Ground- and Excited-State Interactions of a Psoralen Derivative with

Article Cite This: J. Phys. Chem. B 2018, 122, 2277−2286

pubs.acs.org/JPCB

Ground- and Excited-State Interactions of a Psoralen Derivative with Human Telomeric G‑Quadruplex DNA Sneha Paul and Anunay Samanta* School of Chemistry, University of Hyderabad, Hyderabad 500046, India S Supporting Information *

ABSTRACT: G-quadruplex DNA has been a recent target for anticancer agents, and its binding interactions with small molecules, often used as anticancer drugs, have become an important area of research. Considering that psoralens have long been studied in the context of duplex DNA but that very little is known about their potential as G-quadruplex binders and their excited-state interaction with the latter has not been explored, we have studied herein the binding of a planar watersoluble psoralen derivative, 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT), with the 22-mer human telomeric Gquadruplex-forming sequence, AGGG(TTAGGG)3, labeled here as (hTel22), and investigated the consequences of photoexcitation of AMT by calorimetric and spectroscopic techniques. The results show an enthalpy-driven 1:1 binding of AMT with hTel22 via end-stacking mode. Fluorescence quenching experiments on 6-fluorescein amidite-labeled oligomers indicate that the binding site is nearer to the 3′ end of hTel22 in the diagonal loop region. Femtosecond time-resolved transient absorption measurements indicate electron transfer from the guanine moiety of hTel22 to photoexcited AMT, leading to the formation of a radical pair species (AMT•−G•+), which survives for 30 ps and is favored by a parallel/quasi-parallel orientation between the two. The findings reveal psoralens as a prospective class of compounds for the development of anticancer therapeutics by targeting the G-quadruplex DNA.

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INTRODUCTION Development of drugs and therapies to combat deadly disease “cancer” has been an active area of research for quite some time. In recent years, in addition to the canonical duplex DNA, noncanonical DNA motifs are also potential targets for chemotherapeutics. Among the latter, guanine-rich (G-rich) oligonucleotides capable of folding into four-stranded structures, called G-quadruplex, in the presence of metal ions, have attracted enormous attention.1−3 These structures comprise stacks of G-tetrads formed by association of four guanine molecules via cyclic Hoogsteen and Watson−Crick Hbonding.4 The presence of metal ions (typically K+ or Na+) in the central channel of a quadruplex is vital as they neutralize the negative electrostatic effect of the carbonyl groups of guanine and render stability to the assembly.5,6 G-quadruplexes are structurally diverse, and various factors such as ionic strength, pH, molecular crowding of the medium,7−11 oligonucleotide sequence/length, and flanking nucleotides12,13 influence their conformation, which can broadly be classified into three groups: parallel, antiparallel, and (3 + 1) hybrid structures.4 The G-quadruplex structures have been observed in several plant and animal cells.14−18 In humans, they are present in the oncogene-promoter region where they are involved in gene regulation at the transcription19 and translation levels.20 They are also found in the terminal region of human telomere21 and inhibit the activity of enzyme telomerase, which is dormant in © 2018 American Chemical Society

normal cells but shows enhanced activity in carcinogenic cells, leading to uncontrolled cell growth.22,23 This is why there is an ongoing search for small molecules that can interact and stabilize these structures, and the interactions of several derivatives of acridines,24,25 anthraquinones,26 triazenes,27,28 perylenes,29 porphyrins,30 and tetraphenylethene31 and various natural products32−37 have been explored in recent years. Psoralens are furocoumarins that interact with duplex DNA by intercalation and are employed in PUVA therapy for curing psoriasis and other skin diseases.38,39 Their role in apoptosis and control of tumoral cell growth has also been realized.40 They are particularly found to be effective against breast cancer and T-cell lymphoma.39,40 Even though virtual screening approaches have shown the potential of psoralens as Gquadruplex binders,41 a comprehensive experimental study probing the interactions between the two is lacking. Most of the binding studies involving G-quadruplex and small molecules focus on the ground-state interactions between the two, an understanding of which is essential for the development of anticancer drugs. The literature, however, suggests that oxidation and reduction of nucleobases of DNA (either by direct photoexcitation or through photoinduced processes involving small molecules) play an important role in Received: December 19, 2017 Revised: January 18, 2018 Published: January 27, 2018 2277

DOI: 10.1021/acs.jpcb.7b12475 J. Phys. Chem. B 2018, 122, 2277−2286

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The Journal of Physical Chemistry B

Figure 1. (A) Schematic representation of antiparallel folding topology of hTel22 in aqueous buffer containing Na+ ions (red circles). Guanines in syn conformation are shown as light blue squares, and dark blue squares represent guanines in anti conformation. (B) Structure of 4′-aminomethyl4,5′,8-trimethylpsoralen (AMT).

