Tuning of Intercalation and Electron-Transfer Processes between DNA

A series of acridinium derivatives 1−6, wherein steric factors have been varied systematically ... The acridinium derivatives 1 and 2 showed high DN...
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Bioconjugate Chem. 2004, 15, 1230−1235

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Tuning of Intercalation and Electron-Transfer Processes between DNA and Acridinium Derivatives through Steric Effects Joshy Joseph,† Elizabeth Kuruvilla,† Asha T. Achuthan,† Danaboyina Ramaiah,*,† and Gary B. Schuster‡ Photosciences and Photonics Division, Regional Research Laboratory (CSIR), Trivandrum 695019, India, and School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400. Received July 23, 2004; Revised Manuscript Received September 27, 2004

A series of acridinium derivatives 1-6, wherein steric factors have been varied systematically through substitution at the 9 position of the acridine ring, have been synthesized and their DNA interactions have been investigated by various biophysical techniques. The unsubstituted and methylacridinium derivatives 1 and 2 and the o-tolylacridinium derivative 6 exhibited high fluorescence quantum yields (Φf = 1) and lifetimes (τ ) 35, 34, and 25 ns, respectively), when compared with the arylacridinium derivatives 3-5. The acridinium derivatives 1 and 2 showed high DNA binding affinity (K ) 7.3-7.7 × 105 M-1), when compared to the arylacridinium derivatives 3-5 (K ) 6.9-10 × 104 M-1). DNA melting and viscosity studies establish that in the case of the aryl-substituted systems, the efficiency of DNA binding is in the order, phenyl > p-tolyl > m-tolyl .. o-tolyl derivative. The increase in steric crowding around the acridine ring hinders the DNA binding interactions and thereby leads to negligible binding as observed in the case of 6 (o-tolyl derivative). These results indicate that a subtle variation in the substitution pattern has a profound influence on the photophysical and DNA interactions. Further, they demonstrate that π-stacking interactions of the ligands with DNA are essential for efficient electron transfer between the DNA bases and the ligands. These water soluble and highly fluorescent molecules which differ in their DNA binding mode can act as models to study various DNA-ligand interactions.

INTRODUCTION

The study of interactions of various ligands with DNA has been the subject of considerable interest because of its importance in the design of drugs targeted to DNA as well as in understanding the electron and energy transfer properties of DNA (1-10). Of the various types of interactions, the intercalative interactions of ligands with DNA is particularly important because of the fact that many anticancer drugs and antibiotics exert their biological activity through DNA intercalation (11-14). A better understanding of the factors that govern the binding mode of ligands with DNA is not only essential for the design of efficient drugs targeted to DNA and development of probes for various DNA structures but also for the use of DNA in electronic materials applications (15). To investigate how steric and conformational factors influence the mode and extent of binding of small molecules with DNA, we have chosen systems based on the acridine chromophore for study. Acridines are a special class of compounds not only because of their interesting chemical and physical properties but also due to their immense utility in the pharmaceutical and dye industries (16, 17). Acridines were the first chromophore that was extensively studied for its noncovalent DNA interactions. These studies led to the concept of intercalation. The study of the steric and conformational effects on the DNA binding properties of acridine chromophores * To whom correspondence should be addressed. Fax: (+91) 471-2490186, E-mail: [email protected] or [email protected]. † Regional Research Laboratory (CSIR). ‡ Georgia Institute of Technology.

has received less attention (11, 12, 18, 19). In this context, we have designed a series of acridinium derivatives 1-6, wherein we have systematically varied the steric factors through substitution at the 9 position of the acridinium ring (Figure 1) to assess the energetics and nature of their DNA binding interactions. The results of these investigations indicate that subtle variation in the substitution pattern on the acridinium chromophore has a profound influence on their photophysical and DNA binding interactions. These results demonstrate that these water soluble and highly fluorescent molecules can act as models to study various DNA interactions. EXPERIMENTAL PROCEDURES

The equipment and procedure for spectral recordings are described elsewhere (19). The fluorescence lifetimes were measured on an IBH picosecond single photon counting system using a 401 nm IBH NanoLED source and a Hamamatsu C4878-02 MCP detector. The fluorescence decay profiles were deconvoluted using IBH Data Station software V2.1, fitted with a mono-, bi-, or triexponential decay and minimizing the χ2 values of the fit to 1 ( 0.1. The 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 earlier (20). Solution of calf thymus DNA (CT DNA, Pharmacia Biotech, USA) in phosphate buffer was sonicated for 1 h and filtered through a 0.45 µM Millipore filter. The concentrations of the CT DNA solutions were determined by using the average extinction coefficient value of 6600 M-1 cm-1 for a single nucleotide at 260 nm (21). Polynucleotides (Amersham Pharmacia Biotech Inc.) were dissolved in phosphate buffer, and the concentrations were deter-

