mmol) in dry acetonitrile and
purification as in the earlier case gave 2b in 83% yield, mp
245-246 °C: 1H NMR (DMSO-d6, 300 MHz) δ 5.39 (2H, s),
7.16-7.19 (2H, d, J ) 8.6 Hz), 7.41-7.57 (5H, m), 7.79-
7.85 (5H, m), 8.1-8.22 (5H, m); 13C NMR (DMSO-d6, 75
MHz) δ 148.58, 147.40, 145.98, 142.47, 135.54, 132.24, 131.36,
130.24, 129.98, 129.81, 127.36, 126.95, 126.62, 126.01, 125.45,
123.52, 61.01. Exact mol wt calcd for C25H19N2 (M+):
347.1548. Found: 347.1551 (high-resolution mass spectrom-
etry). Anal. Calcd for C25H19BrN2: C, 70.26; H, 4.48; N, 6.56.
Found: C, 70.34; H, 4.50; N, 6.67.
Figure 3. Absorption spectra of 1b (6.8 × 10-5 M), 2b (6.6 × 10-5
M), and o-tolylacridine (3b, 6.4 × 10-5 M) in water. The inset shows
the fluorescence emission spectra of 2b (1.3 × 10-5 M), 1a (1 × 10-5
M), and 1b (1.2 × 10-5 M) in water. Emission spectra of 1a and 1b
are expanded by a factor of 20 for clarity. Excitation wavelength, 355
nm.
Cyclic Voltammetry. The cyclic voltammograms were
recorded on a BAS CV-50W voltammetric analyzer. A glassy
carbon working electrode was used along with platinum
auxiliary and Ag/AgCl (3 M NaCl) reference electrodes. The
redox potentials obtained were calibrated using ferrocene as an
internal standard. The potentials were measured in dry aceto-
nitrile, which contained 0.1 M tetrabutylammonium tetrafluo-
roborate as the supporting electrolyte. Samples were degassed
for 20 min before recording the voltammogram. The sweep rate
used was 100 mV/s. Square wave techniques were used to get
the half-wave potentials under similar conditions. The measured
oxidation potential of 3a was found to be 1.6 V vs SCE while
1-ethylpyridinium bromide and methyl viologen exhibited
reduction potentials of -1.34 and -0.45 V vs SCE, respec-
tively.29,30
TABLE 1: Absorption and Fluorescence Characteristics of
the Tolylacridine Derivativesa
absorption
emission
λ
max, nm (ꢀ, M-1 cm-1
)
102Φfb
τ, nsc
compd methanol
water
359
(12100)
361
methanol water methanol water
1a
359
(11300)
361
0.8
0.60
0.08
1.7
0.4
1.2
1.1
2.9
0.91
5.4
0.4
1b
0.05
(9000)
585
(9100)
10.6
3.9
(2250)
359
(10750)
361
(9150)
356
2a
2b
3b
359
(10300)
360
(8550)
356
3.8
3.2
5.8d
26
32
8.5
Free Energy Calculations. The free energy of the electron-
transfer reaction (∆Gel) from the singlet/triplet excited states
of acridine to the acceptor molecules was calculated using the
Rehm-Weller equation (eq 1)31,32
55.0
7.8
(10450)
(9200)
a Average of more than two experiments. b Fluorescence quantum
yields are calculated using 9-aminoacridine as standard (Φf ) 0.99);16
error ca. (5%. c Picosecond time-resolved fluorescence studies showed
the existence of a short-lifetime species (τ < 50 ps) in the case of
viologen linked acridines which could not be satisfactorily resolved
within the instrument time scale. For details see the Supporting
Information. d Fluorescence quantum yield and lifetime of p-tolylacri-
dine (3a) in methanol are 0.059 and 0.74 ns, respectively.
