Synthesis, characterization, DNA binding and cleaving properties of photochemically activated phenanthrene dihydrodioxin

Article abstract of DOI:10.1016/j.jphotochem.2019.04.014

Dihydrodioxin compounds are formed upon visible light irradiation of a mixture of an ortho-quinone and a substituted alkene. The reverse photochemical reaction can result in the release of a highly reactive ortho-quinone that can induce oxidative DNA damage. A novel DNA binding and cleaving phenanthrene dihydrodioxin compound (1)was synthesized. The photochemical release of 9,10-phenanthrenequinone was studied under different light irradiation wavelengths. Interaction of 1 with DNA caused an increase in the viscosity of DNA as well as hypochromic effect and a bathochromic shift in the absorption of 1. These results indicated the intercalative mode of binding of 1 to DNA. Binding affinities of 1 to several DNA sequences were evaluated, and slightly preferential binding to AT-rich DNA oligomers was observed. Intercalation of 1 between CT-DNA base pairs induced a partial transition from B-form to Z-form of DNA. Significant stabilization of the DNA duplex was revealed by DNA optical melting experiments with 33 % GC 12-mer (?Tm = 9 oC). ΦX174 photocleavage assay showed that 1 is capable of > 90 % single-strand DNA cleavage and ～ 5 % of double-strand DNA breakage. Therefore, 1 further demonstrates the potential of masked orhto-quinones as efficient photoactivated DNA cleaving agents.

Full text of DOI:10.1016/j.jphotochem.2019.04.014

Accepted Manuscript
Title: Synthesis, Characterization, DNA Binding and Cleaving
Properties of Photochemically Activated Phenanthrene
Dihydrodioxin
Author: Anastasiia A. Tikhomirova Nikolai A. Tcyrulnikov
R. Marshall Wilson
PII:
S1010-6030(18)31646-0
DOI:
Reference:
https://doi.org/doi:10.1016/j.jphotochem.2019.04.014
JPC 11803
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
14 November 2018
24 March 2019
10 April 2019
Please cite this article as: A.A. Tikhomirova, N.A. Tcyrulnikov, R.M. Wilson,
Synthesis, Characterization, DNA Binding and Cleaving Properties of Photochemically
Activated Phenanthrene Dihydrodioxin, Journal of Photochemistry and Photobiology
A: Chemistry (2019), https://doi.org/10.1016/j.jphotochem.2019.04.014
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Synthesis, Characterization, DNA Binding and Cleaving Properties of
Photochemically Activated Phenanthrene Dihydrodioxin
Anastasiia A. Tikhomirova, Nikolai A. Tcyrulnikov, R. Marshall Wilson
Center for Pure and Applied Photosciences, Bowling Green State University,
Bowling Green, OH 43403, USA
1. Introduction
DNA molecule remains the most desirable target in anticancer studies [1]. Thus, the
design and characterization of DNA binding and cleaving agents have been an active area of
research for decades [2]. Small molecules can bind to DNA through covalent and non-covalent
interactions [3]. The latter includes three different binding modes: electrostatic interactions with
the phosphate backbone, intercalation between DNA base pairs, and groove binding [4]. Among
these interactions, intercalating binding proved to be one of the most efficient modes for the
development of DNA targeting compounds [5]. Intercalators are small organic molecules with
planar aromatic moieties that insert between DNA base pairs resulting in the modification of the
native DNA structure [6]. The accommodation of an intercalator induces a crucial perturbation in
the DNA molecule: it unwinds the helical twist and causes DNA lengthening [7]. This
deformation might result in the vital errors in such important biological functions as DNA
replication and transcription [8]. Thus, intercalators represent a promising class of compounds
that are used as lead structures for anticancer drugs design [9].
In addition to a distortion of a DNA molecule caused by the intercalation, either thermal
or photoinduced damage can be caused by DNA cleaving agents. Photoswitchable DNA cleavers
were most accurately described by Armitage as ‘compounds whose excited states can initiate a
series of chemical reactions which ultimately lead to nucleic acid cleavage’ [10]. DNA
photocleavers typically absorb at a wavelength longer than 300 nm [11] – the region where
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nucleic acids and the majority of proteins are transparent, which helps to selectively excite a
photocleavage agent. Photoswitchable DNA damaging agents have a number of advantages over
the thermally activated compounds [12]. Among these advantages is the prior binding of a
photocleaver to DNA molecule before the irradiation and ‘light-clicking’ reaction management:
light allows to control the reaction in both spatial and temporal ways [13]. These unique
properties have triggered the search for efficient DNA photocleaving agents. Although
photoswitchable ligand complexes of transition metals have been thoroughly investigated and
their design still remains an extensive research area [14], smaller number of studies cover ‘light-
clicking’ small organic molecules capable of intercalating in DNA structure. Examples of
photoactivated organic compounds include, for instance, enediynes [15], naphthalimide [16] and
pyrene derivatives [17].
Herein, in continuation of our interest in designing new DNA photocleavage agents [18],
we report the synthesis, characterization and DNA binding and cleaving properties of a novel
compound:
trans-N,N-dimethyl-10,11-di(pyridin-4-ium)-10,11-dihydro-9,12-
dioxobenzo[e]phenanthrene tetrafluoroborate (1). As early as in 1947, it was shown by
Schönberg that 9,10-phenanthrenequinone and other ortho-quinone derivatives form a
dihydrodioxin photoproduct when mixed with substituted alkenes under visible light irradiation
[19]. Exposure of dihydrodioxin solutions to UV light can result in the reverse reaction of the
photochemical release of the ortho-quinone and corresponding alkene [20]. This reaction,
therefore, was successfully implemented to photochemically mask highly reactive ortho-quinone
species [18, 21]. It is known that 9,10-phenanthrenequinone, a component of diesel exhaust
particles – carcinogenic air pollutant, can induce the oxidative DNA damage by enhancing the
production of reactive oxygen species [22]. The mechanism of the ortho-quinone release was
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investigated in previous studies [18, 23] in which the formation of an ortho-quinone radical
anion, dihydrodioxin radical cation and Reactive Oxygen Species (ROS) was demonstrated.