10.1021/bc0498222 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/28/2004

Tuning of Intercalation and Electron-Transfer Processes

Figure 1. Structures of acridinium derivatives 1-6 under study.

mined by using the average extinction coefficient value of 7400 M-1 cm-1 at 253 nm for poly(dG).poly(dC) and 6000 M-1 cm-1 at 260 nm for poly(dA).poly(dT) (21). The absorption and fluorescence titrations of the acridinium 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. The binding affinities were calculated using fluorescence quantum yields, according to the method of McGhee and von Hippel by using the data points of the Scatchard plot (22-24). Preparation of the Acridinium Derivatives 1-6. The acridine derivatives 8b-f were synthesized by a modified Bernthsen procedure as shown in Scheme 1 (25). The condensation reaction of diphenylamine with the corresponding carboxylic acid in the presence of anhydrous ZnCl2 yielded the acridine derivatives 8b-f in 50-60% yields. The quarternization of these acridine derivatives with methyl iodide gave the corresponding acridinium derivatives 1-6 in quantitative yields, which were recrystallized from a mixture (4:1) of acetonitrile and methanol (26). 10-Methylacridinium iodide (1): 79%; mp 230-232 °C; 1H NMR (300 MHz, DMSO-d6) δ 4.87 (3H, s), 8.05 (2H, t, J ) 7.5 Hz), 8.48 (2H, t, J ) 8 Hz), 8.63 (2H, d, J ) 8.4 Hz), 8.79 (2H, d, J ) 9.2 Hz), 10.21 (1H, s); 13C NMR (75 MHz, DMSO-d6) δ 150.96, 141.69, 139.43, 131.98, 128.15, 126.60, 119.36, 39.44. Elemental analysis calcd (%) for C14H12IN: C, 52.36; H, 3.77; N, 4.36. Found: C, 52.36; H, 3.51; N, 4.53. 9,10-Dimethylacridinium iodide (2): 76%, mp 248-249 °C; 1H NMR (300 MHz, DMSO-d6) δ 3.48 (3H, s), 4.77 (3H, s), 8.01 (2H, t, J ) 7.2 Hz), 8.41 (2H, t, J ) 7.4 Hz), 8.72 (2H, d, J ) 9.2 Hz), 8.89 (2H, d, J ) 8.2 Hz); 13C NMR (75 MHz, DMSO-d6) δ 149.76, 138.85, 136.22, 127.42, 125.55, 124.71, 120.51, 39.59, 15.36. Elemental analysis calcd (%) for C15H14IN: C, 53.75; H, 4.21; N, 4.18. Found: C, 53.87; H, 4.07; N, 4.18. 9-Phenyl-10-methylacridinium iodide (3): 71%; mp 201-202 °C; 1H NMR (300 MHz, DMSO-d6) δ 4.93 (3H, s), 7.54-7.56 (2H, m), 7.75-7.80 (3H, m), 7.92 (4H, d, J ) 3.7 Hz), 8.44-8.49 (2H, m), 8.88 (2H, d, J ) 9.2 Hz); 13 C NMR (75 MHz, DMSO-d6) δ 160.95, 141.76, 138.97, 133.66, 130.67, 130.35, 130.06, 129.41, 128.49, 126.08, 119.81, 39.52. Elemental analysis calcd (%) for C20H16IN: C, 60.47; H, 4.06; N, 3.53. Found: C, 60.55; H, 4.03; N, 3.27. 9-(4-Methylphenyl)-10-methylacridinium iodide (4): 71%; mp 228-230 °C; 1H NMR (300 MHz, DMSO-d6) δ 2.54 (3H, s), 4.93 (3H, s), 7.47 (2H, d, J ) 8