∆Gel ) E°(D) - E°(A) - E(0,0)
-
e2
1
1
1
1
e2
ꢀsdcc
+
-
-
(1)
(
)(
)
2 rD rA 37 ꢀs
where E°(D) is the oxidation potential of the donor (acridine),
E°(A) is the reduction potential of the acceptor (viologen or
pyridinium), E(0,0) is the singlet/triplet excited-state energy of
acridine (singlet and triplet energies are 3.26 and 1.95 eV,
respectively),33 ꢀs is the dielectric constant of the solvent used,
rD and rA are the radii of the donor and acceptor molecules,
and dcc is the center-to-center distance between the ions. The
values of rD, rA, and dcc were estimated using a computer
modeling program.34 The changes in free energy values
calculated for the electron transfer (∆Gel) from the singlet
excited state of acridine to viologen are found to be -1.24 and
-1.27 eV in methanol and water, respectively. For the pyri-
dinium systems, relatively lower change in free energy values
were observed (-0.36 and -0.40 eV in methanol and water,
respectively).30 Similarly for the electron transfer from the triplet
excited state of acridine to the viologen moiety, ∆Gel values
are calculated to be 0.07 and 0.04 eV in methanol and water,
respectively.
Results and Discussion
Absorption and Fluorescence Emission Properties. Figure
3 shows the absorption spectra of the o-tolylacridine derivatives
1b, 2b, and 3b in water. Similar spectra were obtained in the
case of the p-tolylacridine derivatives 1a, 2a, and 3a. The
absorption spectra of both the viologen and pyridinium linked
tolylacridines 1a,b and 2a,b can be best described as the sum
of the absorption bands of the individual units contained in them.
However, a small red shift of around 3-5 nm was observed in
the absorption maxima of the viologen and pyridinium linked
tolylacridines when compared to those of the model acridine
derivatives. There is no evidence for any ground-state charge-
transfer interactions existing between the acridine and viologen/
pyridinium moieties in these systems in water. Table 1
summarizes the absorption properties of the viologen and
pyridinium linked tolylacridines in water and methanol. The
viologen linked o-tolylacridine derivative 1b in dry methanol,

Properties of Viologen and Pyridinium Linked Acridines
J. Phys. Chem. B, Vol. 107, No. 18, 2003 4447
on the other hand, unusually exhibited a broad and structureless
absorption at around 585 nm with time, in addition to the
absorption due to the acridine chromophore at 361 nm. This
broad band decreased with increasing temperature and solvent
viscosity and was not observed in acidic methanol solution. We
tentatively assign this absorption to the existence of ground-
state charge-transfer interaction between the viologen and
acridine moieties in 1b. By following the change in absorption
of the acridine chromophore with pH, the pKa values were
determined and are found to be 4.9 and 3.5 for 1a and 1b,
respectively. The comparison of pKa values indicates that 1b is
more acidic than 1a and further supports the presence of
effective interactions between the acridine and viologen chro-
mophores in the case of the ortho-derivative 1b.
The inset of Figure 3 shows the fluorescence emission spectra
of the viologen linked tolylacridines 1a and 1b and the
pyridinium linked tolylacridine 2b in water. The fluorescence
properties, including the lifetimes of these compounds in water
and methanol, are summarized in Table 1. The fluorescence
yields of 1a and 1b were found to be very low when compared
to those of the pyridinium linked derivatives 2a,b and the model
tolylacridines 3a,b. For example, the pyridinium linked o-
tolylacridine 2b exhibited a fluorescence quantum yield of 0.32
in water, whereas the viologen linked tolylacridine derivatives
1a and 1b exhibited much lower quantum yields of 0.006 and
0.0008, respectively, indicating thereby that the fluorescence
of the acridine unit is efficiently quenched by the viologen
moiety in these systems. Furthermore, the quantum yields of
1a in water and methanol were nearly 6 times higher than that
of 1b, suggesting an efficient quenching in the later case because
of close proximity of the interacting units. In contrast, a reverse
order was observed in the case of the pyridinium linked
derivatives, where the para-derivative 2a exhibited lower
quantum yields of flourescence than that of the otho-derivative
2b (Table 1). This observation reveals that the viologen moiety
in the ortho-position undergoes a more efficient interaction with
the acridine chromophore than that of the ortho-pyridinium
moiety.
Figure 4. Cyclic voltammograms of (A) 1b and (B) 2b in acetonitrile
showing the reduction peaks.