These species are well known to cause oxidative DNA damage, which may additionally
contribute to DNA damaging properties of 1 [18, 20, 21, 22, 24]. The interaction of 1 with DNA
was described by a number of spectroscopic analytical tools, such as UV-Vis absorption (DNA
titration and DNA optical melting experiments), circular dichroism (CD) and fluorescence.
Intercalative binding mode of 1 was unambiguously demonstrated by viscometric analysis.
Photolysis of the compound in question with both visible and UV light was performed to better
understand the photochemistry of 1 that is involved in DNA cleavage. ФХ 174 photocleavage
assay was used to evaluate DNA damage triggered by 1.
Main advantages of pyridinium containing dihydrodioxins are: a) ease of synthetic
preparation from commercially available compounds; b) dihydrodioxins present a masked form
of reactive ortho-quinones, release of which can be controlled by irradiation by different
wavelengths of light; c) phenanthrene dihydrodioxin has a higher solubility in biological media
comparing to the previously studied more hydrophobic pyrene dihydrodioxin derivative [18]; d)
combination of flat aromatic moiety with positively charged pyridinium rings allows for both
intercalative and electrostatic interactions of 1 with DNA.
2. Experimental
2.1 Materials and methods
All reagents and solvents were used as received from chemical suppliers with no
additional purification unless otherwise noted. Moisture sensitive reactions were performed
under an atmosphere of argon in glassware that was flame-dried under high vacuum. Moisture
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sensitive reagents were handled in a glovebox in an atmosphere of nitrogen. UltraPure Calf
Thymus DNA (CT-DNA, 10 mg/mL) solution was purchased from Fisher. Dilution of the CT-
DNA stock solution was performed using BioPerformance Certified water (Sigma-Aldrich).
Concentrations of all CT-DNA solutions were calculated in the concentration of base pairs (bp),
using the extinction coefficient of 13200 bp cm-1 M–1 at 260 nm [25]. The purity of CT-DNA
was confirmed by high A260/A280 ratio (1.9), which is a strong indication of a negligible presence
of proteins in CT-DNA solution [26]. All DNA experiments were performed in 10 mM
Phosphate Buffer (PB) solution of pH 7.4 (2 mM NaH2PO4, Sigma-Aldrich, anhydrous,
BioPerformance Certified, >99 %; 8 mM Na2HPO4, Sigma-Aldrich , anhydrous, BioXtra, >
99%) with 100 mM NaCl (Sigma-Aldrich, BioXtra, > 99%) added. BioPerformance Certified
water was used for buffers preparation.
Double-stranded DNA oligonucleotides (Integrated DNA Technologies) used in the
current work included the following sequences (one strand is shown): 5’– ATCGACCAAGC –
3’ (11-mer, 54.5 % of GC content); 5’ – GTTAGTATATGG – 3’ (12-mer, 33.3 % of GC
content); 5’ – GCCGCGCGCGG – 3’ (11-mer, polyGC); 5’ – ATATATATATA – 3’ (11-mer,
polyAT). All samples were purified by Polyacrylamide Gel Electrophoresis (PAGE) procedure
o
[27] by the supplier. DNA oligonucleotides were stored at -20 C. To prepare DNA
oligonucleotides stock solutions, the samples were thawed at room temperature, stirred and
dissolved in BioPerformance Certified water. Concentrations of stock solutions were calculated
using the exact molar amounts and extinction coefficients (see SI) of double-stranded DNA
oligonucleotides provided by the supplier.
Proton and carbon NMR were recorded on a Bruker Avance III 500 Spectrometer
equipped with cryoprobe using tetramethylsilane (TMS) or residual solvent peak as the internal
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standard. IR spectra were recorded on Thermo Scientific Nicolet iS5 ATR IR Spectrometer.
Mass spectrometry data wereobtained on a cyclotron resonance Fourier Transform MS using
electrospray ionization.