DNA damage and/or repair.42 Majima et al. studied the holetrapping dynamics in riboflavin-labeled G-quadruplexes and concluded that quadruplex structures of DNA can act as traps of the oxidative damage processes in the genome.43 It is observed that photoinduced oxidation of the guanines in Gquadruplexes by stress inducers and molecules such as porphyrins can be extremely efficient in inhibiting telomerase activity in tumor cells.44,45 These studies thus highlight the need of probing the excited-state interactions of small molecules serving as anticancer drugs and G-quadruplexes and motivate us to undertake the present work on psoralen derivative 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) and 22-mer human telomeric G-quadruplex-forming sequence AGGG(TTAGGG)3 (hTel22), which is known to adopt an antiparallel structure under Na+-rich conditions (Figure 1).4 The choice of AMT is governed by its water solubility, planarity, amino functionalization (which can enhance its interaction with DNA via H-bonding), and commercial usage as a drug in PUVA therapies. We have probed the ground- as well as excited-state interactions between the two using calorimetric, steady-state, and time-resolved spectroscopic techniques. The binding constant (Kb) of AMT with hTel22 (∼105 M−1) is found to be higher than that reported for the G−C duplex DNA (∼104 M−1).46 Additionally, a favorable geometry of AMT with respect to the G-quartet plane is found to facilitate ultrafast electron transfer between them upon photoexcitation of AMT. This leads to the formation of a radical pair, AMT•−G•+. These findings open up newer prospects for psoralens, which have long been known to interact with duplex DNA, but now their ground-state as well as photoinduced interactions with various G-quadruplexes can also be explored to design novel anticancer drugs.

10 mM phosphate buffer (pH 7.2) containing 100 mM NaCl and the solutions were annealed by heating at 95 °C for 10 min and then allowed to cool to room temperature slowly. The solutions were stored at 4 °C for 24 h prior to use. The concentrations of the formed G-quadruplexes were estimated using the molar extinction coefficient value of 228 500 M−1 cm−1 strand−1 at 260 nm.47 The AMT stock solutions were prepared in the same buffer using Milli-Q water. Methods. Steady-State Measurements. Steady-state absorption and fluorescence spectra were recorded on a UV−vis spectrophotometer (Cary 100, Varian) and spectrofluorimeter (Fluorolog 3, Horiba Jobin Yvon), respectively. The UV−vis titration experiments were carried out for a fixed AMT concentration (10 μM) and varying the hTel22 concentrations. For Job’s plot, the total concentration of AMT and hTel22 was fixed at 5 μM and the changes in absorbance were measured by varying the mole fraction of each. Circular Dichroism (CD) Studies. CD spectra were recorded using an Aviv 420SF spectropolarimeter (Aviv Biomedical, Inc., Lakewood, NJ) at 25 °C in the wavelength range of 220−320 nm at 1 nm interval with 1 nm bandwidth in a 1 cm pathlength cuvette having 2 mL sample volume. Each spectrum was an average of three scans collected at a speed of 50 nm min−1 and baseline-corrected using blank buffer. During the titration experiments, the spectra were recorded 5 min after each addition of AMT to a fixed amount of hTel22 for equilibration of the sample. For the CD melting experiments, 3 μM hTel22 was mixed with 20 μM AMT in aqueous buffer and the CD signal at 290 nm was monitored in the temperature range of 10−110 °C at intervals of 5 °C and at a heating rate of 0.5 °C min−1. Isothermal Titration Calorimetry (ITC). ITC experiments were performed in a MicroCal VP-ITC instrument (MicroCal LLC, Northampton, MA) at four temperatures: 15, 20, 25, and 30 °C. All samples and buffer solutions were degassed prior to use. The titration was conducted by placing 20 μM hTel22 in a sample cell (1.44 mL) and injecting 1 mM AMT solution from the syringe (286.5 μL) in 30 aliquots with the first injection volume being 5 μL and the rest being 7 μL, with a resting time of 300 s between each injection. The reference cell was filled with the same buffer that was used for sample preparation. Blank experiments were also conducted at all temperatures by injecting the same concentration of AMT into a buffer solution with identical titration parameters for correction of dilution effects. The corrected isotherms were then analyzed using the