Figure 5. Effect of MV2+ concentration on the fluorescence emission
spectra of o-tolylacridine (3b, 2.9 × 10-5 M) in methanol. [MV2+]:
(a) 0; (b) 5.1; (c) 10; (d) 14.8; (e) 24.1; (f) 32.9; (g) 41.3 mM. Inset
Nanosecond time-resolved fluorescence studies show that the
para-viologen linked tolylacridine derivative 1a and the pyri-
dinium linked derivatives 2a and 2b exhibit single-exponential
decay in both methanol and water (Table 1). In contrast, the
ortho-viologen linked tolylacridine 1b exhibited biexponential
fluorescence decay in these solvents. These observations
establish that 1a, 2a, and 2b exist as single conformers, whereas
1b exists in two conformations in which the viologen moiety
has different orientations with respect to the acridine plane. In
one of the conformations, the viologen moiety lies above the
acridine plane (folded conformation), wherein considerable
spatial interactions exist between the two interacting units. In
another conformer, the viologen is away from the acridine
chromophore (extended conformation), where there is less
chance for the existence of spatial interactions between the donor
and acceptor moieties.18
shows the Stern-Volmer plot for the quenching of 3b with MV2+
Excitation wavelength, 355 nm.
.
whereas the third reduction could be attributed to the acridine
chromophore. Similarly, the para-derivative 1a showed three
reduction waves in the voltammogram with no significant shifts,
when compared to 1b. The pyridinium linked tolylacridine
derivative 2b, on the other hand, showed only two reduction
waves appearing at the half-wave potentials -1.15 and -1.53
V (Figure 4B). These reduction waves can be assigned to the
reduction of the pyridinium and acridine moieties, respectively.
Similar observations were made in the case of the para-
pyridinium linked tolylacridine 2a. The observation of similar
redox potential values for the acridine chromophore in tolyl-
acridines (3a,b), the viologen linked derivatives 1a,b, and the
pyridinium linked derivatives 2a,b indicates that no ground-
state interaction exists between the acridine chromophore and
the acceptor moiety in these systems.
Electron-Transfer Studies. The intermolecular photoinduced
electron-transfer reactions between tolylacridines (3a,b) and
methyl viologen (MV2+) were investigated by a fluorescence
technique. Figure 5 shows the effect of the concentration of
MV2+ on the fluorescence emission spectrum of 3b in methanol,
and the inset of Figure 5 shows the corresponding Stern-Volmer
Cyclic Voltammetry Studies. Figure 4 shows the cyclic
voltammograms of 1b and 2b in acetonitrile. Under similar
conditions, the oxidation of o-tolylacridine (3b) was observed
to occur irreversibly at a half-wave potential of 1.6 V (vs SCE).
Of the three reductions exhibited by the ortho-viologen linked
tolylacridine derivative 1b (Figure 4A), the first two, at the half-
wave potentials -0.35 and -0.80 V, were found to be reversible
while the reduction at the half-wave potential -1.54 V was
observed to be irreversible. On the basis of literature evidence,29
we assign the former two reductions to the viologen moiety

4448 J. Phys. Chem. B, Vol. 107, No. 18, 2003
Joseph et al.
TABLE 2: DNA Binding Constants (K), Intramolecular
Electron Transfer Rate Constants (kET), Sums of Rate
Constants of Singlet Excited State Deactivation (kSI), and
Rate Constants of Quenching by DNA (kDNA) of Viologen/
Pyridinium Linked Tolylacridine Derivatives 1a,b and 2a,ba
compd
K,b M-1
k
ET, s-1
kSI,c s-1
k
DNA, s-1
1a
1b
2a
2b
(1 ( 0.1) × 105
1.16 × 1010 1.17 × 1010 7 × 109
nbd
8.8 × 1010
8.81 × 1010 nbd
(3.3 ( 0.2) × 105 1.4 × 108
2.7 × 108
3 × 109
nbd
0.9 × 108
2.2 × 108
nbd
a Average of more than two experiments. b Calculated on the basis
of fluorimetric titration of the probe against calf thymus DNA in 10
mM phosphate buffer (pH 8) containing 50 mM NaCl. K for 1a at 100
mM NaCl is 4.9 × 104 M-1
.
c kSI ) kd + kET is the sum of radiative
and nonradiative rate constants for deactivation of the acridine singlet
excited state (kd) and the intramolecular electron-transfer rate constant
(kET). d nb ) exhibited negligible binding with DNA.
