Solvent Effects on the Thermodynamics and Kinetics of Coralyne Self

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J. Phys. Chem. B 2009, 113, 188–196

Solvent Effects on the Thermodynamics and Kinetics of Coralyne Self-Aggregation Begonã Garcıá,*,† Saturnino Ibeas,† Rebeca Ruiz,† Jose´ M. Leal,† Tarita Biver,‡ Alessia Boggioni,‡ Fernando Secco,*,‡ and Marcella Venturini‡ Departamento de Quı´mica, UniVersidad de Burgos, 09001 Burgos, Spain, and Dipartimento di Chimica e Chimica Industriale, UniVersita` di Pisa, 56126 Pisa, Italy ReceiVed: September 5, 2008; ReVised Manuscript ReceiVed: NoVember 4, 2008

The role of solvent effects on the thermodynamics and kinetics of the coralyne self-aggregation process has been investigated in ethanol-water mixtures of different compositions. The changes in the UV/visible spectra of coralyne and FAB/LSIMS mass spectrometry agreed well with the formation of a dimer species. 1D and 2D 1H experiments have allowed one to look into the features of the self-aggregation process and to determine the equilibrium constant and the ∆H0 and ∆S0 values for the aggregate formation in 0-50% ethanol-water mixtures. The kinetics of self-aggregation has been investigated by the T-jump chemical relaxation method, and the results have been interpreted in terms of dimer formation. The dependence of the relative viscosity of coralyne solutions on the dye concentration was studied in different ethanol-water mixtures. Finally, it was found that coralyne behaves as a solvatochromic indicator which is preferentially solvated according to the sequence ethanol > ethanol-water > water. All of the results concur in elucidating the relevant role of the hydrophobic interaction process of coralyne stack formation. Introduction The isoquinoline alkaloids constitute an important class of natural products endowed with extensive biological applications.1 This sort of compound is quite widely spread out all over several botanic families. The chief member is berberine, which has a synthetic analogue known as coralyne. Coralyne (8-methyl-2,3,10,11-tetramethoxydibenzo[a,g]quinolizinium), a planar aromatic molecule, bears a positive charge and is a dye compound characterized by four fused aromatic rings; these two relevant features have turned coralyne into a suitable candidate for intercalation reactions into polynucleotides. Coralyne has been shown to hold back the leukemia growth in cells,2 this property being strictly associated with the ability of coralyne to interact with DNA; moreover, it brings about efficient photoinduced DNA damage by single-strand cleavage.3 Coralyne exhibits a noticeable antitumor activity and presents a great deal of promise because of its low toxicity and its ability to inhibit the topoisomerase I function.4 Several studies on the binding of coralyne to DNA have revealed that the solution chemistry of coralyne is not a simple one since, unfortunately, coralyne exhibits a high tendency for aggregation in water, even at low concentrations.5 Therefore, in aqueous solution, coralyne is present in the form of aggregates rather than as separate, independent molecules, and this feature may exert a significant influence, especially on the optical properties of the system. So far, this phenomenon has remained as a major problem in the understanding of the mechanism of coralyne binding to DNA. The intrinsic complexity revealed by this feature suggests that the dye self-aggregation comes about quite easily, despite the positive charge present in coralyne. Self-aggregation has been observed experimentally in coralyne for a long time.5,6 Nevertheless, a recent review7 and a paper8 reporting on the poly(dA) · poly(dT) disproportionation * To whom correspondence should be addressed. † Universidad de Burgos. ‡ Universita´ di Pisa

into a triplex and a single poly(dA) strand make no mention of the dye tendency to self-aggregation. Pal et al.6 have found that the coralyne property to aggregation decreases in the presence of ethanol and have reported that in 30% EtOH, the dye is present essentially as a monomer. Flat aromatic molecules aggregate on stacks, and the stacking is caused by a variety of interactions, hydrophobic interactions, dipole-dipole interactions, π-π interaction, dispersion forces, and other kinds of interaction. Previous investigations, mainly in nucleobase-nucleobase self-aggregation, have shown that the hydrophobic interactions play an important role in the stacking process. However, the hydrophobic/hydrophilic properties of the solvent and the solute give rise to a preferential solvation9,10 which plays an important role in determining the forces that govern the aggregation phenomena. Therefore, in view that self-aggregation may affect the physicochemical features of coralyne and in the framework of our investigations on dye-nucleic acid interactions, we found it useful and interesting to carry out an extensive investigation of the coralyne behavior in ethanol-water mixtures of different compositions. Materials and Methods Computational Details. All the calculations were carried out with Gaussian 03.11 The geometry has been optimized using the DFT-B3LYP method,12 with the 6-31G* basis set. To evaluate the solvent effects, the conductor-like polarizable continuum model (C-PCM)13 has been used. Natural bond orbital theory (NBO)14 was used to calculate atomic charges. Materials. Coralyne chloride was a 99% pure Sigma-Aldrich product and was used as received. The stock solutions were frequently prepared by dissolving weighed amounts of the solid in doubly distilled water. Other chemicals were analytical-grade reagents and were used without further purification. Methods. The pH measurements were made with a PHM84 radiometer Copenhagen pH meter fitted out with a combined glass electrode and a 3 M NaCl solution as a liquid junction. The pH of the solutions was adjusted to the value of 7.0 by