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Hz), 7.58 (2H, d, J ) 7.9 Hz), 7.90-7.99 (4H, m), 8.46 (2H, t, J ) 7.7 Hz), 8.85 (2H, d, J ) 9.3 Hz). 13C NMR (75 MHz, DMSO-d6) δ 161.26, 141.67, 140.41, 138.88, 130.66, 130.40, 130.11, 129.92, 128.35, 126.09, 119.75, 39.62, 21.54. Elemental analysis calcd (%) for C21H18IN: C, 61.33; H, 4.41; N, 3.41. Found: C, 61.60; H, 4.39; N, 3.48. 9-(3-Methylphenyl)-10-methylacridinium iodide (5): 68%; mp 220-222 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.86 (3H, s), 4.95 (3H, s), 7.36 (1H, d, J ) 7.5 Hz), 7.57 (1H, t, J ) 7.3 Hz), 7.63-7.72 (2H, m), 7.78 (2H, d, J ) 8.6 Hz), 7.95 (2H, t, J ) 7.6 Hz), 8.48 (2H, t, J ) 7.9 Hz), 8.90 (2H, d, J ) 9.2 Hz); 13C NMR (75 MHz, DMSO-d6) δ 160.72, 141.76, 139.04, 136.33, 133.30, 131.16, 130.72, 129.92, 129.40, 128.75, 126.78, 125.96, 120.03, 39.63, 19.76. Elemental analysis calcd (%) for C21H18IN: C, 61.33; H, 4.41; N, 3.41. Found: C, 61.08; H, 4.18; N, 3.51. 9-(2-Methylphenyl)-10-methylacridinium iodide (6): 60%; mp 224-225 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.87 (3H, s), 4.97 (3H, s), 7.36 (1H, d, J ) 7.5 Hz), 7.57 (1H, t, J ) 7.3 Hz), 7.63-7.72 (2H, m), 7.78 (2H, d, J ) 8.6 Hz), 7.95 (2H, t, J ) 7.6 Hz), 8.48 (2H, t, J ) 7.9 Hz), 8.90 (2H, d, J ) 9.2 Hz); 13C NMR (75 MHz, DMSO-d6) δ 160.72, 141.76, 139.04, 136.33, 133.30, 131.16, 130.72, 129.92, 129.40, 128.75, 126.78, 125.96, 120.03, 39.63, 19.76. Elemental analysis calcd (%) for C21H18IN: C, 61.33; H, 4.41; N, 3.41. Found: C, 61.55; H, 4.17; N, 3.62. RESULTS AND DISCUSSION

The photophysical properties of the acridinium derivatives were investigated under different conditions, and these results are summarized in Table 1. The acridinium derivatives 1, 2 and the o-tolylacridinium derivative 6 exhibited quantitative fluorescence quantum yields (Φf = 1) with an emission maximum around 500 nm. In contrast, the fluorescence quantum yields of the arylacridinium derivatives 3-5 (Φf ) 0.09, 0.05, and 0.11, respectively) were found to be significantly quenched. The acridinium derivative 1 exhibits a single-exponential decay with a lifetime of 35 ns, whereas the o-tolylacridinium derivative 6 shows a reduced lifetime of 25 ns (Table 1). When compared to 1, 2, and 6, unusually low lifetimes were observed for the arylacridinium derivatives 3-5 (τ ) 1.3, 0.6, and 1.7 ns, respectively). The lower values of the fluorescence quantum yields and lifetimes observed for the arylacridinium derivatives 3-5 can be attributed to enhanced nonradiative decay pathways due to the rotation between the acridinium and the phenyl ring and the favorable quenching of the singlet excited state by the aryl group through an electron-transfer mechanism (26, 30, 31). Interestingly, the excited-state properties of the o-tolylacridinium derivative 6, is very similar to that of the simple acridinium derivatives 1 and 2, probably because of the steric constraints offered by the o-methyl substituent. The acridinium derivatives 1-6 can in principle interact with DNA through intercalation and/or electrostatic modes. Figure 2 shows the change in the absorption spectra of the acridinium derivative 2 with increasing DNA concentrations. The addition of CT DNA to a solution of 2 results in a strong decrease in the absorption of the acridinium chromophore, along with a red shift of around 3 nm. Furthermore, with the increase in DNA concentration, a strong quenching of the acridinium fluorescence emission is observed as shown in Figure 3. Similar changes in absorption and fluorescence emission

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Scheme 1

Table 1. Photophysical and DNA Binding Properties of the Acridinium Derivatives 1-6a compound 1 2f 3 4 5 6

