Novel Bifunctional Acridine−Acridinium Conjugates: Synthesis and

J. Phys. Chem. B 2005, 109, 21997-22002

21997

Novel Bifunctional Acridine-Acridinium Conjugates: Synthesis and Study of Their Chromophore-Selective Electron-Transfer and DNA-Binding Properties Elizabeth Kuruvilla, Joshy Joseph, and Danaboyina Ramaiah* Photosciences and Photonics DiVision, Regional Research Laboratory, TriVandrum 695 019, India ReceiVed: August 5, 2005; In Final Form: September 19, 2005

Novel bifunctional conjugates 1-3, with varying polymethylene spacer groups, were synthesized, and their DNA interactions have been investigated by various biophysical techniques. The absorption spectra of these systems showed bands in the regions of 300-375 and 375-475 nm, corresponding to acridine and acridinium chromophores, respectively. When compared to 1 (Φf ) 0.25), bifunctional derivatives 2 and 3 exhibited quantitative fluorescence yields (Φf ) 0.91 and 0.98) and long lifetimes (τ ) 38.9 and 33.2 ns). The significant quenching of fluorescence and lifetimes observed in the case of 1 is attributed to intramolecular electron transfer from the excited state of the acridine chromophore to the acridinium moiety. DNA-binding studies through spectroscopic investigations, viscosity, and thermal denaturation temperature measurements indicate that these systems interact with DNA preferentially through intercalation of the acridinium chromophore and exhibit significant DNA association constants (KDNA ) 105-107 M-1). Compound 1 exhibits chromophoreselective electron-transfer reactions and DNA binding, wherein only the acridinium moiety of 1 interacts with DNA, whereas optical properties of the acridine chromophore remain unperturbed. Among bifunctional derivatives 2 and 3, the former undergoes DNA mono-intercalation, whereas the latter exhibits bis-intercalation; however both of them interact through mono-intercalation at higher ionic strength. Results of these investigations demonstrate that these novel water-soluble systems, which exhibit quantitative fluorescence yields, chromophore-selective electron transfer, and DNA intercalation, can have potential use as probes in biological applications.

1. Introduction The design of molecules that exhibit strong binding affinity to DNA is a challenging area of research. Such molecules can act as excellent chemotherapeutic reagents that exert their biological activity through interactions with DNA.1-6 Interactions with DNA are not the only the factors that determine the biological activity of these molecules, but their reactivity and selectivity are often correlated with their mode of binding with DNA.5,6 Therefore, a better understanding of the factors that govern the interactions of small molecules with DNA has an important role in the rational design of various DNA-targeted chemotherapeutic agents and molecular probes for DNA. Of these small molecules, the bifunctional derivatives that can undergo photoinduced electron-transfer processes have attracted much attention in recent years for their use in DNA detection, analysis, and cleavage.6-12 There has been widespread interest in bifunctional molecules joined by various linker groups to obtain molecules of higher DNA-binding affinity for use both as chemotherapeutic agents and as probes for nucleic acids.2,13-21 These molecules can, in principle, bind with DNA through intercalation, bis-intercalation, groove binding, and electrostatic interactions, depending on the nature of the chromophore and the spacer group present in them. Le Pecq and co-workers2 have postulated that bis-intercalative binding occurs in a molecule only when the linker chain length is more than 10.2 Å. Such molecules follow the neighbor exclusion principle wherein the DNA interacting chromophores * Author to whom correspondence should be addressed. Phone: (91) 471 2515362. Fax: (91) 471 2490186 or (91) 471 2491712. E-mail: [email protected] or [email protected].