2.2
Synthesis
of
trans-10,11-di(pyridin-4-yl)-10,11-dihydro-9,12-
dioxobenzo[e]phenanthrene
To 9,10-Phenanthrenequinone (Sigma-Aldrich, > 99%, 0.52 g, 2.49 mmol) solution in
160 mL of benzene was added a two-fold access of 1,2-di(4-pyridyl)ethylene (Sigma-Aldrich,
97%, 0.91 g, 5.00 mmol). After all reaction components were completely dissolved, the bright
yellow solution was transferred into a photochemical reaction vessel equipped with a gas
dispersion tube with a porous fritted glass tip. The reaction mixture was purged with argon for
1.5 hours and then placed into a photoreactor. The reaction vessel was irradiated by 419 nm
lamps (Southern New England Ultraviolet Company) inside the photoreactor with constant
o
stirring and argon purging for 3 hours at 20 C (the temperature maintained by Haake A82
Temperature Bath / Recirculator filled with ethanol). After 3 hours, the gas dispersion tube was
removed, and the photochemical reaction vessel was tightly closed with a stopper for continuous
overnight irradiation. After the overnight reaction, the solvent from the resulting pale yellow
solution was evaporated under reduced pressure followed by the addition of 160 mL of
methanol. The immediate formation of the off-white precipitate was observed. The reaction
o
mixture was kept in the dark for 12 hours at 0 C. The precipitate was filtered and thoroughly
washed with methanol and diethyl ether to afford off-white crystals. Further recrystallization of
the
solid
from
acetone
gave
trans-10,11-di(pyridin-4-yl)-10,11-dihydro-9,12-
dioxobenzo[e]phenanthrene as an off-white fine crystalline powder (0.26 g, 26 %). Mp = 207-
210 oC (decomp.) 1H NMR (500 MHz, CDCl3) δ 8.67 (d, J = 9.44 Hz, 2 H), 8.60 (d, J = 6.01 Hz,
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4 H), 8.22 (d, J = 9.45 Hz, 2 H), 7.65-7.62 (m, 4 H), 7.12 (d, J = 6.03 Hz, 4 H), 5.09 (s, 2 H). 13C
NMR (125 MHz, CDCl3) δ 150.13, 144.3, 133.3, 127.0, 126.9, 125.7, 125.6, 122.7, 122.4, 120.8,
79.2. IR (ν = cm-1): 3031, 1600, 1450, 1325, 1248, 1112, 1038, 976, 825, 721, 655, 551. HRMS
+
(ESI): m/z calcd. for C26H19N2O2 [M+], 391.14410, found 391.14410.
2.3. Synthesis of trans-N,N-dimethyl-10,11-di(pyridin-4-ium)-10,11-dihydro-9,12-
dioxobenzo[e]phenanthrene tetrafluoroborate (1)
To
a
stirred
solution
of
trans-10,11-di(pyridin-4-yl)-10,11-dihydro-9,12-
dioxobenzo[e]phenanthrene (0.57 g, 0.15 mmol) in 160 mL of dichloromethane (freshly distilled
from P2O5) was added an excess of trimethyloxonium tetrafluoroborate (Sigma-Aldrich, 95%,
0.14 g, 0.97 mmol). The reaction mixture was stirred at the boiling temperature of
dichloromethane for 3 days under an argon atmosphere. After 3 days, the formation of fine pale
yellow crystals was observed. After cooling to room temperature, most of the solvent was
evaporated under reduced pressure leaving approximately 30 mL of the suspension in the flask.
o
This light brown suspension was cooled down to 0 C in an ice bath, vacuum-filtered and
liberally washed with dichloromethane and a few small portions of diethyl ether. The precipitate
obtained was additionally purified by recrystallization from the chloroform/methanol mixture
(5:1) to afford 1 as a pale yellow crystalline powder (0.62 g, 69 %). Mp = 228-230 oC (decomp.)
1H NMR (500 MHz, CD3CN) δ 8.75 (dd, J = 7.86, 1.3 Hz, 2 H), 8.57 (d, J = 6.61 Hz, 4 H), 8.21
(dd, J = 9.35, 1.7 Hz, 2 H), 8.03 (d, J = 6.53 Hz, 4 H), 7.76-7.61 (m, 4 H), 6.20 (s, 2 H), 4.25 (s,
13
6 H). C NMR (125 MHz, CD3CN) δ 155.5, 146.8, 133.0, 128.7, 128.3, 127.5, 127.1, 126.0,
124.1, 121.5, 76.1, 49.2. IR (ν = cm-1): 3078, 1637, 1501, 1457, 1338, 1287, 1191, 1038, 975,
2+
845, 763, 520. HRMS (ESI): m/z calcd. for C28H24N2O2 [M2+], 420.18268, found 420.18266;
+
-
m/z calcd. for C28H24N2O2BF4 [M2+· BF4 ], 507.18615, found 507.18604.
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2.4 Photolysis of 1
Acetonitrile solution of 1 in 1 cm quartz cuvette (Starna Cells) equipped with a screw cap
was placed inside a photoreactor and irradiated by 350 nm UV lamps. The changes inthe UV
spectrum of 1 were monitored every 20 s during a 5 min. time period, every 30 s during a 15
min. time period, and every 60 s within a 5 min. time period and recorded on an Agilent
Technologies UV-Vis spectrometer. Irradiation of 1 in acetonitrile by 419 nm photochemical
lamps was performed according to the same procedure. The concentration of 1 in acetonitrile
was calculated from the absorbance at 305 nm and the extinction coefficient determined by
Beer’s law (ɛ305 = 11150 cm–1 M–1).
2.5 Studies of interaction of 1 with DNA
2.5.1 Viscosity measurements
Measurements were performed using Ostwald type viscometer (Thomas PHILA, USA) at
o
(24.0 ± 0.1) C. A 1 mM solution of CT-DNA base pairs was prepared in 10 mM PB solution
o
with 100 mM NaCl and transferred into the viscometer and equilibrated to 24 C during 5
minutes. The flow-time of CT-DNA solution was measured by digital stopwatch 4 times until the
flow times fell within ± 0.5 s. The CT-DNA solution was then titrated by incremental additions
of 6.74 mM stock solution of 1 in DMSO. The solution of 1 was prepared in DMSO due to the
lower solubility of the studied compound in aqueous media. After each addition of 1 the resulting
solution in the viscometer was mixed by repeated gentle air suction with a disposable syringe
connected to the rubber tube, and then the solution was equilibrated to 24 oC for approximately 5
minutes. The average flow-time after each addition of 1 was calculated based on at least 3 flow-
time measurements the results of which fell within ± 0.5 s. To evaluate the effect of the additions
7
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of the organic solvent (DMSO) on the viscosity of the DNA solution, 2 additional series of the
control titrations were performed: 1) the flow-times of the 10 mM PB solution with 100 mM
NaCl itself and with incremental additions of pure DMSO were measured; 2) the flow-times of
the 1 mM CT-DNA solution in 10 mM PB with 100 mM NaCl itself and with incremental
additions of pure DMSO were determined.
