Carboxymethylcellulose Gel on the Excimer Formation

J. Phys. Chem. B 2009, 113, 15455–15459

15455

Effect of Water/Carboxymethylcellulose Gel on the Excimer Formation of Polyamine Ligands Functionalized with Naphthalene Laura Rodrı´guez,† Estefanı´a Delgado-Pinar,‡ Alejandra Sornosa-Ten,‡ Javier Alarco´n,§ Enrique Garcı´a-Espan˜a,*,‡ Manoli Cano,† Joa˜o C. Lima,† and Fernando Pina*,† REQUIMTE, Departamento de Quı´mica, Faculdade de Cieˆncias e Tecnologia, UniVersidade NoVa de Lisboa, Portugal, Institut de Cie`ncia Molecular (ICMol), Departament de Quı´mica Inorga`nica, Facultat de Quı´mica, UniVersitat de Vale`ncia, Paterna, Spain, and Departament de Quı´mica Inorga`nica, Facultat de Quı´mica, UniVersidat de Vale`ncia, Burjasot, Spain ReceiVed: August 4, 2009; ReVised Manuscript ReceiVed: September 21, 2009

Formation of intramolecular excimers was studied for the compounds 6,20-bis-naphthalene-2-ylmethyl3,6,9,17,20,23,29,30-octaaza-tricyclo[23.3.1.1] triaconta-1(29),11,13,15(30),25,27-hexane (L1), a bisnaphthalene derivative, and N1-(2-{bis-[2-(3-amino-propylamino)-ethyl]-amino}-ethyl)-propane-1,3-diamine (L2), a tris-naphthalene derivative, incorporated in gels of carboxymethylcellulose sodium salt. Excimers are formed through dynamic processes as well as from ground state dimers. A mathematical treatment including preformed dimers was used to split the static and dynamic contributions in the excimer/monomer emission ratio. In the case of compound L1, the activation energy for excimer formation in water is 11 kJ mol-1 and experimental evidence that the dynamic terms are identical in water and in the gel was achieved. On the other hand, ground state dimers are extremely favorable in the gel with an equilibrium constant of 8.2 at 25 °C. On the contrary, in the case of compound L2, the ground state dimers are observed in water but not in the gel. The results were interpreted as reflecting a balance between specific interactions (hydrogen bond) and confinement effects. Introduction Intramolecular excimer formation is a unimolecular process occurring within a molecule in the first excited singlet state. The formation of the excimer is environment dependent, being affected by both the local solvent viscosity and the local structural limitations. Bischromophoric molecules bearing aromatic hydrocarbons at the ends of a polyamine are versatile molecules able to undergo intramolecular excimer formation in solution, including water.1-5 In the case of bis-naphthalene polyamine systems, the fluorescence emission intensity of the monomer (as well as the lifetime) can be affected by the pH. The fully protonated species exhibits the highest monomer emission intensity and lifetime, and when a proton is removed from one of the polyaminic nitrogens, the fluorescence emission intensity of the monomer (and lifetime) decreases due to quenching by electron transfer from the lone pair of the amine to the excited fluorophore. This quenching can be increased stepwise, upon removal of additional protons, and consequently, the lifetime of the monomer can be tuned by the pH. Another interesting feature of these systems is the pH dependence of the excimer formation rate constant. The rigidity of the chain and ability to interconvert between conformations depends on the electrostatic repulsion of the positive charges created by the successive protonations. Removal of protons confers the polyamine chain larger flexibility, facilitating excimer formation. Thus, the kinetic parameters for excimer formation can be tuned to some extent, allowing one to obtain different regimes for the effect of temperature in * To whom correspondence should be addressed. E-mail: fjp@ dq.fct.unl.pt (F.P.); [email protected] (E.G.-E.). † Universidade Nova de Lisboa. ‡ Institut de Cie`ncia Molecular (ICMol), Universitat de Vale`ncia. § Departament de Quı´mica Inorga`nica, Universidat de Vale`ncia.

excimer formation, either increasing the amount of excimer with temperature (excimer dissociation slower than excimer decay) or decreasing the amount of excimer with temperature (excimer dissociation faster than excimer decay). Zachariasse et al. showed that the excimer/monomer intensity ratio can be used to detect phase transitions and to study the microfluidity in micelles.6 The dependence of the intramolecular excimer formation rate constant on solvent viscosity has been debated among several research groups.7-11 From high-pressure studies, Hara and Yano found that the excimer formation is mainly dependent on the solvent viscosity.11 However, viscosity variation by changing the type of solvent may introduce local variations in the solvent shell interactions with the fluorophore. Therefore, specific and/or steric interactions between a solvent and a fluorophore may also affect the rotational reorientation process and also the stability of the intramolecularly formed excimer. An additional complication arises from the hydrophobic interactions of the chromophores that, depending on the polarity of the environment, can induce preformed dimers which upon excitation give rise to excimer emission without the need of conformational changes, thus insensitive to the viscosity of the medium.12 The effect of temperature in the emission of preformed dimers reflects its dissociation equilibrium constant in the ground state and can mask the dynamic information in the plots of excimer/monomer emission ratio. In the present paper, we study two polyamine based molecules bearing naphthalene chromophores which are able to undergo intramolecular excimer formation and that show a significant change in the amount of preformed excimers, when going from solution to gel phase. A mathematical treatment including preformed excimers was used to split the contribution of static and dynamic contributions in the excimer/monomer emission ratio.