Figure 1. Schematic representation of DNA interactions of a small molecule bearing two intercalating motifs: (A) bis-intercalation, (B) mono-intercalation, and (C) bis-intercalation with the violation of the neighbor exclusion principle.

occlude two base pairs between them (Figure 1A). When the spacer group is rigid or insufficient to span two base pairs, such molecules prefer mono-intercalation as shown in Figure 1B.22-24 Very few examples are reported in the literature that exhibit bis-intercalation, with the violation of the neighbor exclusion principle as represented in Figure 1C.15,24,25 Although the interactions of small molecules with DNA have been extensively investigated, the competitive interactions of covalently linked bifunctional molecules bearing two different DNA-binding motifs have received less attention. In this context, we have designed novel acridine-based bifunctional conjugates, not only because of their interesting chemical and physical properties but also due to their immense utility in the pharmaceutical and dye industries.13-14,18-19,26 The derivatives of

10.1021/jp0543532 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/26/2005

21998 J. Phys. Chem. B, Vol. 109, No. 46, 2005

Figure 2. Structures of bifunctional acridine-acridinium conjugates 1-3 and the model compounds, 9,10-dimethylacridinum iodide (4) and 9-methylacridine (5) under investigation.

acridine exhibit a broad range of chemotherapeutic properties against prokaryotic and eukaryotic cells, because of the facility with which they interact with DNA targets. Although the antitumor acridines are known to exert their activity through DNA intercalation,5,6 their interaction with topoisomerase II and inhibition of DNA processing enzymes such as polymerases and telomerase might also contribute to the overall biological activity.27,28 In the present paper, we report the synthesis and results of photophysical and competitive DNA-binding studies of a few water-soluble bifunctional conjugates 1-3 and for comparison model compounds 4 and 5 (Figure 2). These derivatives with polymethylene spacers linked directly to the 9-position of the acridine ring are easier to synthesize and are different from the previously reported bisacridines, where the spacers are linked through the amino functionality.14 Our results reveal that bifunctional conjugate 1 exhibits unusually chromophore-selective electron-transfer processes and binding interactions with DNA occur preferentially through the intercalation of the acridinium chromophore. However, compounds 2 (r ) 7.6 Å) and 3 (r ) 12.6 Å), which undergo mono- and bis-intercalation, respectively, at low ionic strength (2 mM NaCl), were found to exhibit only mono-intercalative interactions at the higher ionic strength (100 mM NaCl). These bifunctional systems, which show quantitative fluorescence yields and exhibit chromophore-selective and spacer-length- and ionic-strength-dependent interactions with DNA, can have potential applications as fluorescent probes in biology. 2. Experimental Section 2.1. General Techniques. The equipment and procedure for spectral recordings are described elsewhere.10,11,29-30 The fluorescence spectra were recorded on a SPEX-Fluorolog F112X spectrofluorimeter. The fluorescence quantum yields were measured by the relative methods using optically dilute solutions, with 10-methylacridinium trifluoromethane sulfonate in water (Φf ) 1) as the standard.30 The nanosecond fluorescence lifetime studies were carried out using an Edinburgh FL900CD single photon counting system. Fluorescence lifetimes were determined by deconvoluting the instrumental function with a mono- or biexponential decay and minimizing the χ2 values of the fit to 1 ( 0.1. The thermal denaturation temperature (Tm) of the oligonucleotide duplex was obtained in 10 mM phosphate buffer (pH 7.4) by using a thermoelectrically coupled PerkinElmer spectrophotometer, interfaced to a PC-XT computer for the acquisition and analysis of experimental data. DNA-binding studies of the bifunctional derivatives were carried out in 10 mM phosphate buffer (pH 7.4) containing either 2 mM or 100 mM NaCl. A solution of calf thymus DNA was sonicated for 1 h and filtered through a 0.45 µM Millipore filter. The concentrations of DNA solutions were determined by using