The viscosity of CT-DNA solution before the addition of 1 was calculated according to
the formula ηo = (t – to)/to
where to – flow-time of the buffer without CT-DNA and t – flow-
time of the buffer with CT-DNA added. To calculate the viscosities of the CT-DNA solution
upon incremental additions of 1, the following equation was utilized η = (t1 – to)/to where t1 –
flow-time of the CT-DNA solution with DMSO solution of 1 added. The effect of the organic
solvent on the viscosity of the CT-DNA solution was considered and the viscosities values were
corrected by using the flow-times of the PB (to) and CT-DNA (t) solutions from the control
titrations with pure DMSO. The results of viscometric titrations of the CT-DNA solution by 1
were presented as relative viscosity of CT-DNA, (η/ηo)1/3, plotted vs the ratio of molecules of 1
to the base pairs of CT-DNA, r (1/CT-DNA) [28].
2.5.2 UV-Vis titration
DNA binding was studied by monitoring changes of the UV-Vis absorption spectrum of
1 upon addition of double-stranded DNA oligonucleotides: 11-mer, polyGC, polyAT. Absorption
spectra were recorded in the 200-600 nm range on an Agilent Technologies UV-Vis
spectrometer. PB solutions with a constant concentration of 1 (20-30 µM was used in each
experiment, for exact concentrations see SI) in 1 cm quartz cuvette were titrated by incremental
additions of the DNA solutions until no further absorption changes of 1 were observed. After
each DNA addition, the cuvette was stirred for 60 s. The concentration of 1 in PB was calculated
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from the absorbance at 305 nm and extinction coefficient determined experimentally (ɛ305 = 8979
cm–1 M–1).
Binding constants were determined by plotting the difference in apparent extinction
coefficient of the DNA binder in question vs DNA oligonucleotides concentration [29]. The
following equation of a half-reciprocal plot was used [30]
ꢀ
ꢁ
ꢀ
ꢁ
DNA
DNA
1
=
+
(1)
∆εapp
∆ε
∆εK
where ∆εapp= |εa - εf|, ∆ε= |εb - εf|, and εa , εf and εb are apparent, free (unbound) and bound
extinction coefficient of the DNA binder, correspondingly; [DNA] – molar concentration of
DNA oligonucleotides in solution, K – equilibrium binding constant of DNA with 1. The slope
of the [DNA ]/∆εapp vs. [DNA] plot equals to 1/∆ε, while Y-axis intercept is equivalent to 1/∆εK.
DNA binding constant is therefore defined as the ratio of the graph slope to the intercept.
2.5.3 UV-Vis optical melting experiments
Melting curves were recorded in the absence and presence of incremental additions of 1.
DNA absorbance at 260 nm was monitored on Cary-Bio UV–Vis spectrophotometer (Varian
Inc.) equipped with a thermo controlling cell. Solutions of DNA oligonucleotides in 1 cm quartz
o
o
cuvette were thermostated at 20 C for 3 min. Stirred solutions were heated from 20 C to 60 oC
at 0.5 oC/min. rate. Melting temperatures of pure DNA oligonucleotides, Tm (DNA), and in the
presence of the binder, Tm (DNA-binder), were determined from the first derivative curves
(dA260/dT vs T). Shifts in DNA melting temperatures, ∆Tm, induced by the addition of 1 were
evaluated as the difference between the melting temperature of the DNA-binder mixture and the
melting temperature of the DNA itself.
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2.5.4 Circular Dichroism (CD) spectroscopy
A solution of CT-DNA (30 µM) in 1 cm quartz cuvette was placed in thermoelectrically
controlled cell holder of an AVIV 62DS Circular Dichroism Spectrometer. Spectra of CT-DNA
solution itself and solutions of CT-DNA with increasing amounts of 1 were recorded under a
nitrogen atmosphere at 22.5 oC. Addition of the DNA binder continued until no further change in
the CD spectrum of CT-DNA was observed [31]. CD changes of the CT-DNA solutions were
monitored in the 190-350 nm range.
2.5.5 Fluorescence measurements
A solution of 1 (33 µM) in PB was excited at the 308 nm isosbestic point. Emission
spectra of 1 itself and its mixtures with 11 µM CT-DNA were recorded. After CT-DNA addition
the sample was stirred for 180 s. Fluorescence intensities were monitored at 410 nm.
Measurements were taken in 1 cm quartz cuvette at room temperature on Edinburgh FLS920-stm
steady state spectrofluorimeter (Edinburgh Instruments). Emission was monitored in 360-520 nm
range.