10.1021/jp907490w CCC: $40.75  2009 American Chemical Society Published on Web 10/20/2009

15456

J. Phys. Chem. B, Vol. 113, No. 47, 2009

Rodrı´guez et al.

Figure 1. (A) Fluorescence emission of the compound L1 (λexc ) 260 nm), 1 × 10-5 M in water/ethanol 70:30, [NaCl] ) 0.15 M; traced line pH 1.0, full line pH 5.0. Inset: Mole fraction distribution of the different protonation states obtained by potentiometry superimposed to the emission maximum of the monomer, 335 nm (b), and excimer, 430 nm (O). (B) Stevens-Ban plot for L1 in water/ethanol 70:30 at pH 5. (C) Stevens-Ban plot for L1 in carboxymethylcellulose sodium salt gel at pH 5.

Experimental Section The synthesis of the compounds 6,20-bis-naphthalene-2ylmethyl-3,6,9,17,20,23,29,30-octaaza-tricyclo[23.3.1.1] triaconta-1(29),11,13,15(30),25,27-hexane (L1) and N1-(2-{bis-[2(3-amino-propylamino)-ethyl]-amino}-ethyl)-propane-1,3diamine (L2) has been carried out as reported elsewhere.13,14 The samples that were handled as L1 · 6HCl and L2 · 6HCl salts gave satisfactory characterization and elemental microanalysis. The gel samples were prepared by dissolving 0.4 g of carboxymethylcellulose sodium salt in 8 mL of the ca. 1 × 10-5 M solutions of L1 or L2, and the final pH (measured on a MeterLab 240 pH meter) slightly readjusted before gelification with the addition of small amounts of NaOH or HCl. Absorption spectra were recorded at 25 °C on a Varian Cary 100 Bio UVspectrophotometer and fluorescence emission spectra on a Horiba-Jobin-Yvon SPEX Fluorolog 3.22 spectrofluorimeter with an external Thermo NESLAB RTE7 water bath. The solution measurements were made in 0.15 M NaCl (in pure water or water/ethanol 70:30). Temperature ramps of both solutions and gels were recorded at pH 5 for L1 and pH 4 for L2, after confirmation that the fluorimetric titration in solution and gel showed the same trend (same pKa values and same plateau regions) within the experimental error. At the chosen pH values, the emission maxima could be attributed to only one species in both cases (H4L in the case of L1 and H6L in the case of L2). The fluorescence decays were obtained using home-built equipment that has been described elsewhere15 and were analyzed using the method of modulating functions implemented by Striker.16 All of the measurements were made in the presence of oxygen. The excimer and monomer emissions were taken after Gaussian fitting of the excimer emission band and subtraction of the fitted Gaussian from the experimental emission spectrum (containing both monomer and excimer emission overlapping bands); in this way, it was possible to correct for the excimer emission contribution at the monomer emission wavelength (335 nm); see Figure S1 in the Supporting Information. Results Photophysical Studies of Compound L1. The fluorescence emission spectra of compound L1 in water/ethanol 70:30 at pH 1 and pH 5 are reported in Figure 1A. Two distinct bands, corresponding to naphthalene emission (ca. 330 nm) and,

similarly to other bis-naphthalene compounds, naphthalene excimer emission, can be observed at ca. 430 nm. Changes in the pH give rise to changes in intensity of both emission bands, and the inset of Figure 1A shows the fluorimetric titration followed at 335 and 430 nm, superimposed to the molar fraction distribution obtained by potentiometric titration. The species H6L corresponds to the protonation of the six aliphatic amines; no evidence was found from 1H NMR to consider protonation of the pyridine moieties. Successive deprotonation gives rise to a quenching effect on the naphthalene emission due to the electron transfer from the lone pairs of the amines to the excited fluorophore. The species H4L is the dominant form between pH 4 and pH 6 and corresponds to the complete protonation of the secondary amines. It is worth noting that the excimer emission intensity decreases upon protonation of the tertiary amines, which are the closest ones to the fluorophores. The electrostatic repulsion in the fully protonated form either favors a conformer where the excimer formation is not possible or significantly increases the activation energy for the encounter of the two chromophores. Only after removing the protons from these nitrogens the two fluorophores can approach, independently if the dimer is formed during the excited state or is preformed in the ground state.