Kuruvilla et al. the average extinction coefficient value of 6600 M-1 cm-1 for a single nucleotide at 260 nm.31 The absorption and fluorescence titrations of the bifunctional derivatives with DNA were carried out by adding small aliquots of DNA solution containing the same concentration of the compound as that in the test solution. DNA association constants were calculated using fluorescence quantum yields, according to the method of McGhee and von Hippel by using the data points of the Scatchard plot.32-33 2.2. Materials. Calf thymus DNA (Amersham Pharmacia Biotech, Piscataway, NJ) was used without further purification. 9,10-Dimethylacridinium iodide (4, mp 248-249 °C),34 9-methylacridine (5, mp 116-117 °C),34 1,5-bis(acridin-9-yl)pentane (mp 219-220 °C),35 and 1,10-bis(acridin-9-yl)decane (mp 158159 °C)35 were prepared by modifying the reported procedures (Supporting Information, Schemes S1-S4). Oligonucleotides (DNA1, 5′-CGT GGA CAT TGC ACG GTA C-3′; DNA2, 5′GTA CCG TGC AAT GTC CAC G-3′) were synthesized on an Applied Biosystems DNA synthesizer as described earlier36 and were kindly provided by Professor Gary B. Schuster (Georgia Institute of Technology, Atlanta, GA). Commercially available starting materials and solvents were purified and distilled before use. Petroleum ether used was the fraction boiling at 60-80 °C. 2.3. Synthesis of Bifunctional Acridinium Derivatives 1 and 2. To a solution of 1,5-bis(acridin-9-yl)pentane (1 mmol) in dry acetonitrile (20 mL) was added methyl trifluoromethane sulfonate (1 mmol) dropwise over a period of 30 min with stirring at 25 °C. The reaction mixture was refluxed for 4 h, and the residue obtained after evaporation of the solvent was chromatographed over silica gel. Elution of the column with a mixture (1:4) of methanol and ethyl acetate gave 46% of monoacridinium derivative 1, mp 232-233 °C, after recrystallization from a mixture (1:4) of ethyl acetate and acetonitrile. 1H NMR (300 MHz, DMSO-d , δ): 1.80-1.88 (6H, m), 3.83 6 (2H, t, J ) 7.5 Hz), 3.96 (2H, t, J ) 7.6 Hz), 4.79 (3H, s), 7.81 (2H, t, J ) 7.6 Hz), 7.97 (2H, t, J ) 7.5 Hz), 8.14 (2H, t, J ) 7.5 Hz), 8.25 (2H, d, J ) 8.5 Hz), 8.40 (2H, t, J ) 7.6 Hz), 8.60 (2H, d, J ) 8.3 Hz), 8.74 (2H, d, J ) 9 Hz), 8.83 (2H, d, J ) 8.4 Hz). 13C NMR (75 MHz, DMSO-d6, δ): 29.49, 30.31, 32.42, 34.44, 40.56, 124.34, 127.42, 127.69, 129.06, 129.86, 130.39, 132.33, 138.81, 141.64, 147.16, 158.63. Mass spectrometry (electron impact, MH+) Calcd for C33H29N2F3O3S: 442.2409. Found: 442.2404. Further elution of the column with a mixture (1:1) of methanol and ethyl acetate gave 31% of bisacridinium derivative 2, mp 260-261 °C, after recrystallization from a mixture (1:4) of ethyl acetate and acetonitrile. 1H NMR (300 MHz, DMSO-d6, δ): 1.82-1.89 (6H, m), 3.97 (4H, t, J ) 8.1 Hz), 4.81 (6H, s), 8.0 (4H, t, J ) 7.8 Hz), 8.43 (4H, t, J ) 8 Hz), 8.77 (4H, d, J ) 9.3 Hz), 8.68 (4H, d, J ) 8.5 Hz). 13C NMR (75 MHz, DMSO-d6, δ): 29.30, 30.15, 32.54, 44.91, 124.42, 127.73, 129.86, 130.39, 136.33, 141.64, 158.63. Anal. Calcd for C35H32N2O6F6S2: C, 55.70; H, 4.27; N, 3.71; S, 8.50. Found: C, 55.45; H, 4.07; N, 3.78; S, 8.40. In a separate reaction, 1,5-bis(acridin-9-yl)pentane (1 mmol) was dissolved in dry dichloromethane (20 mL), and methyl trifluoromethane sulfonate (2.5 mmol) was added slowly with stirring at room temperature. The reaction mixture was refluxed for 4 h, and the residue obtained after recrystallization from a mixture (1:4) of ethyl acetate and acetonitrile gave bisacridinium derivative 2 in 74% yield, mp 260-261 °C (mixture mp). 2.4. Synthesis of Bifunctional Derivative 3. To a solution of 1,10-bis(acridin-9-yl)decane (1 mmol) in dry acetonitrile (20 mL) was added methyl trifluoromethane sulfonate (2.5 mmol)