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EXPERIMENTAL SECTION Materials and Sample Preparation. 4′-Aminomethyl4,5′,8-trimethylpsoralen (AMT) hydrochloride, NaCl, NaH2PO4, and Na2HPO4 were purchased from Sigma-Aldrich and used without purification. The 22-mer unlabeled human telomeric sequence 5′-AGGGTTAGGGTTAGGGTTAGGG3′ and 6-fluorescein amidite (FAM)-labeled oligomers, 5′FAM-AGGGTTAGGGTTAGGGTTAGGG-3′ and 5′AGGGTTAGGGTTAGGGTTAGGG-FAM-3′ (HPLC-purified), were procured from reGene Biologics, Hyderabad, India, and used as received. To prepare the G-quadruplex DNA, the oligomers (labeled/unlabeled) were suspended in a 2278


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Figure 2. (A) CD spectra of hTel22 (5 μM) in aqueous buffer (pH = 7.2) containing 100 mM NaCl in the absence (green) and increasing concentrations of AMT (red to pink). The CD spectrum of only AMT (300 μM) is shown in black. (B) CD melting curves of hTel22 (3 μM) alone and in the presence of AMT (20 μM). The solid lines represent sigmoidal fits to the data.

Figure 3. (A) UV−vis absorption spectra of AMT (solid line, 10 μM) and hTel22 (squares, 100 μM) in aqueous buffer (pH = 7.2) containing 100 mM NaCl. (B) Change in the absorption profile of AMT (10 μM) with increasing concentrations of hTel22 (0−100 μM). (C) Job’s plot for binding of AMT to hTel22 by monitoring absorbance changes at 340 nm. χAMT is the mole fraction of AMT.

single-site binding model and nonlinear least-squares fitting algorithm available in the built-in Microcal LLC ITC software to obtain the thermodynamic parameters of the binding event. Time-Resolved Emission and Transient Absorption (TA) Studies. Time-resolved fluorescence decay was recorded on a time-correlated single photon counting spectrometer (5000, Horiba Jobin Yvon IBH). Nano LED (330 nm, 1 MHz repetition rate, and 980 ps pulse width) and MCP photomultiplier (Hamamatsu R3809U-50) were used as an excitation source and detector, respectively. The transient absorption measurements were carried out on a femtosecond pump−probe setup, which consisted of a modelocked Ti:sapphire oscillator (Mai Tai, Spectra Physics) producing femtosecond pulses (fwhm < 100 fs, ∼2.5 W at 80 MHz). These pulses centered at 800 nm were directed to a regenerative amplifier (Spitfire Ace, Spectra Physics), which

was pumped by a frequency-doubled Nd:YLF laser (Empower, Spectra Physics) to produce amplified pulses. The major part of the output from the amplifier was passed through an optical parametric amplifier (TOPAS-Prime, Spectra Physics) to generate pump wavelength of 350 nm for excitation of the sample. The remaining amplified beam was directed through a CaF2 crystal for generation of white light used as the probe beam, which was passed through an optical delay line (∼4 ns). The instrumental resolution was ∼80−90 fs. Additional details of the setup along with analysis of the data are reported elsewhere.48

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RESULTS AND DISCUSSION Structural Characterization: Circular Dichroism (CD) Studies. CD is an established tool for predicting the topology of G-quadruplexes and investigating the influence of ligand 2279


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Figure 4. (A) Raw ITC titration profile of 0.02 mM hTel22 titrated against 1 mM AMT in aqueous buffer (pH = 7.2) containing 100 mM NaCl at 288 K. (B) Binding isotherm obtained by integration of peak area and correction of heats of dilution as a function of molar ratio. The solid line is the fit to the experimental data.