10.1021/jp807894a CCC: $40.75  2009 American Chemical Society Published on Web 12/15/2008

Thermodynamics and Kinetics of Coralyne Self-Aggregation

J. Phys. Chem. B, Vol. 113, No. 1, 2009 189

Figure 1. Optimal Geometry of the coralyne molecule in the gas phase, water, EtOH, DMSO, acetone, and CCl4. Further information on the molecule is contained in the Supporting Information.

additions of cacodylic acid/sodium cacodylate (3 × 10-3 M), and the ionic strength was adjusted with NaCl. Viscometric measurements were performed using a Micro-Ubbelohde viscometer, which is able to work using 3.00 mL of solution. Mass spectrometry FAB/LSIMS measurements were recorded on a VG AutoSpec apparatus using an ionization liquid secondary ion mass spectrometer (LSMIS+) and Cs and nitrophenyl alcohol as the matrix. The isotope simple distribution was deduced using a homemade program. The NMR measurements were performed with a Unity Inova Varian 400 instrument at 9.4 T (operating at 399.941 MHz). The spectra were recorded using a spectral window of 15 ppm, over the 20-70 °C temperature and 0-50% ethanol-water ranges and a constant 0.005 M coralyne concentration. The acquisition time was 13.2 µs, and a pulse angle of π/2 was employed. All measurements are referred to the corresponding (solvent + TMS) system. The 1H-1H 2D NOESY spectra at 40 °C were recorded under the following conditions: relaxation delay 1.000 s, mixing 0.500 s, acquisition time 0.236 s, 2D width 4333.5 Hz, 16 repetitions, 2 × 200 increments, observe 1H, and 399.9409828 MHz. Data processing: Gauss apodization 0.109 s. F1 data processing: Gauss apodization 0.043 s and FT size 2048 × 2048. Unfortunately, the low solubility of coralyne made it unfeasible to collect the spectra at dye concentrations above 0.005 M and temperatures below 20 °C. The kinetic study of self-aggregation was carried out using a Dialog T-jump instrument in the absorbance detection mode. To minimize coralyne photo bleaching, the sample was illuminated only for the short time needed for measurements. The spectrophotometric measurements have been performed using a Perkin-Elmer Lambda 35 and a HP 8453 photodiode array instrument. Results and Discussion Theoretical Calculations. The theoretical study has shown that, interestingly, the positive charge of coralyne (Figure 1) is shared by the peripherical H-atoms, whereas the O-atoms and even the nitrogen atom bear negative charge densities both in the gas phase and in solvents of different polarity. In Table 1S (Supporting Information) are reported the charge distributions

Figure 2. Set of spectra of coralyne recorded in 20% ethanol at increasing dye concentration. CD ) 5.1 × 10-6, 2.3 × 10-5, 4.5 × 10-5, and 6.8 × 10-5 M (from top to bottom at 300 nm). I ) 0.1 M(NaCl), pH ) 7.0, T ) 25 °C.

in the gas phase and in the presence of water, ethanol, DMSO, acetone, and CCl4. Spectral Analysis of Aggregation. Figure 2 shows the set of spectra of coralyne recorded in 20% ethanol, pH ) 7.0 (sodium cacodylate) and I ) 0.10 M (NaCl), for different dye concentrations. Changes of the buffer concentration did not affect the spectral behavior. The dependence of the apparent absorptivity on wavelength reveals the presence of different coralyne forms in equilibrium since the spectra do not coincide and an inversion of the two maxima centered at about 300 and 310 nm comes about when the dye concentration is increased. The principal component analysis (PCA) of coralyne solutions (not shown) reveals that at least two components are needed to reproduce the experimental spectra. This conclusion was born out by the absorbance versus dye concentration plots. The deviations from Lambert-Beer’s behavior, also reported previously,6 indicate that the aggregate formation has commenced already at dye concentrations below 3 × 10-5 M (Figure 3). A set of spectra of a coralyne solution was also recorded at different temperatures, from 10 to 90 °C (Figure 4); the monomer-aggregate equilibrium displacement is clearly observed, indicating that the more energetic of the two maxima at ∼300 nm should be assigned to the aggregate.