The ratio of excimer to monomer fluorescence intensities (IE/ IM) is related to the rate constants for monomer and excimer interconversion and decay in the excited state.17 If a large enough temperature range is studied, a plot of ln(IE/IM) vs 1/T is expected to yield a bell shaped curve with two well-defined limits: the

Excimer Formation of Polyamine Ligands

J. Phys. Chem. B, Vol. 113, No. 47, 2009 15457

Figure 2. Fluorescence decay times and amplitudes at 335 nm for H4L1 in water/ethanol 70:30 (closed symbols) and in carboxymethylcellulose gel (open symbols) at pH 5. The lines are fittings with the Birks kinetic scheme to the data in water/ethanol.

high-temperature limit, HTL, identified by a positive slope, and a low-temperature limit, LTL, identified by a negative slope (see ref 17 and the Supporting Information). Inspection of Figure 1B shows that in the water/ethanol mixtures (70:30) at pH 5 the excimer of L1 shows a linear trend with negative slope at low temperatures (characteristic of LTL) and a plateau at higher temperatures characteristic of the transition between the LTL and HTL regimes. The HTL cannot be seen in this temperature range (only at higher temperatures, inaccessible in the case of water/ethanol mixtures due to ethanol evaporation). From the slope of the linear part of Figure 1B, it is possible to retrieve the activation energy (ref. Stevens-Ban) for the excimer formation in water/ethanol 70:30 at pH 5, Ea ) 11 kJ mol-1. The LTL regime corresponds to a limiting situation where the temperature is low enough to decrease the excimer dissociation rate constant in an extent that it becomes negligible for the excimer deactivation pathway, i.e., much smaller than the intrinsic decay of the excimer (kd , kE). Going to a higher viscosity medium is expected to take the system further into the LTL limit. Inspection of Figure 1C, where the same plot is represented for the case of L1 in carboxymethylcellulose gel at pH 5, shows that the plateau characteristic of the LTL to HTL regime transition is almost missing. At first sight, this result is consistent with a more viscous medium; however, the activation energy for excimer formation, retrieved from the negative slope of Figure 1C, is in the same range (7.5 kJ mol-1) as the value obtained in water/ethanol 70:30 at the same pH. The first conclusion is that the gel does not affect the dynamic formation of excimer; i.e., the viscosity of the microenvironment felt by the molecules able to undergo dynamic excimer formation is close in viscosity to the solution phase. This is clearly seen from the fluorescence decay times obtained in both media (Figure 2) as a function of temperature. Either in water/ethanol 70:30 or in carboxymethylcellulose gel at pH 5, the decays are well fitted with sums of two exponentials, and are identical within the experimental incertitude. Differences are only observed in the amplitudes. According to the Birks kinetic scheme,18 the two observed decay times can be related to the rate constants for excimer formation, ka, dissociation, kd, and intrinsic rates for monomer, kM, and excimer, kE, decays by

X + Y ( √(X - Y)2 + 4kakd 1 ) τ1,2 2

(1)

where X ) kM + kd and Y ) kE + kd. The pre-exponential factors of the double exponential decays can also be related to the rate constants by

1 τ1 a1 ) ; 1 1 τ2 τ1 X-

1 -X τ2 a2 ) 1 1 τ2 τ1

(2)

The above set of equations was used to fit the experimental data in water/ethanol, and the lines in Figure 2 represent the best fits of the decay times and amplitudes. From the fitting of the time-resolved fluorescence data, the activation energy retrieved for the excimer formation is 12 kJ mol-1, while the obtained activation energy for excimer dissociation was 21 kJ mol-1, yielding an excimer binding energy of 9 kJ mol-1. Since the temperature dependence of the decay times in the gel is very similar to that observed in solution, it is reasonable to assume that the same activation energies will be found for the excimers that are formed dynamically in the gel, while the differences observed in amplitudes will reflect the presence of static dimers, i.e., preformed ground state dimers. An interesting part is that at 20 °C the excimer dissociation rate constant, kd, is of the order of 1.4 ns-1, while the intrinsic excimer decay time is 0.26 ns (kE ) 3.85 ns-1), and consequently, kd < ke and the system is entering the LTL limit. The monomer lifetime was found to span between 5 and 3 ns, decreasing with temperature. Thus, the difference in the plots of Figure 1B and C does not lie in the dynamic excimer formation but in the presence of ground state dimers. In water/ethanol 70:30 at pH 5, the excitation spectra collected at the monomer (320 nm) and excimer (450 nm) emission bands reproduce the monomer absorption, and show no evidence for the presence of preformed ground state dimers. On the contrary, in the gel, the excitation spectra collected under the same conditions are not identical, and a significant amount of ground state dimers must be preformed, Figure 3. If the presence of ground state dimers is responsible for the difference in the Stevens-Ban plots over all temperatures studied, a more complete formalism must be used to account for this contribution (see full formalism in the Supporting Information). When accounting for the presence of preformed dimers, it is also possible to include LTL and HTL limits, and also in this case, the presence of a negative slope identifies the

15458

J. Phys. Chem. B, Vol. 113, No. 47, 2009

Rodrı´guez et al.