Bifunctional Acridine-Acridinium Conjugates

J. Phys. Chem. B, Vol. 109, No. 46, 2005 21999 TABLE 1: Photophysical and DNA-Binding Properties of Bifunctional Derivatives 1-3 and Model Compounds 4 and 5a λab, nm compound (, M-1 cm-1) λem, nm Φf b τ, ns KDNA (M-1)c,d 1 2 3 4f

Figure 3. Absorption spectra of compounds 1 (21.4 µM), 2 (9.6 µM), and 4 (12 µM) in water and 5 (15 µM) in methanol. The inset shows the fluorescence spectra of these derivatives under same conditions. The fluorescence spectrum of 5 is magnified by a factor of 4 for clarity. The excitation wavelength is 355 nm (optically matched solutions with OD ) 0.1 at 355 nm).

slowly over a period of 30 min with stirring at 25 °C. The reaction mixture was refluxed for 4 h, and the residue obtained after evaporation of the solvent was recrystallized from a mixture (1:4) of ethyl acetate and acetonitrile to yield 76% of the bisacridinium derivative 3, mp 255-256 °C. 1H NMR (300 MHz, DMSO-d6, δ): 1.15-1.78 (16H, m), 3.98 (4H, t, J ) 8 Hz), 4.81 (6H, s), 8.02 (4H, t, J ) 8.3 Hz), 8.42 (4H, t, J ) 8.3 Hz), 8.76 (4H, d, J ) 9.2 Hz), 8.88 (4H, d, J ) 8.8 Hz). 13C NMR (75 MHz, DMSO-d6, δ): 29.30, 30.15, 32.54, 44.91, 124.42, 127.73, 129.86, 130.39, 136.33, 141.64, and 158.63. Anal. Calcd for C40H42F6N2O6S2: C, 58.24; H, 5.13; N, 3.40; S, 7.77. Found: C, 58.20; H, 5.03; N, 3.42; S, 7.70. 3. Results and Discussion 3.1. Synthesis. The synthesis of bifunctional conjugates 1 and 2 was achieved in good yields by quaternization of 1,5bis(acridin-9-yl)pentane with methyl trifluoromethane sulfonate in acetonitrile. Quaternization using a 1:1 molar ratio of 1,5bis(9-acridinyl)pentane and methyl trifluoromethane sulfonate gave 46% of bifunctional derivative 1, along with bisacridinium derivative 2 in 31% yield. In contrast, compound 2 was isolated as an exclusive product (74%) when the same reaction was carried out with the reactants in the molar ratio of 1:2.5. The similar reaction when carried out between 1,10-bis(acridin-9yl)decane and methyl trifluoromethane sulfonate in the ratio of 1:2.5 yielded bifunctional derivative 3 in 76% yield. These bifunctional conjugates were purified and characterized on the basis of analytical results and spectral evidence. For example, the 1H NMR spectrum of conjugate 1 showed a peak corresponding to the methyl group at δ 4.79, whereas the aliphatic protons corresponding to the pentamethylene spacer appeared as multiplets in the region between δ 1.80 and 3.96. Similarly, for bifunctional derivatives 2 and 3, two methyl groups linked to two quaternary nitrogens of the acridinium moieties appeared as a singlet at δ 4.81, while the aliphatic protons of the spacer group appeared as multiplets in the region between δ 1.82 and 3.97 and δ 1.15 and 3.98, respectively. Characteristically, bifunctional derivative 1 showed 16 carbons in the 13C NMR spectrum, while bisacridinium derivative 2 with symmetrical structure exhibited fewer carbons (11 carbons) when compared to bifunctional system 1. 3.2. Investigation of Absorption and Fluorescence Emission Properties. Figure 3 shows the absorption and fluorescence emission spectra (inset) of representative bifunctional derivatives 1 and 2 and their model compounds 4 and 5 in aqueous medium, while their photophysical properties are summarized in Table 1. The acridine chromophore exhibits absorption in the region