λem, nm 491 493 508 508 508 501

Φfb 1 1 0.09 0.05 0.11 1

τ, ns

K (M-1)c

nd

kDNA (s-1)e

35 34 1.3 0.6 1.7 25

7.7 × 7.3 × 105 1.1 × 105 7.1 × 104 6.9 × 104 g

2.5 2.5 6 7 6.5 g

1.2 × 1010 1.1 × 1010 5 × 1010 6.5 × 1010 3 × 1010 g

105

a Average of more than two experiments and error ca. (5%. The photophysical and DNA binding properties were examined in water and phosphate buffer (10 mM, pH 7.4), respectively. b Fluorescence quantum yields were calculated using 10-methylacridium trifluoromethane sulfonate as the standard (Φf ) 1) (26, 27). c DNA association constants determined by the Scatchard analysis of fluorescence titration data. d Number of nucleotides occluded by a bound ligand. e Rate of static quenching by DNA calculated as reported earlier (28, 29). f Compound 2, under similar conditions exhibited DNA association constants (K) 8.5 × 105 M-1 and 4.12 × 105 M-1 and electron-transfer rate constants (kDNA) 3.8 × 1010 s-1, and 0.24 × 1010 s-1 with poly(dG).poly(dC) and poly(dA).poly(dT), respectively. g Negligible binding.

Figure 2. Change in absorption spectra of 2 (32 µM) in phosphate buffer (10 mM; pH 7.4) with increasing concentration of CT DNA. [DNA] (a) 0, (b) 0.05, (c) 0.12, (d) 0.19, and (e) 0.34 mM. Inset shows the change in absorption spectra of 6 (42.5 µM) in phosphate buffer (10 mM; pH 7.4) with increasing concentration of CT DNA. [DNA] (a) 0, (b) 0.17, (c) 0.34, and (d) 0.42 mM.

spectra were observed for the acridinium derivatives 1 and 3-5 (Supporting Information; Figures S1-S4). Among all the acridinium derivatives, the o-tolylacridinium derivative 6 showed an interesting variation. Insets of Figures 2 and 3 show the changes in the absorption and fluorescence emission spectra of 6, respectively, with increasing concentration of DNA. Both the absorption

Figure 3. Change in fluorescence spectra of 2 (32 µM) in phosphate buffer (10 mM; pH 7.4) with increasing concentration of CT DNA. [DNA] (a) 0, (b) 0.05, (c) 0.09, (d) 0.12, (e) 0.16, (f) 0.19, (g) 0.26, (h) 0.34, and (i) 0.42 mM. Inset shows the change in fluorescence spectra of 6 in phosphate buffer (10 mM; pH 7.4) with increasing concentration of CT DNA. [DNA] (a) 0, (b) 0.17, (c) 0.34, (d) 0.42, and (e) 1 mM. Excitation wavelength, 363 nm.

and fluorescence studies indicate that the ortho-derivative 6 exhibits negligible interactions with DNA. With a view to understanding the effect of various DNA sequences, we have investigated the interaction of the acridinium derivatives with polynucleotides such as poly(dG)‚poly(dC) and poly(dA)‚poly(dT). The addition of poly(dG)‚poly(dC) to a solution of 2 resulted in a strong decrease in the absorption of the acridinium chromophore, along with a red shift of around 5 nm with an isosbestic point at 450 nm. Similar observations were made with poly(dA)‚poly(dT) and with other acridinium derivatives (Supporting Information; Figure S5). Furthermore, with the increase in poly(dG)‚poly(dC) concentration, a strong quenching of the acridinium fluorescence emission is observed as shown in Figure 4. The poly(dG).poly(dC) exhibits an efficient fluorescence quenching of the acridinium derivative 2 when compared to CT DNA and poly(dA)‚poly(dT) (inset of Figure 4), indicating thereby the preferential interactions of these molecules with DNA rich in guanosine sequences. The DNA association constants (K) of the complexes formed between the acridinium derivatives 1-5 and DNA were determined based on fluorescence titration experiments, according to the method of McGhee and von Hippel by using the data points of the Scatchard plot (9) and are reported in Table 1. For example, 10-methylacridinium (1) shows a binding constant of 7.7 × 105 M-1,

Tuning of Intercalation and Electron-Transfer Processes

Figure 4. Change in fluorescence spectra of 2 (32 µm) in phosphate buffer (10 mM; pH 7.4) with increasing concentration of poly(dG).poly (dC). [poly(dG).poly(dC)] (a) 0, (b) 0.08, (c) 0.16, (d) 0.24, (e) 0.30, (f) 0.38, (g) 0.5 mM. Inset shows the change in fluorescence spectra of 2 in phosphate buffer (10 mM, pH 7.4) with increasing concentration of poly(dA).poly(dT). [poly(dA).poly(dT)] (a) 0, (b) 0.08, (c) 0.17, (d) 0.25, (e) 0.33, (f) 0.48, (g) 0.63 mM. Excitation wavelength, 363 nm.