190 J. Phys. Chem. B, Vol. 113, No. 1, 2009

Figure 3. Absorbance of coralyne as a function of CD, the total dye concentration, in aqueous solution; I ) 0.10 M (NaCl), pH ) 7.0, λ ) 414 nm, T ) 25 °C. The straight line represents the Lambert-Beer behavior.

Figure 4. Absorbance spectra of coralyne in aqueous solution recorded with increasing temperatures (10 to 90 °C from bottom to top at 300 nm). CD ) 1.26 × 10-5 M, I ) 0.10 M (NaCl), pH ) 7.0.

Figure 5. Mass spectrum of coralyne (CD ) 0.005 M) in aqueous solution. Nitrophenyl alcohol matrix.

FAB/LSIMS Spectra. The presence of a dimer species in solution was revealed by high-resolution mass spectrometry FAB/LSIMS (fast atom bombardment /liquid secondary ion mass spectrometry). Figure 5 shows the mass spectrum recorded. The intense signal at m/z 364.08 is assumed to correspond to the coralyne monomer species (M+) accompanied by a series of fragment ions, including the methyl loss [M - CH3]•+ at m/z 349.05 and [M - Me - N + H]•+ at m/z 336.08. The feeble signal at 727.14 is assumed to correspond to the [2M - H]+, where one proton of the dimer is missed. The theoretical isotopic

Garcıá et al. distribution shown in Figure 6 bears out the assumptions made for the monomer and the dimer signals. Viscosity Experiments. The effect of the ethanol addition on the dye self-aggregation can be rationalized by the dependence of the viscosity ratio (η/ηo) (where ηo is the viscosity of the pure solvent and η is that of the solution) on the dye concentration (CD) observed for different ethanol-water mixtures (Figure 7). Addition of coralyne to water brings into play no significant effect on the viscosity of pure water. In the presence of ethanol, the relative viscosity decreases as the dye content is increased, this effect becoming more and more pronounced as the ethanol content rises. Given that coralyne tends to be preferentially solvated by ethanol (see below, solvatochromic effect), such preferential solvation can modify the energy of the ethanol-water hydrogen-bond network, thus inducing a viscosity decrease. Moreover, an increase in the ethanol content brings about the displacement of the monomer-dimer equilibrium in favor of the monomer species (see NMR experiments), thus providing a further contribution to the readjustment of the solvent structure. 1 H NMR Signal Assignment and Structural Considerations. Figure 8 shows the 1H-1H 2D NOESY spectrum of the aromatic protons of coralyne in D2O. It should be noted that in the NOESY spectra, the spin decoupling could only be observed between the 1H nuclei and over a distance of about 4 Å.15 The spectrum consists of two doublets and five singlets, each corresponding to the aromatic protons labeled 1-7. Protons 1 and 2 each have given rise to a doublet because they are coupled to only one adjacent H nucleus. As expected, a spin decoupling effect was observed between 1 (8.05 ppm) and 2 (7.25 ppm) (interproton distance, 2.422 Å). A NOESY effect was also observed between 2 (7.25 ppm) and 7 (6.66 ppm) (interproton distance, 2.4786 Å), which enables an unambiguous signal assignment of the proton labeled as 7. Similarly, proton 3 (7.82 ppm) displays a decoupling effect regarding the protons labeled as 6 (6.67 ppm; interproton distance, 2.4511 Å) and 4 (6.76 ppm; interproton distance, 1.9501 Å). Finally, proton 5 does not display any NOESY effect because it is located further than 4 Å from the other aromatic protons (the 1-5 interproton distance is 4.8873 Å). Several groups of workers have demonstrated the usefulness of the NMR method in the elucidation of aromatic stacking.16 Actually, the stacking of aromatic rings results in an upfield shift in the proton resonance, compared with the nonstacked ones. Figure 9 shows the 1H spectra of coralyne in the region of the aromatic protons recorded in deuterated water and different temperatures between 20 and 70 °C. Table 2S (Supporting Information) lists the chemical shift values of signals 1-7, recorded between 20 and 70 °C in five different 0-50% ethanol-water mixtures. The temperature rise brings about downfield displacements of all signals, though to somewhat different extents. In all cases, the displacement of signals 3 and 4 is most pronounced; the displacements of these signals with temperature can be accounted for on the basis of only the presence of aggregates.16 Similar conclusions can be reached from the analysis of the chemical shifts induced by addition of ethanol (Figure 10); here, too, the protons labeled as 3 and 4 undergo the more pronounced shifts upon increasing the ethanol content, thus revealing that ethanol favors the aggregate dissociation Figure 11 shows the chemical shift of signal 3 at different EtOH contents. It can be observed that a increase in temperature and ethanol amounts diminishes the stacking interactions, thus