2.6 ФX174 photocleavage assay
A solution of 0.5 µL ФX174 RF I DNA (1 mg/mL, 5386 base pairs length, New England
Biolabs (NEB) was transferred in a 0.6 mL Eppendorf tube (Fisher Scientific, 02-681-300). Then
1 µL of PB solution of 1 of the required concentration was added to the tube and stirred. Three
different concentrations of 1 were used: 1 molecule of 1 per 50 base pairs of ФX174 RF I DNA
(1:50 bp), 1:10 bp and 1:2 bp. The samples were incubated in the dark for 10 minutes. After
incubation, each sample was placed at the 20 cm distance from a He/Cd CW laser (56 series
Melles Griot Co, 70 mW) and irradiated by 442 nm light. Irradiation was performed for up to 60
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minutes in 15 minutes intervals. After irradiation, samples were diluted with 10 µL of PB. DNA
photocleavage was visualized by gel electrophoresis (1 % agarose gel prestained with 0.01% of
GelRed Nucleic Acid Stain, (Biotium) /1X TAE buffer/Blue Juice Loading dye (Thermo
Fisher)/1kb DNA ladder (NEB). Typically gel electrophoresis was run for approximately 3 hours
at 60 V. Gels were visualized using a 302 nm transilluminator (TR-302, Ultraviolet) and
photographed by a digital camera through an ethidium bromide filter. Densitometric analysis of
the DNA bands was performed in Image Studio Lite software.
3. Results and discussion
3.1 Photolysis of 1
Scheme 1. Synthesis and structure of 1.
Phenanthrene dihydrodioxin 1 was synthesized based on the Schönberg-Mustafa reaction
followed by the methylation reaction to produce the final pyridinium salt photoproduct (Scheme
1). To analyze the photochemical properties of 1 and to monitor the 9,10-phenanthrenequinone
release, we performed steady-state photolysis using both UV (350 nm) and visible light (419 nm)
irradiation. Extinction coefficients of 1 in acetonitrile at two different irradiation wavelengths
were determined experimentally as ɛ350 = 1960 cm-1 M–1 and ɛ419 = 550 cm-1 M–1. The 9,10-
11
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phenanthrenequinone, similar to other ortho-quinones has a very distinctive visible n-π*
absorption. Visible absorption band of 9,10-phenanthrenequinone has a small overlap with the
absorption of 1, which allows one to clearly visualize the photochemical release of 9,10-
phenanthrenequinone (Figure 1).
Figure 1. A) Photolysis of 37 µM acetonitrile solution of 1 upon 350 nm UV light irradiation,
(red line – 9,10-phenanthrenequinone, blue line – N,N-dimethyl-1,2-di(pyridin-4-ium)ethylene
tetrafluoroborate (N-methylated alkene); B) Results of the 350 nm light irradiation of a 305 µM
acetonitrile solution of 1; C) Time evolution of the 425 nm absorption band of 9,10-
phenanthrenequinone in 305 µM solution of 1.
12
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Upon UV light irradiation of 1, a pronounced increase of the absorption bands at 320 nm
and 425 nm was observed (Figure 1, A). The enhancement of the 425 nm absorption band can be
attributed to the release of 9,10-phenanthrenequinone which has a distinctive absorption band at
this wavelength (Figure 1, A: red line). The photorelease of 9,10-phenanthrenequinone upon 350
nm UV light irradiation was additionally supported by 1H NMR experiment (See SI). The increase
of the 320 nm absorption band is associated not only with the photochemical release of 9,10-
phenanthrenequinone, but also with the formation of the other product of the reverse Schönberg-
Mustafa reaction – methylated salt of the substituted alkene, trans-N,N-dimethyl-1,2-di(pyridin-
4-ium)ethylene tetrafluoroborate (Figure 1, A: blue line). A clear decrease of the 255 nm band of
1 is related to the transformation of the initial dihydrodioxin into corresponding photoproducts
upon UV light irradiation. Quantum yield of the 9,10-phenanthrenequinone release under UV
light irradiation was determined as 1.8 % (for full details, see SI). Time evolution of the 425 nm
absorption band of 9,10-phenanthrenequinone (Figure 1, C) shows the progress of the ortho-
quinone release caused by UV light irradiation.
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Figure 2. A) Photolysis of 40 µM acetonitrile solution of 1 upon visible light irradiation (419
nm) in acetonitrile; B) 312 µM acetonitrile solution of 1 irradiated by 419 nm light; C)
Comparison of the time evolution of the 425 nm absorption band of 9,10-phenanthrenequinone at
419 nm and 350 nm excitation wavelengths.
Visible light irradiation (419 nm) of 1 resulted in the slower 9,10-phenanthrenequinone
release (Figure 2, C). This difference can be explained by a higher molar absorptivity of 1 at
shorter wavelengths (ɛ350 = 1960 cm-1 M–1 compared to ɛ419 = 550 cm-1 M–1). Although 9,10-
phenanthrenequinone release might take longer time upon visible light irradiation of 1, the
visible light is a less harsh source of stimulus compared to UV light so it could be used in DNA
damaging experiments. It was also previously shown that the formation of the quinone radical
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anion which triggers DNA damage is an autocatalytic process: the system is activated under
visible light irradiation (absorption by the released quinone) and facilitates DNA damage upon
continued light irradiation [18a, b].
3.2 DNA binding mode of 1 studied by viscometry
Viscometry is considered to be one of the least ambiguous and the most conclusive tests
to elucidate the binding mode of a binding agent to DNA [32]. It is well known that classical
DNA intercalators induce the DNA base pairs to separate significantly in order to accommodate
the binding molecule [33]. This separation causes the DNA helix lengthening upon the addition
of intercalative DNA binder. In contrast, the interaction of groove binders with DNA typically
results in a bend in the DNA helix that reduces the effect of the groove binders on the DNA helix
length which either remains constant or decreases insignificantly upon the addition of the groove
binder [34]. Thus, to distinguish the binding mode of 1, the hydrodynamic method sensitive
towards the changes of DNA helix length, i.e. viscometry, was chosen.
15
Page 15 of 45

Figure 3. Change of the relative viscosity of a 1 mM CT-DNA solution in 10 mM PB with 100
mM NaCl (pH = 7.4) at 24 oC upon the increasing concentration of 1 dissolved in DMSO.