5g

358 (18 500) 421 (4200) 358 (33 200) 421 (9450) 358 (33 600) 418 (10 100) 357 (19 100) 416 (5000) 356 (8300)

ne

4.7 × 10

496

1.7 19.2 0.91 38.9

4.3 × 106

2.7

496

0.98 33.2

2.5 × 107

2

490

1

7.31 × 105

2.5

418

0.08

h

h

478

0.25

33.8 7.9

5

2.5

a

The data are the average of more than two experiments, and the error is ca. (5%. The photophysical and DNA-binding properties were examined in aqueous medium and 10 mM phosphate buffer containing 2 mM NaCl (pH 7.4), respectively. b Fluorescence quantum yields were calculated using 10-methylacridinium trifluoromethane sulfonate as the standard (Φf ) 1).30 c DNA association constants were determined by the Scatchard analysis of fluorescence titration data. d DNA association constants (KDNA) for 1, 2, and 3 are KDNA ) 1.1 × 104, 4.2 × 105, and 4.31 × 105 M-1, respectively, in 10 mM phosphate buffer containing 100 mM NaCl (pH 7.4). e Number of nucleotides occluded by a bound ligand. f Data for compound 4 taken from ref 30. g In methanol. h Not determined.

300-400 nm, whereas the acridinium moiety possesses absorption that extends up to 470 nm. The investigation of the absorption spectra of bifunctional derivative 1 in various solvents indicated that there is no ground-state interaction existing between the acridine and the acridinium moieties present in it. As shown in the inset of Figure 3, bifunctional derivative 1 exhibits fluorescence emission in the range 425-550 nm with significantly reduced quantum yields when compared to bifunctional derivatives 2 and 3 (not shown in the figure) and model acridinium derivative 4. The fluorescence quantum yield (Φf) of 1 is found to be 0.25, in aqueous medium, while bisacridinium derivatives 2 and 3 and model acridinium derivative 4 exhibited quantitative fluorescence quantum yields (Φf ) 0.91, 0.98, and 1.0, respectively). Nanosecond timeresolved fluorescence studies indicated that bifunctional derivative 1 exhibits a biexponential decay with lifetimes of 1.7 (12%) and 19.2 ns (88%), whereas compounds 2 and 3 showed singleexponential decays with lifetimes of 38.9 and 33.2 ns (Supporting Information, Figure S1), respectively (Table 1). 3.3. Study of Chromophore-Selective Photoinduced Electron Transfer. In comparison with model compounds 4 (τ ) 33.8 ns) and 5 (τ ) 7.9 ns), the short- and long-lived species observed in the case of bifunctional derivative 1 can be assigned to the acridine and acridinium chromophores, respectively. However, the observation of decreased lifetimes of these moieties in 1 clearly indicates that these chromophores undergo reversed intramolecular fluorescence quenching, and the mechanism of quenching is assigned to the exothermic electron transfer from the acridine chromophore to the acridinium moiety. In support of this view, we have calculated the change in free energy values (∆Gel) for the electron-transfer processes using the redox potentials (Eox and singlet energy of the acridine chromophore are 1.6 and 3.25 eV, and Ered and singlet energy of the acridinium moiety are -0.57 and 2.75 eV, respectively; Supporting Information) and confirmed it by intermolecular fluorescence quenching and laser flash photolysis studies. We have obtained ∆Gel ) -0.95 and -0.58 eV for the electron transfer involving the singlet excited states of the acridine and acridinium chromophores, respectively, suggesting thereby that such processes are indeed energetically feasible.

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Kuruvilla et al.

Figure 5. Change in absorption spectra of bifunctional derivative 1 (38.4 µM) in 10 mM phosphate buffer (pH 7.4) containing 2 mM NaCl, with increasing concentration of CT DNA. The inset shows the corresponding changes in the fluorescence spectra. [DNA]: (a) 0, (b) 0.046, (c) 0.069, (d) 0.09, (e) 0.11, (f) 0.14, (g) 0.16, and (h) 0.2 mM. The excitation wavelength is 335 nm.