Figure 6. Widening of the mass spectrum of coralyne (A, B) and theoretical isotopic distribution (A′, B′) of the monomer and dimer species, respectively. The ordinates are normalized with respect to the most abundant isotope.

TABLE 1: Self-Aggregation Constant Values of Coralyne at 25°C and Related Thermodynamic Parameters, Evaluated from 1H Measurements, in Different EtOH/H2O Mixtures % EtOH 0 10 20 30 40 50

Figure 7. Relative viscosity (η/η0) of coralyne solutions vs total dye concentration. (9) 0, (0) 5, (b) 10, (O) 20, and (() 30% EtOH. I ) 0.1 M (NaCl), pH ) 7.0, T ) 25 °C.

disfavoring the formation of aggregates. This behavior suggests that the coralyne monomers do aggregate in stacks, where optimal overlapping involves protons 3 and 4. It is interesting to note that passing from 0 to 50% EtOH brings about a δ shift much larger than that induced by the temperature increase. This behavior suggests that some factors other than π-π interactions are at work, such factors being in close connection with hydrophobic interactions like electrostatic interactions between π-face and substituent17 (and + - π/+ - π interactions).18 A very interesting article on genuine π-π interactions has been recently published.19 Thermodynamics of Aggregate Formation. The NMR experiments have required rather high coralyne concentrations. Under these circumstances, one could expect that the aggregation process does not stop at the dimerization stage; instead, more

ε

K(M-1)

78.43 8693 ( 102 73.88 2987 ( 90 68.91 1959 ( 58 63.60 917 ( 35 58.01 740 ( 30 52.23 494 ( 18

ln Κ

∆H0 ∆S0 (kcal mol-1) (cal K-1 mol-1)

9.03 8.00 7.58 6.82 6.60 6.20

-10.7 ( 1.4 -10.5 ( 1.2 -10.4 ( 1.5 -10.7 ( 1.1 -11.2 ( 1.4 -8.8 ( 0.9

-17.8 ( 1.8 -19.4 ( 2.2 -20.0 ( 2.4 -22.2 ( 2.5 -24.6 ( 2.3 -17.3 ( 1.2

complex aggregates can be expected. Therefore, the 1H data used to evaluate the self-aggregation equilibrium have been analyzed according to an isodesmic model that has already been put forward to analyze stacking interactions of nucleobases.20 Such an analysis requires knowledge of the chemical shifts of the monomer, δ1. These parameters have been assumed to coincide with the chemical shifts measured in pure DMSO where, based on the lack of dependence with temperature of the 1H signals, it can be assumed that only the monomer form is present (Figures 1S and 2S of the Supporting Information). The other parameter to be employed in the analysis is δi, the chemical shift corresponding to the i aggregate species, with ∆ ) δ - δ1 and ∆i ) δi - δ1; ∆2 and ∆3 are the relative chemical shifts in the dimer and trimer complexes (“dimer and trimer shifts”). According to Dimicoli and Helene,21 we are using γ ) ∆3/∆2 ) 2; the δ3 value was estimated for each H signal by extrapolating the data obtained at different temperatures down to the freezing point. An attempt to analyze the data by setting ∆3 ) ∆222 was unsuccessful. The details needed for the derivation of the equations employed to evaluate the equilibrium constant K ) an+1/(an × a1) as well as the meaning of the


Figure 8.

Garcıá et al.