Table 1. Thermodynamic Parameters for the Binding of AMT to hTel22 at Different Temperatures As Obtained from ITC Experiments T (K) 288 293 298 303

Kb × 105 (M−1)

n 1.11 1.21 1.06 1.14

± ± ± ±

0.02 0.04 0.06 0.03

1.04 0.98 0.92 0.83

± ± ± ±

0.32 0.12 0.08 0.02

ΔH (kcal mol−1) −8.61 −9.28 −10.11 −11.05

± ± ± ±

1.12 1.05 0.08 0.06

binding on their structures.49,50 The different polymorphs of Gquadruplexes (parallel, antiparallel, and 3 + 1 hybrid) vary in the glycosidic bond angles of the guanine residues in the Gtetrads and the sugar phosphate backbone, leading to alterations in their base-stacking geometries, giving rise to distinct CD signals for each of them.51 Figure 2A shows the CD spectra of hTel22 in aqueous buffer rich in Na+ ions in the presence of different quantities of AMT. The observed spectra with positive signals at 290 and 245 nm and a negative signal at around 260 nm are indicative of an antiparallel conformation.50 It is evident that the addition of AMT does not perturb the structure of hTel22 much and the antiparallel conformation is maintained. The enhancement of CD signal at 290 and 245 nm with increasing amounts of ligand indicates stacking interactions between AMT and the outer Gtetrad of hTel22, leading to stabilization of the antiparallel structure (as achiral molecule AMT does not exhibit any CD signal and cannot contribute to the observed enhancement of the signal). The CD melting studies (Figure 2B), which show an increase in melting temperature (Tm) of hTel22 by 6 °C from 58 to 64 °C on addition of AMT, support this explanation. UV−Vis Absorption Studies. Figure 3A shows the steadystate absorption spectrum of AMT and hTel22 in aqueous buffered solution containing 100 mM NaCl. The ground-state interaction between AMT and hTel22 is evaluated by performing UV−vis titration experiments with different amounts of hTel22 keeping the concentration of AMT constant. A hypochromicity of about 20% is observed at 330 nm, and an isosbestic point at around 355 nm (Figure 3B) is also seen, which suggests that a two-state transition process may possibly be involved in the interaction.52 A separate titration experiment was performed by monitoring the absorbance of a set of samples with varying concentrations of AMT and hTel22 but keeping the total number of moles constant in each set from which Job’s plot was constructed,

TΔS (kcal mol−1)

ΔG = ΔH − TΔS (kcal mol−1)

−2.27 −3.16 −3.91 −4.67

−6.34 −6.12 −6.20 −6.38

analysis of which yielded a 1:1 binding stoichiometry. This also suggests a single site for binding of AMT in hTel22. Isothermal Titration Calorimetric (ITC) Studies. ITC is an indispensable technique to understand the energetics of binding of small molecules to biomacromolecules. It directly measures the heat changes that occur during the binding event and provides valuable information about the binding affinity, stoichiometry, binding sites, and nature of forces involved in binding.53 The raw ITC profile for interaction of AMT with hTel22 at 288 K is shown in Figure 4A, where the individual peaks correspond to each injection of AMT. The heat liberated on successive addition of AMT was obtained by integrating the area under the peaks, and these values were then corrected by subtracting the heat of dilution obtained by titrating identical amounts of AMT into buffer alone. Figure 4B shows the plot of corrected heats of injection versus the molar ratio of AMT to hTel22. To obtain a clearer picture of the molecular forces driving these binding interactions, similar titrations and analysis were performed at three other temperatures (293, 298, and 303 K), the ITC profiles of which have been provided in the Supporting Information (Figure S1). The thermodynamic parameters, binding constants, and stoichiometries obtained from fitting the experimental data have been summarized in Table 1, for different temperatures. The experimental data fits well to the “one set of sites” model of binding, and a stoichiometry of 1:1 is obtained at all temperatures, indicating that only one binding event occurs predominantly. This finding is in accordance with our results from Job’s plot analysis. At 288 K, a Kb value on the order of 105 suggests moderate binding between AMT and hTel22. This value decreases slightly with increasing temperatures, which is common for other small aromatic molecules that bind to the Gquadruplex DNA via stacking interactions.54 The thermodynamic parameters reveal that the binding of AMT and hTel22 is characterized by negative ΔH and TΔS values and the reaction is enthalpically driven in the 2280


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Figure 5. (A) Enthalpy−entropy compensation plot for association of AMT and hTel22 along with a linear fit to the data points. (B) Variation of ΔH of the binding process with temperature. The slope of the linear fit to the data gives the value of ΔCp.