Figure 6. Transient absorption spectrum obtained immediately after
355 nm laser excitation (pulse width 20 ns) of an argon saturated
solution of o-tolylacridine (3b, 5.1 × 10-5 M) in the presence of MV2+
(1.3 mM). Inset shows the growth of the reduced methyl viologen
radical cation at 395 nm under (a) argon saturated and (b) air saturated
conditions and the decay of the acridine triplet excited state at 440 nm
under (c) argon saturated and (d) air saturated conditions.
plot. The quenching rate constants were calculated by employing
the Stern-Volmer equation (eq 2)
I0/I ) 1 + Ksv[Q]
(2)
and
to 1a and 1b. This fact was further substantiated by the
observation of lower change in free energy values for the
pyridinium systems (∆Gel ) -0.36 and -0.40 eV, in methanol
and water, respectively), when compared to the viologen linked
derivatives (∆Gel ) -1.24 eV and -1.27 eV, in methanol and
water, respectively). These observations indicate that the pyri-
dinium moiety is less efficient in oxidizing the acridine
chromophore when compared to the viologen and that the spacer
group in these systems exhibits a control over the rate of
electron-transfer reactions by positioning the donor and acceptor
moieties at different distances.
Ksv ) kqτ0
(3)
where I0 and I are the fluorescence intensities in the absence
and presence of the quencher (Q), Ksv is the Stern-Volmer
constant, τ0 is the fluorescence lifetime of the tolylacridine in
the absence of quencher, and kq is the quenching rate constant.
On the basis of fluorescence titration data, the calculated
bimolecular fluorescence quenching rate constants for 3a and
3b are found to be 2.51 × 1010 and 2.54 × 1010 M-1 s-1
,
respectively. The observed high fluorescence quenching rate
constants and the calculated change in free energy (∆G ) -1.24
eV in methanol) for the electron transfer from the singlet excited
state of tolylacridine to MV2+ indicate that both these acridine
derivatives (3a and 3b) are capable of donating electrons to
the viologen moiety very efficiently.
Laser Flash Photolysis Studies. Laser flash photolysis
experiments were carried out under different conditions to
characterize the transient intermediates involved in these
systems. For example, Figure 6 shows the transient absorption
spectrum obtained immediately after laser excitation (355 nm,
pulse width 20 ns) of o-tolylacridine (3b) in methanol and in
the presence of methyl viologen (MV2+; 1.3 mM). On the basis
of quenching experiments with molecular oxygen, the transient
species with absorption maxima at 440 nm could be assigned
to the triplet excited state of 3b. As per the literature evidence,35
the other transient with two absorption maxima at 395 and 610
nm could be assigned to the reduced methyl viologen radical
In the case of the covalently linked systems 1a,b and 2a,b,
an estimate of the intramolecular electron-transfer rate constants
(kET) can be made on the basis of the fluorescence quantum
yields and the lifetime of the corresponding model compound
and using eq 4,
kET ) [(Φref/Φ) - 1]/τref
(4)
where kET represents the intramolecular electron-transfer rate
constant, Φref and Φ are the relative fluorescence quantum yields
of the corresponding model compound and the acceptor linked
tolylacridine, respectively, and τref is the fluorescence lifetime
of the model compound. The intramolecular electron-transfer
rate constants for the viologen and pyridinium linked tolylacri-
dine derivatives 1a,b and 2a,b in water were estimated and are
summarized in Table 2.
As shown in Table 2, the ortho-derivative 1b exhibits higher
electron-transfer rate constants in both methanol and water (kET
) 1.3 × 1011 and 8.8 × 1010 s-1), when compared to those of
the para-derivative 1a (kET ) 6.9 × 1010 and 1.16 × 1010 s-1).
This can be attributed to the existence of strong through-space
interactions between the acridine and viologen units in 1b,
whereas no such possibility exists in the case of 1a. The
pyridinium linked tolylacridine derivatives 2a (kET ) 5.8 × 108
and 1.4 × 108 s-1 in methanol and water) and 2b (kET ) 9 ×
108 and 0.9 × 108 s-1), on the other hand, showed nearly 2-3
orders lower electron-transfer rate constants, when compared
cation formed by the electron-transfer reaction from 3b to MV2+
.