whereas the arylacridinium derivatives 3-5 show relatively lower values of DNA association constants. These association constants were also found to be sensitive to the DNA sequences. For example, the association constant of the acridinium derivative 2 with poly(dG)‚poly(dC) was found to be around 1.2 and 2 times greater than that of CT DNA and poly(dA)‚poly(dT). Interestingly, the binding site size (n) was found to be greater for the arylacridinium derivatives 3-5 (n ) 6-7) than the acridinium derivatives 1 and 2 (n ) 2.5). Thus, a comparison of the DNA binding constants indicate that the acridinium derivatives 1 and 2 bind to DNA by nearly one order greater efficiently when compared with the arylacridinium derivatives 3-5. In the case of the arylsubstituted derivatives, the efficiency was found to be in the order, phenyl > p-tolyl > m-tolyl .. o-tolyl, wherein the increase in steric factors around the acridine ring hinders the DNA binding interactions and finally leads to a negligible binding as observed in the case of the o-tolylacridinium derivative 6. Picosecond time-resolved fluorescence studies in the presence and absence of DNA indicate that the acridinium derivative 2 exhibits a single-exponential decay in buffer with a fluorescence lifetime of 34 ns (Figure 5), while in the presence of DNA it shows a triexponential decay with lifetimes of 35 ps, 0.8 ns and 33 ns. The short lifetimes observed (35 ps and 0.8 ns) may be attributed to the intercalative binding of the acridinium chromophore at two different base sequence environments, whereas the third lifetime could be due to the unbound molecules. On the other hand, the ortho-derivative 6, having a fluorescence lifetime of 25 ns in buffer, exhibited negligible changes in the presence of DNA (inset of Figure 5). These observations confirm that the acridinium chromophore in the case of 6 undergoes neither intercalative nor groove binding interactions with DNA. Figure 6 shows the first derivative plots for the thermal denaturation curves obtained for the duplex DNA1 and DNA2 in the presence and absence of the acridinium derivatives 2-6. The oligonucleotide duplex melts at 52 °C in the absence of ligands, whereas the presence of 20 µM of acridinium derivative 2 stabilizes the duplex by 9 °C. As expected, the arylacridinum derivatives 3-5, under similar conditions, stabilize the duplex less (6-7

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Figure 5. Fluorescence decay profiles of 2 (32 µM) in the presence and absence of DNA in phosphate buffer (10 mM; pH 7.4). [DNA] (a) 0 and (b) 0.34 mM. Inset shows the fluorescence decay profiles of 6 in the presence and absence of DNA under similar conditions. [DNA] (a) 0 and (b) 0.34 mM. Excitation wavelength, 401 nm.

Figure 6. First derivative plots for the thermal denaturation curves of the DNA duplex, DNA1.DNA2 in the presence and absence of the acridinium derivatives 1-6 (20 µM) in 10 mM phosphate buffer (pH 7.4). DNA1: 5′-CGT GGA CAT TGC ACG GTA C-3′; DNA2: 5′-GTA CCG TGC AAT GTC CAC G-3′.

°C), whereas, the ortho-derivative 6 provides negligible stabilization to the DNA duplex. The absorbance changes observed at 260 nm, corresponding to DNA during the thermal denaturation of DNA in the presence and absence of the acridinium derivatives, clearly reflect the extent and nature of binding interactions of these derivatives with DNA. Similar absorption changes were observed when monitored at 355 nm corresponding to the acridinium chromophore, which further supports the binding trends observed in the case of the substituted acridinium derivatives. Figure 7 shows the results of the viscometry studies of representative examples which indicate that the viscosity of DNA increases proportionally with the binding affinity of the acridinium derivatives 2 and 4, whereas negligible viscosity changes are observed for the orthoderivative 6. The change in viscosity could be attributed to the π-stacking of the acridinium derivatives between DNA base pairs, which results in the increase in the viscosity of DNA as in the case of typical intercalators (32, 33). On the other hand, the ortho-derivative 6 shows negligible binding with DNA by intercalation as reflected from the physical and spectroscopic studies. The intercalative interactions of several acridinium conjugates with DNA have been well characterized in the literature by various biophysical and NMR techniques (34-37).

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Io/I ) 1 + kDNA/kSI

Figure 7. Effect of increasing concentrations of the acridinium derivatives 2 (a), 4 (b), and 6 (c) on the relative viscosity of CT DNA (0.7 mM) at 26 ( 0.2 °C in phosphate buffer (10 mM, pH 7.4).