H-1H NMR 2D NOESY spectra of the coralyne aromatic protons in deuterated water. CD ) 0.005 M, T ) 40 °C, I ) 5 × 10-3 M.

1

TABLE 2: Polarity/Polarizability Index, π*, for the Ethanol/Water System Deduced from Equation 2 Using the Measured Frequenciesa x1 0.00000 0.01554 0.03220 0.05049 0.06827 0.09193 0.11316 0.14074 0.17245 0.19413 0.23228 0.27013 0.31241 0.36087 0.40570 0.47199 0.54635 0.62826 0.73036 0.85107 1.00000 a

10-3ν, cm-1 23.720 23.668 23.636 23.633 23.609 23.578 23.555 23.524 23.502 23.484 23.465 23.451 23.441 23.430 23.421 23.410 23.398 23.391 23.370 23.350 23.316

TABLE 3: Kinetic Parameters for the Coralyne Self-Aggregation Process at Different Ethanol Concentrationsl I ) 0.10 M (NaCl), pH ) 7.0, T ) 25°C % EtOH xC2H5OH 10–8 kf (M-1 s-1) 10–4 kd, (s-1) 10–3 KD, (M-1)

π* b

1.09 1.02 0.98 0.97 0.94 0.90 0.87 0.83 0.81 0.78 0.76 0.74 0.73 0.72 0.71 0.69 0.68 0.67 0.64 0.61 0.54b

The x1 is the ethanol mole fraction. CD ) 3 × 10-5 M.

0 5 10 15 20 25

b

Ref

27.

relative symbols can be found in the article by Schimmack et al.20 The analyses have been carried out for all seven aromatic protons, and the K value has been averaged. Table 1 contains the values of K(i) for the ith signal, measured under the different conditions of temperature and solvent composition used. Table 1 lists the results at 25 °C using different ethanol contents, as well as the dimerization enthalpy and entropy values.

0.00 0.02 0.03 0.05 0.07 0.09

0.48 0.80 1.16 1.21 1.31 1.33

0.99 1.08 1.17 1.51 2.06 3.22

5.15 7.78 10.0 8.28 6.31 4.13

The decrease of K could not be entirely ascribed to the change of the dielectric constant, ε. Actually, application of the Fuoss equation in a logarithmic form (ln K versus 1/ε) would result in a straight line, contrary to the experiment. The ∆H0 and ∆S0 values shown in Table 3 are similar to the corresponding parameters obtained by Poerschke and Eggers for self-aggregation of N6,N9-dimethyladenine investigated by osmometry23 (∆H0 ) (-8.7 ( 1.5) kcal mol-1 and ∆S0 ) (-21.6 ( 3) cal K-1 mol-1). In agreement with the conclusions of the above authors, we propose here that the rather negative ∆H0 and ∆S0 values could be explained in terms of surface solute-solvent interactions that change during the stack formation. Actually, the diminution of the equilibrium constant with the rise in the ethanol content should be related to the entropy variation, which reveals the role played by the hydrophobic interactions in the self-aggregation process. Solvatochromic Effect. Changes in solvent polarity may induce changes in the UV/vis spectrum of an absorbing molecule. In mixed solvents, such as the ethanol-water system employed in this work, the observed displacements of the coralyne absorption band with the solvent composition provide information on the intermolecular solute-solvent interactions,


Figure 9. 1HNMR spectra of aromatic protons of coralyne in deuterated water at different temperatures. CD ) 0.005 M, I ) 5 × 10-3 M.

which, in turn, can shed light into the role played by the solvation effects in the self-aggregation process. Actually,


Figure 10. 1HNMR spectra of aromatic protons of coralyne in deuterated water in different ethanol-water mixtures. CD ) 0.005 M, T ) 20 °C, I ) 5 × 10-3 M.

organic molecules displaying this property are also known as solvatochromic indicators. In the course of the spectrophotometric investigation of the binding equilibria, the coralyne band in water centered at 422


Garcıá et al.

Figure 11. Temperature and ethanol content effects on the chemical shifts of signal 3; (9) water and (0) 10, (b) 20, (O) 30, ([) 40, and (]) 50% EtOH, CD ) 0.005 M.

Figure 14. Reciprocal relaxation time, 1/τ, versus the free coralyne concentration, [D], at different ethanol contents, (9) 0, (0) 5, (b) 10, (O) 15, (() 20, and (]) 25%; pH ) 7.0, I ) 0.1 M (NaCl), T ) 25 °C.