Figure 4. Schematic representation of the photoinduced intramolecular electron-transfer reaction in bifunctional derivative 1 involving (A) from the excited state of the acridine chromophore to the acridinium moiety and (B) from the acridine moiety to the excited state of the acridinium chromophore.

The rate of intramolecular electron transfer (ket) in bifunctional derivative 1 can be calculated by taking into account the change in lifetimes of the acridine and acridinium chromophores as per the eq 1

ket ) 1/τ - 1/τ0

(1)

where τ is the fluorescence lifetime of the linked chromophore and τ0 is the fluorescence lifetime of the model compound. In the case of 1, we obtained ket ) 0.47 × 109 and 0.23 × 108 s-1, respectively, for the electron-transfer reaction from the acridine chromophore to the acridinium moiety, wherein the excited state involved is the acridine (Figure 4A) or the acridinium (Figure 4B) chromophores, as shown in Figure 4. These values are in good agreement with the theoretically calculated change in free energies (∆Gel) and confirm that in the case of 1 the electron transfer can occur from the excited states of both the chromophores. Of the two chromophores, the reaction involving the excited state of the acridine chromophore (Figure 4A) is found to be more favorable (nearly 20-fold) than the reaction mediated from the excited state of the acridinium chromophore (Figure 4B). 3.4. Investigation of Chromophore-Selective Interactions with DNA. To evaluate the potential applications of bifunctional conjugates 1-3 as probes in biology, we have investigated their efficiency and chromophore-selective interactions with DNA using various spectroscopic and biophysical techniques. Figure 5 shows the changes in the absorption spectra of 1, with increase in concentration of calf thymus DNA (CT DNA). By the addition of DNA, compound 1 showed significant hypochromicity at the absorption corresponding to the acridinium chromophore, along with a red shift of 2 nm, as observed for the intercalating acridinium derivatives.13-15 Similarly, the fluorescence spectra of 1 showed an efficient quenching with the increase in DNA concentration (inset of Figure 5). At higher concentrations of DNA (>0.16 mM), we observed the complete quenching of the fluorescence originating from the acridinium moiety, indicating that the acridinium chromophore of 1 preferentially undergoes facile interactions with DNA. The fluorescence spectra of 1, at and above 0.16 mM of DNA, were

Figure 6. Change in absorption and fluorescence emission spectra of bifunctional derivative 2 (11.4 µM) in 10 mM phosphate buffer (pH 7.4) containing 2 mM NaCl, with increasing DNA concentrations. [DNA]: (a) 0, (b) 0.024, (c) 0.048, (d) 0.072, (e) 0.096, (f) 0.12, (g) 0.14, (h) 0.17, (i) 0.28, and (j) 0.47 mM. The inset shows the change in absorbance of 2 with increasing concentration of CT DNA above 0.14 mM. [DNA]: (g) 0.14, (h) 0.17, (i) 0.28, and (j) 0.47 mM. The excitation wavelength is 363 nm.

Figure 7. Change in absorption spectra of bifunctional derivative 3 (12.8 µM) in 10 mM phosphate buffer (pH 7.4) containing 2 mM NaCl, with increasing concentration of CT DNA. [DNA]: (a) 0, (b) 0.024, (c) 0.048, (d) 0.072, (e) 0.096, and (f) 0.14 mM. The inset shows the corresponding changes in the fluorescence emission spectra. The excitation wavelength is 363 nm.

found to be characteristic of the acridine chromophore, suggesting that the photophysical properties of the acridine chromophore remain unaffected in the presence of DNA (Supporting Information, Figure S2). The changes in the absorption and fluorescence emission properties of bifunctional conjugates 2 and 3 with added DNA concentrations are shown in Figures 6 and 7, respectively. Bisacridinium derivative 2, interestingly, showed significant hypochromicity in the absorption spectrum up to a [DNA]/ [ligand] ratio of 12 (0.14 mM of DNA), after which a small