Figure 8. Minimum energy conformations of p- and o-tolylacridinium derivatives 4 and 6, obtained by AM1 calculations.

The differences observed in DNA binding affinity of these acridinium derivatives could be rationalized in terms of the steric effects offered by the substituents. The structural analysis of the arylacridinium derivatives reveals that the acridinium as well as the aryl substituent are out of plane to each other and the dihedral angle (φ) according to the minimum energy conformations for 3-6 are 64.8°, 64°, 64.7°, and 77.5° (Figure 8) (38). To achieve effective intercalation of the acridinium chromophore, the aryl group has to undergo a rotation during the process of intercalation. The feasibility of this rotation is the deciding factor in effective binding of these molecules to DNA. In the case of the ortho-derivative 6, the rotation of the tolyl group is sterically hindered and hence no significant binding with DNA was observed. The π-stacking of the acridinium chromophore between the DNA bases as in the case of the simple acridinium derivatives 1 and 2 and arylacridinium derivatives 3-5 causes a strong quenching of the fluorescence of the molecule due to the possible electron-transfer processes from DNA bases (39). Whereas, in the case of the orthoderivative 6, which does not intercalate, the electron transfer is not an efficient process and hence the fluorescence emission properties remain unchanged in the presence of DNA. On the basis of the limiting fluorescence intensity (under conditions where >99% of the substrate is bound to DNA), the rate constant for the static quenching of the singlet excited state of the acridinium chromophore by DNA (kDNA) could be evaluated using eq 1,

(1)

where Io and I are the fluorescence intensities in the absence and presence of DNA and kSI is the sum of rate constants (kr + knr) of the singlet excited-state deactivation (28, 29). As shown in Table 1, for the acridinium derivatives 1-5, which bind with DNA through intercalation, the rate constant for static quenching was found to be in the order of 1010 s-1. This observation confirms that the intercalative mode of binding is crucial to observe an efficient quenching of fluorescence of ligands through the electron transfer process from the DNA bases. Moreover, the electron transfer rate constant in the case of the polynucleotide enriched with GC content, poly(dG)‚poly(dC), was found to be around 3.5 and 16 times greater than that of CT DNA and poly (dA)‚poly(dT), respectively (Table 1). This can be explained in terms of lower oxidation potential of guanosine (39) and hence greater feasibility of electron transfer from guanosine to the singlet excited state of the acridinium moiety. In conclusion, we have demonstrated that by introducing subtle steric factors one may alter the DNA binding properties of acridinium derivatives. It was observed that the unsubstituted and methylacridinium derivatives 1 and 2 bind with DNA by nearly one order more efficiently when compared to the bulky arylacridinium derivatives 3-5. In the case of the aryl-substituted derivatives, the efficiency of binding was found to be in the order, phenyl > p-tolyl > m-tolyl .. o-tolyl, wherein the increase in steric factors around the acridine ring hinders the DNA binding interactions and finally leads to a negligible binding as observed in the case of the o-tolylacridinium derivative 6. Furthermore, these results support the fact that intercalative π-stacking interactions of the ligands with DNA are essential for observing efficient electron transfer reactions between the DNA bases and the ligands. Studies related to the use of these highly fluorescent molecules for the recognition of various DNA structures and duplexes containing mismatches and base bulges are in progress in our laboratory. ACKNOWLEDGMENT

This work was supported by the Council of Scientific and Industrial Research (CSIR) and the Department of Science and Technology (DST), India, and National Science Foundation (NSF), USA. We acknowledge the National Centre for Ultrafast Processes (NCUFP), Chennai, for lifetime measurements. This is contribution number RRLT-PPD(PRU)-177 from the Regional Research Laboratory, Trivandrum. Supporting Information Available: Absorption and emission spectra. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Waring, M. J. (1981) DNA modification and cancer. Annu. Rev. Biochem. 50, 159-192. (2) Tomasz, M. (1994) The mitomycins: Natural cross-linkers of DNA. Molecular Aspects of Anticancer-Drug Interactions (Neidle, S., and Waring, M., Eds.) Vol. 2, pp 312-349, CRC Press, Boca Raton, FL. (3) Wilson, W. D. (1999) DNA intercalators. DNA and Aspects of Molecular Biology (Kool, E., Ed.) Vol. 7 in Comprehensive Natural Products Chemistry (Barton, D., and Nakanishi, K., Eds.), Chapter 12, Pergamon Press, Elsevier.

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