The parameters of eq 1 have been obtained by linear regression of the data of Table 4S (Supporting Information), which reports the π* values for different solvents,27 their values being νo ) (22.86 ( 0.06) × 103 cm-1 and s ) (0.79 ( 0.09) × 103 cm-1. These parameters have, in turn, been used to evaluate the π* values for the different ethanol-water mixtures employed in this work. The π* values obtained for these mixtures are reported in Table 2. The mixing solvatochromic parameter ∆π* has been evaluated using eq 2

∆π* ) π* - π1*x1 - π2*x2 Figure 12. Wavenumbers, ν, of the absorption maxima of coralyne in EtOH-water mixtures versus x1, the EtOH mole fraction; the continuous line was obtained according to the preferential solvation model based on eqs 5 and 6.

(2)

where π1* refers to ethanol and π2* to water. The preferential solvation of coralyne in the binary solvent used was analyzed assuming a simple solvent exchange model9,10 according to eqs 3 and 4

I(S1)2 + 2S2 T I(S2)2 + 2S1

(3)

I(S1)2 + 2S2 T I(S12)2 + 2S1

(4)

where I stands for the indicator species, S1 and S2 for the pure solvents, and S12 for the mixed solvent. I(S1) represents the indicator solvated by the S1 component, I(S2) the indicator solvated by the S2 component, and I(S12) the indicator solvated by the S12 mixed solvent. The solvatochromic properties of the system were evaluated by a global minimization procedure introducing a simulated annealing algorithm.28,29 according to eqs 5 and 6 Figure 13. T-jump experiment (absorbance mode) showing the coralyne aggregation; CD ) 5.2 × 10-5 M, I ) 0.10 M (NaCl), pH ) 7.0, T ) 25 °C, λ ) 420 nm, rise time ) 5 µs, and heating time ) 1.6 µs. The continuous line displays the fitted monoexponential kinetic effect.

Y)

Y1(1 - x2)2 + Y2f2/1x22 + Y12f12/1(1 - x2)x2 (1 - x2)2 + f2/1x22 + f12/1(1 - x2)x2

+ ∆Y

(5) nm underwent a bathochromic shift upon increasing the ethanol content. This observation reveals that coralyne displays negative solvatochromism. The frequency of the absorption maximum ν, depends on the π* parameter, which accounts for the solvent polarity/polarizability. According to Kamlet-Taft, 24-26 the simplest equation that relates ν and π* is

ν ) νo + sπ*

(1)

∆Y )

kf2/1x22[(1 - x2)2 + f12/1(1 - x2)x2 /2] [(1 - x2)2 + f2/1x22 + f12/1(1 - x2)x2]2

(6)

where Y stands for ν, π*, and ∆π*. The constants f2/1 and f12/1 refer to the process described by eqs 3 and 4, respectively, and are defined according to eqs 7 and 8


f2/1 ) (xs2 /xs1)/(x2 /x1)2

(7)

f12/1 ) (xs12 /xs1)/(x2 /x1)2

(8)

where xsi is the mole fraction of the solvent i in the solvation sphere of the indicator, and xi represents the bulk mole fraction. The f12/2 parameter, corresponding to the 12/2 exchange, was evaluated with eq 9

f12/2 )

f12/1 f2/1

(9)

The continuous line in Figure 12 shows the values of Y ≡ ν calculated according to the above model. The properties Y1 ≡ ν1 ) (23.316 ( 0.006) × 103 cm-1 and Y2 ≡ ν2 ) (23.707 ( 0.006) ×103 cm-1 of the pure components have also been evaluated by the fitting procedure. These values agreed quite nicely with the experimental ones (Table 2), thus reinforcing the validity of the procedure. Similar results have been obtained by applying eq 5 to Y ) π* and Y ) ∆π*. The parameter f2/1 ) (0.26 ( 0.1) provides a measure of the preferential solvation of the solvatochromic indicator by water compared to ethanol, whereas the parameter f12/1 ) (0.86 ( 0.4) provides a measure of the preferential solvation of the mixed solvent with respect to ethanol. Finally, f12/2 ) (3.3 ( 2) provides a measure of the preferential solvation of the mixed solvent compared to that of water. The above results indicate that the preferential solvation of coralyne varies in the order ethanol > ethanol-water > water, thus suggesting that this dye is remarkably sensitive to the solute/solvent hydrophobic interactions. Therefore, it turns out that the monomer species becomes stabilized when the EtOH content is increased, and hence, the affinity of the aggregation process should decrease, as the NMR measurements bear out. Kinetic Analysis of Coralyne Self-Aggregation. The effect of the presence of ethanol on the kinetics of the dye aggregation was investigated by chemical relaxation spectrometry. The T-jump experiments performed in 0-25% ethanol-water mixtures displayed a monoexponential behavior (Figure 13). The kinetic results can be rationalized in terms of a simple dimerization process of the dye, D, according to eq 10 kf