The results of the viscometric titrations of the CT-DNA solution with 1 are shown in
Figure 3. It was established that the incremental additions of 1 induce a gradual increase of the
relative viscosity of the CT-DNA solution and, thus, the lengthening of the DNA helix. These
results allow one to suggest the intercalative mode of binding of 1 to DNA. The linear fit of the
plotted data yields a slope of 0.89 when set to intercept the Y-axis at 1 (relative viscosity in the
absence of the intercalator). According to Cohen and Eisenberg [35], similar viscometric studies
of classical intercalators should give a slope value close to 1 which is in a reasonable agreement
with the data obtained for compound 1.
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To further support the intercalative binding mode of 1 and investigate the interactions of
this compound with DNA in more details, several spectroscopic techniques were applied to study
the changes induced upon DNA binding.
3.3 DNA binding studied by UV-Vis absorption and fluorescence spectroscopy
Interactions of a binder with DNA cause electronic perturbations in the DNA-binder
complex. The most common tool applied to evaluate these changes is UV-Vis absorption
spectroscopy. A conclusion regarding the intercalative binding mode of compound 1 drawn after
the viscosity measurements can be further supported based on the changes in the UV-Vis
absorption spectrum of the binder solution upon gradual addition of DNA. For instance,
absorption decrease (hypochromic effect) and red shift of absorption (bathochromic shift) have
been previously observed for a number of compounds with intercalative mode of binding [36].
Hypochromic effect is thought to originate from π-π stacking interactions between DNA base
pairs and the aromatic moiety of an intercalator [37]. The bathochromic shift is related to the
coupling between π orbitals of the intercalated part of a molecule and π orbitals of DNA base
pairs followed by the decrease in the π-π* energy gap [38]. Phenanthrene derivatives are known
to intercalate between DNA base pairs [39] owing to the aromaticity of phenanthrene. Thus, UV-
Vis absorption spectroscopy was chosen to further investigate the interaction of 1 with DNA.
The intercalation process was monitored by observing the changes in the absorption
spectrum of 1 upon gradual addition of different double-stranded DNA oligomers: 11-mer,
polyGC, and polyAT. In each case, a hypochromic effect and bathochromic shift were observed
and evaluated (Table 1). Values of both hypochromic effects and bathochromic shifts are
comparable with the values reported for different DNA intercalating agents [40, 18a]. In addition
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to the DNA intercalation, positively charged pyridinium groups of 1 can electrostatically interact
with the negatively charged phosphate backbone of DNA [18a].
Table 1. Binding constants, hypochroism and bathochromic shifts of 1 upon its titration with
several double-stranded DNA oligomers derived from UV-Vis absorption experiments.
Kb (M-1) per
Kb (M-1) per
DNA bp
Bathochromic
shift (nm)
DNA Sequence
DNA sequence
Hypochroism (%)
polyAT
polyGC
11-mer (54 %
GC)
(1.9 ± 0.3) · 105 (1.7 ± 0.3) · 104
(1.5 ± 0.2) · 105 (1.4 ± 0.2) · 104
26
30
6
5
(1.4 ± 0.1) · 105 (1.3 ± 0.1) · 104
24
6
To determine whether 1 exhibits preferential binding to either type of DNA base pairs,
we performed two UV-Vis absorption titration experiments with polyAT and polyGC DNA
sequences. The gradual addition of these double-stranded DNA oligomers to a solution of 1
resulted in the decrease of absorption of 1 at 305 nm (by 26 and 30 % for polyAT and polyGC
correspondingly) and red shift of the 305 nm characteristic peak (by 6 and 5 nm for polyAT and
polyGC correspondingly) (Figure 4 and 5). A distinctive isosbestic point at 308 nm in both
titrations was observed. The presence of an isosbestic point, a single wavelength at which the
absorption coefficients of two species present in the solution are equal [41], is strong evidence of
the coexistence of two forms of 1: unbound to DNA and bound to DNA. Afore described results
are an additional indication of the intercalative mode of binding of 1 to DNA [42].
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Half-reciprocal plots (Figure 4 and 5, inset C) were obtained based on the data derived
from UV-Vis absorption titration experiments of 1 by solutions of polyAT and polyGC (Figure 4
and 5). Binding constants were calculated according to the equation (1) [29, 30] and gave values
of 105 order of magnitude when estimated per DNA sequence and of 104 when calculated per
base pair (Table 1). No significant difference in hypochromic effects and bathochromic shifts
was observed upon interaction of 1 with polyAT and polyGC. However, binding of 1 to polyAT
appeared to be slightly stronger than to polyGC (KpolyAT/KpolyGC ~ 1.3). Majority of intercalators
demonstrate preferential binding towards GC rich DNA sequences [43], and fewer intercalators
exhibit selective binding to AT-rich DNA sequences. It was previously reported in the literature
that phenanthrene derivatives exhibit higher AT binding specificity [44] compared to GC which
is in agreement with the results observed in this work. Thus, phenanthrene derivatives might be
well suited for the design of photocleavers that target AT sites.
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Figure 4. A) UV-Vis absorption titration of 1 (34 µM) in phosphate buffer by a solution of
polyAT; B) Enlargement of the 290 nm – 330 nm area to examine the isosbestic point; C) Half-
reciprocal plot of 1 binding with polyAT DNA determined form the absorption titration data. For
full details, see SI.