Figure 6. (A) Fluorescence quenching of 3′ (squares) and 5′ (circles) FAM-labeled hTel22 (λex = 490 nm, λem = 520 nm) with increasing amounts of AMT in aqueous buffered solution (pH = 7.2) containing 100 mM NaCl. F0 and F represent the fluorescence intensity of FAM-labeled hTel22 in the absence and presence of AMT, respectively. The solid lines are sigmoidal fits to the experimental data. (B) Schematic representation of the preferable binding site of AMT (yellow disc) in hTel22.

temperature range investigated here. The reaction becomes more exothermic with the increasing temperature, but the ΔG values are not affected much due to opposing enthalpic and entropic effects. A plot of ΔH versus TΔS gives an idea about the extent of the entropy−enthalpy compensation, where a slope of unity indicates complete compensation of entropy by enthalpy or vice versa. Figure 5A shows such a ΔH versus TΔS plot for AMT−hTel22 interaction, yielding a slope of 1.2, confirming that the reaction is enthalpically favored.55−57 The negative value of ΔH suggests H-bonding and van der Waals interactions between AMT and hTel22 as these are known to contribute majorly to the enthalpy of a system.58 The unfavorable (negative) TΔS values arise due to loss in translational and rotational degrees of freedom of AMT upon binding to hTel22. According to the literature, small molecules with a less extended aromatic surface are likely to interact with G-quadruplex DNA via the end-stacking mode in enthalpically driven binding events.59,60 Furthermore, it is also known that groove binding of ligands leads to expulsion of groove-bound water, resulting in an increase in the entropy of the system and the binding being entropically driven.61,62 However, in our case, an enthalpically driven process with negative entropy terms eliminates the possibility of groove binding and substantiates the end-stacking mode of binding. It is also possible to obtain information on the mode of binding from the heat capacity changes (ΔCp) of a system, which are estimated from the variation of ΔH with temperature (eq 1).

ΔCp =

δ(ΔH ) δT

(1)

ΔCp values are indicative of the structural changes that occur on ligand binding.63 These values also provide an idea about the hydrophobic interactions; a negative value indicates burial of nonpolar surfaces and expulsion of water.64,65 Ha et al. proposed a semiempirical relationship between ΔCp and the driving force (ΔGhyd) of hydrophobic interactions66 given by eq 2. ΔG hyd = 80ΔCp

(2)

The plot of ΔH versus temperature in Figure 5B yields a slope (ΔCp) of −0.08 kcal mol−1 from which the ΔGhyd turns out to be −6.4 kcal mol−1. The small negative value of ΔCp suggests minor structural alterations of hTel22 upon ligand binding, which is in accordance with our CD results, indicating that the antiparallel structure of hTel22 is retained. The value also suggests moderate hydrophobic interactions owing to partial burial of the nonpolar aromatic surface of AMT upon binding. Such a situation can arise when a ligand stacks on the G-tetrad plane as demonstrated earlier.54 Probable Location of Binding Site: Fluorescence Quenching Studies of Labeled hTel22. To further investigate the preferred binding site of AMT in hTel22, we used oligomers 5′ FAM-hTel22 and 3′ FAM-hTel22, wherein 6-fluorescein amidite (FAM) dye is covalently attached to the 2281