2D {\} D2

(10)

kd

The concentration dependence of the relaxation time can be expressed by eq 11

1/τ ) kd + 4kf[D]

(11)

The analysis of the experimental data pairs can be administered by an iterative procedure. To a first approximation, if one considers that [D] ≈ CD, then the monomer concentration at equilibrium, [D], can be evaluated using the KD ) kf/kd ratio, and then, the calculation is iterated until the convergence is attained. The 1/τ versus [D] plots are linear (Figure 14) and yield both the forward (kf) and the backward (kd) rate constants of the reaction in eq 10. Table 3 shows the reaction parameters obtained from the analysis of the kinetic data.

J. Phys. Chem. B, Vol. 113, No. 1, 2009 195 The rate constant for the dimer dissociation process, kd, remains almost constant up to 10% EtOH but displays a remarkable increase above that level. This behavior concurs with the NMR results, which have shown that increasing the EtOH concentration favors the monomer formation. The rate constant for the dimer formation, kf, increased as the EtOH level was raised up to 10% and then remained nearly constant. As a consequence, the equilibrium constant, KD, changes with the ethanol content, and one can note that it displays a maximum value at xC2H5OH ) 0.03. This feature could be correlated with the minimum value of the partial molar excess activation free energy observed at xC2H5OH ) 0.04 by dielectric relaxation measurements performed in ethanol-water mixtures10 and attributed to the structural enhancement of the ethanol-water hydrogen bond network, the so-called hydrophobic hydration. Conclusions The UV/vis measurements have unambiguously demonstrated that coralyne undergoes self-aggregation in ethanol-water mixtures. Mass spectrometry experiments reveal that the most abundant aggregated compound is the dimer species. NMR experiments show that the monomer aggregation comes about by stacking interactions; their importance becomes still more evident by the change of the stacking equilibrium and kinetic parameters, which do not follow the dielectric constant dependence. The solvatochromic study shows that coralyne displays negative solvatochromism and is preferentially solvated by ethanol; this feature suggests that the forces involved in the stacking process are mainly hydrophobic in nature. The T-jump experiments, performed at low dye concentration (where the monomer should prevail), have shown remarkable variations both in the kinetic parameters and in the self-aggregation equilibrium constant with the ethanol content. A sharp change in the reaction parameter values appears at xC2H5OH ) 0.03 (10% ethanol); this feature reveals that the dimer formation is sensitive also to the solvent structure, which is endowed with a minimum partial molar excess activation free energy at xC2H5OH ) 0.04 (roughly 12% ethanol).30 Maxima such as that shown by coralyne (Table 3) have been already observed31 and were attributed to the fact that, once a certain EtOH concentration was reached (in the present case, 10% EtOH), more ethyl residues were available than needed to solvate the dimer. Consequently, they began to solvate the less hydrophobic monomer, inhibiting in this way self-aggregation. The observed difference in the values deduced for the formation constants of coralyne stacks using NMR and T-jump measurements can be put down mainly to the difference in the concentration levels required by these techniques; under the conditions used, in the T-jump experiments, only monomers and dimers are present in equilibrium, whereas in the NMR experiments, monomers, dimers, and even trimers were considered. Acknowledgment. The financial support by Ministerio de Educacioń y Ciencia, Project CTQ2006-14734/BQU, supported by FEDER, and the Junta de Castilla y Leoń, Project BU 001A06, Spain, are gratefully acknowledged. Supporting Information Available: Figure 1S (1HNMR spectra of coraline in DMSO), Figure 2S (chemical shift of the coraline protons in DMSO), Table 1S (charge distribution), Table 2S (chemical shift of the coralyne protons at different temperatures), Table 3S (equilibrium parameters for coralyne self-aggregation), and Table 4S (frequency values and polarity/ polarizability indices of coralyne). This material is available free of charge via the Internet at http://pubs.acs.org.

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Solvent Effects on the Thermodynamics and Kinetics of Coralyne Self

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