Changes of the absorption of 1 upon addition of the third kind of DNA oligonucleotide
was also monitored by means of UV-Vis absorption spectroscopy (Figure 6). 11-mer DNA
sequence containing 54 % of GC base pairs was chosen to compare its binding constant to 1 with
the corresponding values for polyAT and polyGC oligonucleotides and to calculate the binding
site size of 1. Value of the binding constant of 1 to 54% GC 11-mer was close to the binding
constant of 1 to polyGC and slightly smaller than the one for polyAT (Table 1, also see SI). This
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data correlates with the assumption on the marginally preferential binding of 1 towards AT-rich
DNA sequences.
Figure 5. A) UV-Vis absorption titration of 1 (28 µM) in phosphate buffer by a solution of
polyGC; B) Enlargement of the 290 nm – 330 nm area to examine the isosbestic point; C) Half-
reciprocal plot of 1 binding with polyAT DNA determined form the absorption titration data. For
full details, see SI.
To evaluate the DNA binding site size (n), i.e. the number of DNA base pairs spanned by
one molecule of 1, the absorbance of 1 upon titration with 11-mer DNA sequence was plotted vs
the ratio of DNA concentration in base pairs to the concentration of 1 (Figure 6: B). Upon
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increasing the concentration of DNA, the 11-mer sequence becomes saturated by the intercalator
1. After the point of saturation further addition of 11-mer to 1 results in no significant change of
the absorbance of 1 at 305 nm. The ratio of 11-mer concentration to the concentration of 1 that
corresponds to the saturation point was evaluated by the intersection of two linear plots derived
from UV-Vis absorption titration and expressed in the number of base pairs of the DNA
sequence spanned by one molecule of 1. The DNA binding site size of 1 was evaluated as
3.7±0.1 base pairs. The binding site size of 1 is comparable to other intercalators, such as
phenanthroline based cobalt complexes (n ~ 3 bp) [45], crown ether-annelated styryl dyes (n =
3.7 bp) [46] and the derivative of 1 with pyrene aromatic moiety instead of phenanthrene (n = 4.2
bp) [18a]. A possible reason for the relatively high value of the binding site size of 1 is the
presence of two relatively bulky pyridinium positively charged substituents which extend beyond
the phenanthrene plane. This may prevent denser packing of 1 within DNA base pairs stack.
Values of binding constants of 1 to different DNA sequences are of the same order of magnitude
(~104
–
105 M-1) as binding constants of typical intercalators described in literature: pyrene-
guanidiniocarbonyl-pyrrole cation [17], hydroxybenzo[b]quinolizinium derivatives [2b] and thio-
heterocyclic fused naphthalimides containing aminoalkyl side chains [47]. Based on these data
obtained from UV-Vis absorption studies one can conclude that the binding mode of 1 to DNA is
intercalative.
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Figure 6. A) Determination of the binding site size of 1: titration of a 34 µM solution of 1 in
phosphate buffer by a solution of 11-mer (final concentration of 11-mer was 30 µM); B)
Decrease of the 305 nm absorption plotted vs. the ratio of the 11-mer concentration in base pairs
to the concentration of 1. For full details, see SI.
To support the conclusion of the intercalative nature of the binding mode of 1 to DNA,
fluorescence spectroscopy experiment was performed. The emission spectrum of 1 was collected
after the excitation at the isosbestic point (308 nm) established previously by UV-Vis absorption
titrations of 1 with different double-stranded DNA sequences. This excitation wavelength was
chosen to avoid changes in the extinction coefficient of 1 at the excitation wavelength [2b].
Addition of CT-DNA caused the fluorescence quenching of 1 with no significant deviation of the
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emission maxima peak at 390 nm (Figure 7). The decrease of the fluorescence intensity is
consistent with the formation of 1-CT-DNA complex through a non-covalent interaction
resulting in the insertion of the aromatic planar moiety of 1 between DNA base pairs causing the
quenching of the fluorescence signal of 1 [48].
Figure 7. Comparison of the UV-Vis absorption changes (solid lines) with the emission changes
of a 33 µM solution of 1 in phosphate buffer (dotted lines) upon addition of CT-DNA. Red
curves correspond to the saturation of CT-DNA by 1.
Distinctive hypochromic effect, bathochromic shift and isosbestic point at 308 nm
(Figure 7) observed in the absorption of 1 upon CT-DNA addition along with a pronounced
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quenching of the fluorescence of 1 by CT-DNA strongly support our previous conclusion of the
intercalative nature of the binding mode of 1 to DNA molecules.
3.4 Conformational changes in DNA upon binding with 1 monitored by Circular
Dichroism (CD) spectroscopy
Circular dichroism (CD) spectroscopy is capable of monitoring the absorption difference
between right and left circularly polarized light, which makes this technique particularly useful
for studies of active chiral compounds, such as DNA [49]. Addition of small molecules that can
interact with DNA duplex might trigger DNA conformational changes that can be monitored by
CD Spectroscopy [50]. Double-stranded DNA may exist in several different forms, such as A-
form, Z-form and B-form. The latter is the most common form of DNA double helical structure.
The CD spectrum of this form typically consists of two distinctive bands: a positive band at 260-
280 nm and a negative band at 245 nm [51]. The recorded CD spectrum of CT-DNA solution
(Figure 8) consisted of a positive band at 275 nm which is attributed to the base pairs stacking
and a negative band at 245 nm which arises from helicity of CT-DNA [52]. Positions of these
characteristic bands in the CD spectrum of CT-DNA indicated that CT-DNA exists in a B-form.
While groove binding of the small molecules to DNA have an insignificant effect on the
conformation of DNA and thus no changes in CD spectrum, intercalation of a molecule between
DNA base pairs results in the disturbance of the DNA base pair stacking and its helicity that can
be observed by distinctive changes in a CD spectrum of DNA [53]. Changes in the CD spectrum
of CT-DNA triggered by the interaction with 1 were therefore monitored by means of circular
dichroism spectroscopy (Figure 8).