Bifunctional Acridine-Acridinium Conjugates enhancement in the absorption spectrum was observed (inset of Figure 6). Similarly, a strong fluorescence quenching of 2 was observed upon increasing DNA concentration up to 0.14 mM; thereafter the addition of DNA induced a negligible effect as shown in Figure 6. The fluorescence quenching of the bifunctional derivatives is attributed to the favorable electron transfer from the DNA bases to the excited acridinium chromophore.30,37 The observed changes in the absorption and fluorescence emission spectra of bisacridinium conjugate 3 in the presence of DNA (Figure 7) were significantly higher when compared to the changes observed for bisacridinium derivative 2 (Figure 6). Bisacridinium derivative 2 with polymethylene spacer n ) 5 showed ca. 20% hypochromicity in the absorption spectrum in the presence of 0.14 mM DNA. In contrast, bisacridinium conjugate 3 with polymethylene spacer n ) 10 exhibited ca. 41% hypochromicity under similar conditions. Similarly, contrasting effects were observed in the fluorescence properties of 2 and 3 in the presence of DNA. Bisacridinium derivative 3 undergoes efficient fluorescence quenching upon binding with DNA when compared to conjugate 2. At any DNA concentration, the fluorescence quenching observed in the case of bisacridinium derivative 3 is more than twice that of 2, suggesting its greater affinity for DNA. The DNA association constants (KDNA) of 1-3 were determined based on the fluorescence titrations and according to the method of McGhee and von Hippel using the data points of the Scatchard plot (Supporting Information, Figure S3). Bifunctional derivative 1 showed KDNA ) 4.7 × 105 M-1, which is nearly 2 times less than that observed for model compound 4 (KDNA ) 7.3 × 105 M-1) (Table 1). Bifunctional conjugate 2, however, exhibited nearly a 10-fold higher value of the association constant (KDNA ) 4.3 × 106 M-1) when compared to bifunctional derivative 1 and around a 6-fold lower value than that of bisacridinium derivative 3 (KDNA ) 2.5 × 107 M-1). The lower value of KDNA observed in the case of 1 could be partly due to the steric effects of the bulky non-interacting acridine unit present in it. In contrast, the second acridinium moiety of 2 can undergo electrostatic interactions with the negatively charged sugar-phosphate backbone of DNA, thereby resulting in the enhancement of KDNA. The involvement of the electrostatic interactions in 2 was confirmed by the observation of a lower value of KDNA in the buffer containing 100 mM NaCl. The binding constant for bisacridinium derivative 2 (KDNA ) 4 × 105 M-1) was found to be 1 order less in buffer containing 100 mM NaCl when compared to the value obtained in 2 mM NaCl (Table 1). Viscometric studies were carried out to understand the preferred mode of interactions of these derivatives with DNA. In the case of bifunctional derivative 3, the viscosity of DNA changes to a greater extent than that observed for derivative 2 (Figure 8). The π-stacking of the acridinium derivatives of 2 and 3 between DNA base pairs results in the increase in the viscosity of DNA as in the case of the typical intercalators.38 The intercalative interactions of several acridinium conjugates with DNA have been well-characterized in the literature by various biophysical and NMR techniques.39,40 The derivative with the longer space length (n ) 10) preferentially undergoes bis-intercalation, resulting in a marked increase in the DNA viscosity when compared to mono-intercalating derivative 2 with shorter spacer lengths of n ) 5 and model compound 4. Figure 9 shows the thermal denaturation (Tm) curves and first derivative plots obtained for the duplex DNA1 and DNA2 in the presence and absence of bifunctional derivative 2. The oligonucleotide duplex melts at 52 °C in the absence of ligands,

J. Phys. Chem. B, Vol. 109, No. 46, 2005 22001

Figure 8. Effects of increasing concentrations of bifunctional derivatives 2 and 3 and model compound 4 on the relative viscosity of CT DNA (0.72 mM) at 26 ( 0.2 °C in 10 mM phosphate buffer (pH 7.4).