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The Journal of Physical Chemistry B 5′ end and 3′ end, respectively. The fluorescence of FAM is known to be quenched upon direct interaction with organic molecules44,67 and hence the strongest binding site is determined by titrating a known amount of labeled preformed G-quadruplex (5 nM) with varying amounts of AMT. Figure 6 shows efficient fluorescence quenching of FAM with increasing concentrations of AMT, suggesting binding near the diagonal loop region (Figure 6B), where the labeling site is located. Furthermore, it is observed that the fluorescence of 3′ FAMhTel22 is quenched at a lower concentration of AMT as compared to that for 5′ FAM-hTel22, implying that the strongest binding site is closer to the 3′ end of the Gquadruplex. It should be noted that covalent attachment of FAM does not disturb the conformation of hTel22, as revealed by CD measurements (Figure S3). Steady-State Emission Studies. To obtain an idea about the excited-state interaction between AMT and hTel22, we monitored the steady-state emission behavior of photoexcited AMT with increasing concentration of hTel22. Figure 7 depicts

induced electron transfer between psoralens and duplex DNA is known.46,68 Transient Absorption (TA) Studies. To understand the nature of interaction between photoexcited AMT and hTel22, we have studied the TA spectrum of the system. Figure 8A shows the TA spectra upon 350 nm excitation of AMT at indicated delay times. The broad positive absorption with maximum at around 390 nm can be attributed to the S1−Sn excited-state absorption,68 and the weak negative signal at ∼490 nm is clearly due to the stimulated emission of AMT as its fluorescence peak appears at 460 nm (Figure 7). The kinetics at 390 nm is best described by a rising component of 250 fs, which presumably represents the vibrational relaxation in the S1 state and a decay time constant of greater than 1 ns. The latter is close to the radiative lifetime (1.5 ns) of AMT in aqueous solutions, as estimated by time-resolved emission experiments (Figure S4). The other positive signal at longer wavelengths (>500 nm) does not seem to decay in the monitored time window of 1 ns. Considering that the triplet−triplet absorption band of psoralens appears at around 500 nm,69 we assign this long-lived signal to the triplet-state absorption of AMT. In the presence of hTel22, the broad positive absorption at around 400 nm (Figure 8B) is found to decay much faster with time constants 30 ps (87%) and 1 ns (13%) (Figure 9B). The negative signal due to stimulated emission is not discernible anymore and is masked by the positive absorption signal. Furthermore, the long-lived signal beyond 500 nm is replaced by a broad positive absorption band, which decays almost completely in 500 ps. This absorption is attributed to AMT•−G•+ based on the literature,46 and we conclude that this species is formed by photoinduced electron transfer from guanine of hTel22 to AMT. Even though this species also absorbs at 390 nm, as the singlet−singlet absorption of AMT interferes at this wavelength, we investigated the kinetics at 530 nm to extract the time constants of the electron transfer reaction. Biexponential decay with a 35 ps component (94%) and a ∼1 ns component (6%) along with a growth component of 1 ps is observed (Figure 9C). The decay time constants are similar to those obtained from the kinetic analysis at 390 nm with increased amplitude of the shorter time constant (Table 2). We therefore assign the 30 ps component to the decay time of the radical species and the 1 ns to the lifetime of free AMT present in the system. Furthermore, the rising component is attributed to the formation time of the radical species. The measured decay and rise parameters are summarized in Table 2 from which a scheme has been proposed (Scheme 1). The

Figure 7. Steady-state emission spectrum (λex = 355 nm) of 10 μM AMT with increasing concentration of hTel22 in aqueous buffer (pH = 7.2) containing 100 mM NaCl.

a drastic decrease (∼80%) in emission intensity of AMT on addition of hTel22. This observation is not due to any change in absorbance at the excitation wavelength (355 nm) as the excitation is made at the isosbestic point (Figure 3B). The fluorescence quenching thus arises from some photoinduced process, which in most likelihood involves an electron transfer reaction between the two. Energy transfer can easily be ruled out due to lack of overlap between the absorption spectrum of hTel22 and emission spectrum of AMT. Moreover, photo-

Figure 8. Transient absorption spectra of (A) free AMT (0.1 mM) and (B) AMT in the presence of hTel22 (1 mM) in aqueous buffer (pH = 7.2) containing 100 mM NaCl at λex = 350 nm. 2282


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Figure 9. Transient absorption decay profiles of free AMT at 390 nm (A) and AMT bound to hTel22 monitored at 390 nm (B) and 530 nm (C). The insets in (A) and (C) show the kinetics at shorter time scales. The solid lines are the fits to the experimental data.