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Figure 8. Titration of a 30 µM CT-DNA in phosphate buffer by a solution of 1 monitored by
circular dichroism spectroscopy. The molar ratio of 1 to DNA was increased from 0 to 0.5 with
0.05 step (some curves omitted for clarity, for full details, see SI). Arrows show the evolution of
CT-DNA CD bands upon the increasing concentration of 1. Inset – change of the 245 nm CD
band intensity plotted vs the 1/CT-DNA molar ratio.
It is clear from Figure 8 that the gradual addition of 1 to CT-DNA caused major
perturbations in the CD spectrum of CT-DNA. The intensity of the pronounced band at 245 nm
decreased dramatically by more than 60 % and approached more positive values of ellipticity (Y-
axis, expressed in millidegrees or mdeg). After the final addition of 1 to the DNA solution, the
245 nm band was red shifted by 3 nm. The second characteristic CD band of CT-DNA at 275 nm
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demonstrated a similar trend. The intensity of the band decreased by > 40 %, and the peak
maxima red shifted by ~ 9 nm. However, comparing to the 245 nm absorption, this band
decreased towards more negative values of ellipticity during the experiment.
Table 2. Comparison of the characteristic CD bands of B-form of CT-DNA, CT-DNA saturated
with 1, and Z-form of DNA [50, 54].
B-form of CT-DNA
245 nm
CT-DNA after titration by 1
251 nm
Z-form
~ 260 nm
Positive
Negative: -5.0 mdeg
275 nm
Less negative: -2.0 mdeg
286 nm
~ 290 nm
Negative
Positive: 4.1 mdeg
Less positive: 2.5 mdeg
Based on the data derived from the spectroscopic experiment (Table 2), it is reasonable to
assume that the incremental addition of 1 triggers a partial CT-DNA conformational transition
from B-form to Z-form. Among the distinctive features of DNA Z-form are zig-zag phosphate
backbone, left-bended helicity, and characteristic positive CD band at ~ 260 nm and negative
band at ~ 290 nm [50, 54]. In general, a CD spectrum of DNA Z-form represents inversed and
slightly red shifted version of a CD spectrum of DNA B-form. Interaction of 1 with CT-DNA
might induce this inversion as it was previously reported for several other DNA binders [55]. In
this particular case, the transition from B-form to Z-form occurred only partially. A partial
transition from one DNA form to another could be attributed to the CT-DNA reaching a
saturation point at 0.27 ratio of 1/CT-DNA (3.7 ratio of CT-DNA/1, Figure 8, inset). After
achieving this ratio, CT-DNA cannot accommodate more molecules of 1, therefore, no changes
are observed in the CD spectrum of CT-DNA upon further addition of the intercalator. Thus,
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only a partial transition from B-form to Z-form is observed. The number of DNA base pairs
spanned by one molecule of 1 was derived from CD spectroscopic data and calculated as (3.7 ±
0.2). This result correlates well with the binding site size of 3.7 bp obtained from the UV-Vis
absorption binding experiments (Figure 6). After reaching the saturation, the UV-Vis spectrum
of 1 showed no red shifts nor absorption decrease (bathochromic shift and hypochromic effect,
Table 1) upon further addition of the DNA solution. Thus, the values of the binding site size
obtained by means of different spectroscopic methods are in good agreement with each other.
Furthermore, a small induced CD peak at ~ 310 nm, a wavelength corresponding to the
absorption of 1, was observed at higher ratios of 1/CT-DNA. The appearance of a new CD peak
at a wavelength corresponding to the absorption of an intercalator is considered to be extra
evidence of an intercalating binding mode [2b].
3.5 Optical melting experiments
Interaction of small molecules with DNA might result in a number of different effects,
such as conformational changes in DNA structure as discussed in the previous section. The other
effect caused by the intercalation of a molecule between DNA base pairs is the stabilization of
DNA duplex towards double strand separation. One of the main characteristics of the DNA
stabilization is the DNA melting temperature (Tm), which is measured in optical melting
experiments. Tm is defined as a temperature at which 50% of DNA double strands are detached
into the corresponding single strands. By monitoring this parameter upon addition of small
organic molecules one can obtain the information about the nature of DNA binding mode.
Hence, strong binding molecules may cause a significant increase in the melting temperature of
DNA [56]. DNA duplex itself is stabilized by hydrogen bonds between two strands and base
pairs stacking. Intercalators can improve the stabilization of DNA double helix by the insertion
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between base pairs resulting in the additional π-π stacking interactions between DNA base pairs
and the aromatic moiety of the intercalator. Intercalators typically induce an increase of Tm by 8-
12 o C [57].
To evaluate the DNA duplex stabilization by the molecules of 1, we monitored changes
of the 260 nm absorption of the double-stranded DNA 12-mer (5’ – GTTAGTATATGG – 3’, 33
% GC) upon gradual increase of temperature at different concentrations of the intercalator
(Figure 9). The stabilizing effect of 1 was studied for 12-mer/1 ratios ranging from 0 (Tm of DNA
itself) to 21/1, 10.5/1, 5/1 and 3.5/1. Thermal melting curves of DNA-1 complexes displayed a
pronounced shift towards higher values of Tm upon the increasing concentration of the
intercalator. The difference between the Tm of the DNA itself and Tm of the DNA complexes with
1 was therefore found to strongly depend on 1/DNA ratio (Figure 9, inset).
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