Interestingly, the reduced methyl viologen radical cation formed
is found to be quite stable in methanol under argon atmosphere.
The inset of Figure 6 shows the decay of the acridine triplet
and the growth of the methyl viologen radical cation, under
argon saturated and air saturated conditions.
The reduced methyl viologen radical cation can form by
electron-transfer reaction, from either the singlet or the triplet
excited states of acridine to MV2+. To estimate the contributions
of both these excited states, we have examined the electron-
transfer reactions as a function of MV2+ concentration. When
the concentration of MV2+ used was very low (∼10-5 M), the
growth of the methyl viologen radical cation was found to be
concomitant with the decay of the triplet excited state of
acridine. Its formation by quenching of the singlet excited state
would be <5% at these concentrations. At higher concentrations
of MV2+, the growth of the methyl viologen radical cation
became clearly biexponential with a sudden growth immediately
after the laser pulse followed by a slow growth. Under air

Properties of Viologen and Pyridinium Linked Acridines
J. Phys. Chem. B, Vol. 107, No. 18, 2003 4449
Figure 8. Fluorescence emission spectra of 1a (3.2 × 10-5 M) in the
presence of CT DNA in 10 mM phosphate buffer (pH 8) containing
50 mM NaCl. [DNA]: (a) 0; (b) 0.07; (c) 0.14; (d) 0.33; (e) 0.44; (f)
0.64; (g) 0.82; (h) 1.4 mM. Inset shows the Scatchard plot for the
binding of 1a to CT DNA. Excitation wavelength, 355 nm.
Figure 7. Absorption spectra of 1a (3.2 × 10-5 M) in the presence of
CT DNA in 10 mM phosphate buffer (pH 8) containing 50 mM NaCl.
[DNA]: (a) 0; (b) 0.07; (c) 0.14; (d) 0.20; (e) 0.33; (f) 0.44 mM. Inset
shows the absorption spectra of 1b (4.4 × 10-5 M) in the presence of
CT DNA under analogous conditions. [DNA]: (a) 0; (b) 0.14; (c) 0.38;
(d) 0.60; (e) 1 mM.
saturated conditions, on the other hand, the triplet excited state
of acridine was quenched by molecular oxygen, and therefore,
we observed negligible formation of the reduced methyl
viologen radical cation (inset of Figure 6).
Direct 355 nm laser excitation of the viologen linked
tolylacridines 1a and 1b in water or methanol did not show
any transients. However, in the presence of an external donor
such as guanosine, both these derivatives showed transients
corresponding to the reduced methyl viologen radical cation.
This observation can be attributed to the existence of a fast
charge recombination reaction in these covalently linked systems
and the presence of sacrificial donors found to stabilize the
charge-separated species. Interestingly, the laser flash studies
of 1a and 1b in the presence of calf thymus DNA showed
contrasting results. Of these two derivatives, only the para-
derivative 1a, which showed strong affinity for DNA (see
section DNA Binding Studies), exhibited the transient formation
corresponding to the reduced methyl viologen radical cation in
the presence of DNA. These results reveal that effective binding
of the viologen linked tolylacridines to DNA is very essential
for the oxidation of DNA and also for the generation of charge-
separated species.
DNA Binding Studies. To find out how the substitution
affects the DNA intercalating and groove binding properties of
the acridine and viologen chromophores, we have studied the
DNA binding properties of the viologen and pyridinium linked
tolylacridines 1a,b and 2a,b in 10 mM phosphate buffer using
absorption and fluorescence techniques. Figure 7 shows the
change in absorption spectrum of the viologen linked p-
tolylacridine 1a with increasing in concentration of DNA. Upon
increasing DNA concentration, the absorption spectra corre-
sponding to the acridine chromophore showed strong hypo-
chromism (∼40%) as well as a 3 nm bathochromic shift,
suggesting the interaction of the acridine moiety with DNA by
intercalation, whereas, in the case of the ortho-derivative 1b,
the absorption spectra showed negligible changes with the
increase in DNA concentration, as shown in the inset of Figure
7. Similar observations were made in the case of the pyridinium
linked tolylacridine derivatives 2a and 2b, where only the para-
derivative 2a undergoes interaction with DNA, while the otho-
derivative 2b showed negligible affinity for DNA.