Table 2. Rise and Decay Parameters of Free and hTel22-Bound AMT Monitored at Different Wavelengths

a

system

monitoring wavelength (nm)

τ1 (ps)

AMT AMT + hTel22

390 390 530

>1000 32 ± 0.8 35 ± 0.2

a1a 0.87 ± 0.05 0.94 ± 0.05

τ2 (ps) 1200 1200

a2a

τrise (ps)

0.13 ± 0.02 0.06 ± 0.005

0.25 ± 0.06 0.51 ± 0.04 1.1 ± 0.02

ai’s represent the amplitude associated with the decay components.

acceptor in the photoinduced electron transfer process and show that the extent of π-stacking plays an important role in determining the efficiency of the electron transfer reaction.70 Furthermore, Manet and co-workers showed that although 1:1 and 1:2 complexes are formed between doxorubicin and Gquadruplex DNA, only the former participates in ultrafast electron transfer due to favorable quasi-parallel geometry.71 In the present work, the ITC results show that AMT binds to hTel22 via the end-stacking mode of binding that provides appropriate parallel/quasi-parallel geometry for electron transfer to occur efficiently. The observation of ultrafast photoinduced electron transfer in AMT−hTel22 leading to oxidation of guanine brightens the potential of psoralens to be employed and further developed as anticancer drugs.

Scheme 1. Schematic Representation of the Photoinduced Processes Occurring in Free (A) and Bound (B) AMT along with Their Time Constants

observed time constant for the forward electron transfer (1 ps) is slightly faster than that observed in duplex DNA primarily due to lower oxidation potential of guanine in the G-quartets (H-bonded as well as stacked with other guanines) of Gquadruplexes when compared with isolated guanine or only two/three stacked guanine bases.43 The electron transfer reaction observed here is thermodynamically feasible with a driving force of −0.51 eV for the charge separation and −3.14 eV for the charge recombination process. However, it is well known that the relative orientation of the drug molecule with respect to the host is also an important factor in the electron transfer processes in DNA. Wasielewski and co-workers recently demonstrated the importance of the π-stacked arrangement of the donor and

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CONCLUSIONS While psoralens have long been studied in the context of duplex DNA, very little is known about their potential as a Gquadruplex binder and their excited-state interaction with the same. It is found that water-soluble and planar psoralen derivative AMT interacts with hTel22 in a 1:1 stoichiometric ratio with a binding constant on the order of ∼105 M−1, which is enthalpically driven. The binding event does not disturb the antiparallel conformation of hTel22 but renders stability to it by enhanced stacking interactions. These observations are indicative of an end-stacking mode of ligand binding. 2283


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Fluorescence quenching experiments on FAM-labeled hTel22 reveal that the binding site of AMT is nearer to the 3′ end in the diagonal loop region of hTel22. Upon photoexcitation of AMT, one observes ultrafast electron transfer (1 ps) from the guanine moiety of hTel22, leading to the formation of a radical species (AMT•−G•+), which survives for about 30 ps. The fast electron transfer process in photoexcited AMT−hTel22 suggests that AMT and the guanine moiety of hTel22 are in a favorable parallel/quasi-parallel geometry with each other, substantiating our conclusion of the end-stacking mode of interactions. The study highlights psoralens as a prospective class of compounds for the development of anticancer therapeutics by targeting G-quadruplex DNA.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b12475. ITC profiles at different temperatures, CD spectra of labeled oligomers, time-resolved fluorescence decay of AMT, and calculations for free energy changes of the electron transfer reaction (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Anunay Samanta: 0000-0003-1551-0209 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by Research Grant No.: EMR/2015/ 000582 and J.C. Bose Fellowship (to A.S.) from the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), India. We also acknowledge UGC-UPE for financial support to our University. The transient absorption measurements were carried out using the femtosecond laser facility of the School. S.P. thanks University Grants Commission for the BSR Fellowship. The authors thank Debparna Datta and Prof. M.J. Swamy for their help with the ITC experiments.

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REFERENCES

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Ground- and Excited-State Interactions of a Psoralen Derivative with

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