Figure 9. Thermal denaturation curves (Tm) for the DNA duplex (DNA1/DNA2) in the absence (a, Tm ) 52 °C) and presence (b, Tm ) 64 °C) of bifunctional derivative 2 (20 µM) in 10 mM phosphate buffer (pH 7.4). The inset shows the first derivatives of the melting curves. DNA1: 5′-CGT GGA CAT TGC ACG GTA C-3′. DNA2: 5′-GTA CCG TGC AAT GTC CACG-3′.

whereas in the presence of 20 µM of acridinium derivative 2 the DNA duplex is stabilized by 12 °C. A similar extent of stabilization was observed in the case of bifunctional derivative 3. In contrast, bifunctional derivative 1 under similar conditions provides a stabilization of the duplex by only 2 °C (Supporting Information, Figure S4). The absorbance changes observed at 260 nm, corresponding to DNA during the thermal denaturation of DNA in the presence and absence of the derivatives, clearly reflects the extent and binding interactions of these derivatives with DNA. Similar absorption changes were observed when monitored at 355 nm, corresponding to the acridinium chromophore. Bifunctional derivatives 2 and 3 contain two DNA-binding motifs, acridinium chromophores separated by a distance of 7.6 and 12.6 Å, respectively. The observation of a higher DNA association constant (ca. 36-fold) for 3, when compared to model compound 4, clearly suggests that both the acridinium moieties present in 3 undergo interactions with DNA through bisintercalation. In contrast, bifunctional system 2 exhibited only ca. 6-fold higher value of KDNA than that of model compound 4, indicating thereby that 2 interacts with DNA through monointeracalation. The length of the spacer group in this case is not sufficient enough to allow the interaction of both acridinium chromophores through bis-intercalation. Furthermore, a comparison of the absorption and fluorescence changes of 2 and 3, in the presence of DNA at different ionic strengths (Figure 10), clearly indicates that these two molecules exhibit different modes of binding interactions with DNA.40,41 Interestingly, at lower ionic strength (2 mM NaCl), bisacridinium derivative 2 exhibits relatively small changes in the absorption as well as fluorescence properties with increasing DNA concentration, when compared

22002 J. Phys. Chem. B, Vol. 109, No. 46, 2005

Figure 10. Change in fluorescence intensity of bifunctional derivatives 2 and 3 with increasing concentration of DNA in 10 mM phosphate buffer (pH 7.4) containing 2 mM NaCl. The inset shows the changes in fluorescence intensity of 2 and 3 at higher ionic strength (100 mM NaCl).

to that of 3. The observed saturation in the fluorescence changes and significant enhancement in the absorption bands at higher concentrations of DNA show that at the lower ionic strength of the buffer 2 binds through mono-intercalation, whereas 3 interacts through bis-intercalative interactions. In contrast, at the higher ionic strength of the buffer medium (inset of Figure 10), both these derivatives exhibited identical saturation behavior as well as DNA association constants, confirming thereby that both these systems undergo preferentially mono-intercalating interactions with DNA under these conditions. 4. Conclusions In summary, we have, for the first time, demonstrated the chromophore-dependent electron transfer and competitive DNAbinding interactions of systems containing two DNA-binding motifs. Bifunctional conjugate 1 undergoes preferential electrontransfer reactions from the excited state of the acridine chromophore to the acridinium moiety, while its acridinium moiety alone undergoes intercalating interactions with DNA. However, bifunctional derivatives 2 and 3 at lower ionic strengths exhibit, respectively, mono-intercalative and bis-intercalative interactions with DNA, but both undergo only mono-intercalative interactions at the higher ionic strength of the buffer medium. These novel bifunctional systems, which are highly soluble in aqueous medium, stable under irradiation conditions and exhibit significant fluorescence quantum yields and interesting chromophoreselective electron transfer and interactions with DNA, can have potential applications as fluorescent probes. Acknowledgment. This work was supported by the Council of Scientific and Industrial Research and the Department of Science and Technology, Government of India. This is contribution number RRLT-PPD-184 from the Photosciences and Photonics Division of the Regional Research Laboratory, Trivandrum, India. Supporting Information Available: Calculation details of the change in free energy and DNA association constants, synthetic schemes, fluorescence decay profiles, and emission spectra in the presence and absence of DNA, Scatchard plots, and DNA thermal denaturation curves. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Lerman, L. S. J. Mol. Biol. 1961, 3, 18-30. (2) Le Pecq, J.-B.; Bret, M. L.; Barbet, J.; Roques, B. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 2915-2919.

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