Figure 9. Fluorescence emission spectra of 2a (3.7 × 10-5 M) in the
presence of CT DNA in 10 mM phosphate buffer (pH 8) containing
50 mM NaCl. [DNA]: (a) 0; (b) 0.07; (c) 0.14; (d) 0.20; (e) 0.26; (f)
0.38; (g) 0.5; (h) 0.69; (i) 1.22; (j) 1.5 mM. Inset shows the Scatchard
plot for the binding of 2a to CT DNA. Excitation wavelength, 355
nm.
Furthermore, the interaction of these bifunctional molecules
with DNA was studied using the fluorescence technique. Figures
8 and 9 show the change in fluorescence emission spectra of
the viologen linked p-tolylacridine derivative 1a and the
corresponding pyridinium derivative 2a, respectively, with
increasing concentration of DNA.
Only the p-tolylacridine derivatives 1a and 2a exhibited strong
interaction with DNA, while the fluorescence emission proper-
ties of the ortho-derivatives 1b and 2b remained unchanged in
the presence of DNA, indicating thereby the negligible binding
of the ortho-derivatives with DNA. The association constant
(K) of the complex formed between the para-derivatives 1a and
2a and DNA was calculated according to the method of McGhee
and von Hippel and using the data points of the Scatchard plot
(insets of Figures 8 and 9).22-24 The pyridinium linked derivative
2a exhibited a greater association constant (K ) (3.3 ( 0.2) ×
105 M-1) when compared to that of the viologen linked
derivative 1a (K ) (1 ( 0.1) × 105 M-1), indicating thereby
that even the remote steric factors of the substituents perturb
the intercalating interactions of the acridine moiety with DNA.
On the basis of limiting fluorescence intensity (under condi-
tions where >99% of the substrate is bound to DNA), the rate

4450 J. Phys. Chem. B, Vol. 107, No. 18, 2003
Joseph et al.
constant for the static quenching of the singlet excited state of
the photophysical and DNA binding properties of these mol-
ecules. Furthermore, the viologen linked p-tolylacrdine deriva-
tive 1a, which exhibited greater affinity for DNA and oxidizes
DNA very efficiently, can have potential use as a DNA probe
and as a DNA cleaving agent that functions through the
cosensitization mechanism.
the acridine chromophore in these molecules by DNA (kDNA
)
was evaluated using eq 5,
I0/I ) 1 + kDNA/kSI
(5)
where I0 and I are the fluorescence intensities in the absence
and presence of DNA and kSI is the sum of the rate constants
(kd + kET) of the singlet excited-state deactivation.16 The kDNA
values obtained for these derivatives are summarized in Table
2. The static quenching rate constant (kDNA) for 1a was found
to be 7 × 109 s-1, whereas a lower value of 3 × 109 s-1 was
observed for the pyridinium linked derivative 2a. This observa-
tion indicates that the viologen linked p-tolylacridine derivative
1a, though, binds less efficiently but is more efficient in
oxidizing the DNA bases when compared to the corresponding
pyridinium derivative 2a.
Acknowledgment. We are grateful to the Council of
Scientific and Industrial Research (CSIR) and Department of
Science and Technology, Government of India, for the financial
support of the work. This is contribution no. RRLT-PPD(PRU)-
159 from the Regional Research Laboratory, Trivandrum.
Supporting Information Available: Fluorescence decay
profile of 1a. This material is available free of charge via the
Internet at http://pubs.acs.org.
References and Notes
The calculated change in free energy value for the electron-
transfer reactions using oxidation potentials of DNA bases36 and
the reduction potential of acridine (∆G ) -0.64, -0.51, -0.33,
and -0.23 eV for guanine, adenine, cytosine, and thymine,
respectively) shows that all DNA bases can quench the
fluorescence of the acridine moiety by an electron-transfer
mechanism. The extent of fluorescence quenching offered by
DNA depends on the type of interaction of these molecules
undergo with DNA. In the case of the ortho-derivatives 1b and
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2a, the rotation around these interacting units is facile when
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and hence intercalation of the acridine chromophore was
observed only in the case of the para-derivatives.
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