Figure 3. Excitation spectra collecting at 320 nm (monomer) and 450 nm (excimer) wavelength.

LTL regime. However, the equation describing the LTL limit has a complex form (eq 3 and eq S19 in the Supporting Information):

( ) ( ) {

ln

}

Ea IE kfEτE ελ(D)Keq + ελ(M-M) ) ln + ln IM*-M kfM ελ(M-M) RT (3)

The term ln{[ελ(D)Keq + ελ(M-M)]/ελ(M-M)} in eq 3 accounts for the preformed ground state dimers. The remaining terms of the equation are the ones found in the simpler expression that account only for dynamic excimers. If the terms corresponding to dynamic excimer formation are identical in ethanol/water 70:30 and in the carboxymethylcellulose gel, the difference between the plots obtained in both media is contained in the term ln{[ελ(D)Keq + ελ(M-M)]/ελ(M-M)}. Equation 3 is hard to use in the fitting of the experimental data of Figure 1C, without fixing any parameters. However, the term corresponding to the ground state dimers is more amenable, since it simplifies to ln(aKeq + 1), where a is the ratio of dimer to monomer extinction coefficient, a term independent of the temperature, and the ground state dimerization constant, Keq, will have an exponential dependence with temperature of the type Keq ) e∆S/Re-∆H/RT. This equation describes adequately the experimental difference between the Stevens-Ban plots in the gel and solution in the LTL regime, Figure 4. As can be observed in Figure 4, the decrease in the temperature favors the dimerization in the gel. It will also decrease the observed slope in Figure 1C, and consequently, the activation energy observed in steady state fluorescence would be slightly lower (7.5 kJ mol-1) than the one retrieved by timeresolved fluorescence (12 kJ mol-1). The fitting procedure reported in Figure 4 also allows one to retrieve approximate values for the intramolecular dimerization enthalpy and entropy in the ground state ∆H ) -35 ( 5 kJ mol-1 and ∆S ) -100 ( 25 J K-1 mol-1. From the enthalpy and entropy values, we can estimate an equilibrium constant of 8.2 for the ground state dimer formation at 25 °C. The conclusion is that in carboxymethylcellulose gel, at 25 °C, 90% of the observed “excimer” emission is in fact the emission of preformed dimers. The bulk viscosity of the gel is not felt by the molecule, that can undergo

Figure 4. Experimental difference between the ln(IE/IM) curves obtained in gel and water (open circles) and the curve describing the temperature dependence of the term containing the ground state dimerization constant in eq 3 (solid line).

excimer formation in a medium of viscosity close to water/ ethanol solution, while conformations where the dimer is preformed in the ground state are strongly favored. It is expected that the presence of microdomains in gels and the partition of the molecule for these small domains will favor the more compact conformation, thus favoring the formation of intramolecular interactions and preformed dimers. If such is the case, a different molecule in which the transition between stretched and compact conformation would involve a bigger difference in molecular volume would be expected to form ground state dimers even more efficiently. Photophysical Studies of Compound L2. Compound L2 has a tripodal structure also able to form intramolecular excimers, and it is expected that the change from the full extended conformation to a compact conformation will be favored in the gel.

The intensity fluorescence dependence with the pH in water was previously reported.14,14 The species H6L2 (with all secondary amines protonated) is the dominant form between pH 3 and

Excimer Formation of Polyamine Ligands

J. Phys. Chem. B, Vol. 113, No. 47, 2009 15459 the concentration of preformed dimers. The formation of intramolecular ground state dimers in L1, on the other hand, will only depend on the confinement felt by the molecule. Conclusions

Figure 5. Stevens-Ban plots of H6L2 in water (closed circles) and carboxymethylcellulose gel (open circles), at pH 4, λexc ) 280 nm, [L2] ) 1 × 10-5 M.

pH 6 in water, and the same dependence of the fluorescence intensity with the pH was found in carboxymethylcellulose gel (see Figure S2 in the Supporting Information). In contrast with H4L1, in the case of H6L2, the Stevens-Ban plots in water or in carboxymethylcellulose are in the HTL regime (see Figure 5). This highly protonated species presents negligible quenching of the fluorophores by the polyamine chain, and the excimer decays slowly (≈100 ns) enough for a considerable fraction of excimer to dissociate and yield excited monomer (at 30 °C, the dissociation constant kd ) 0.028 ns-1 is more than double the intrinsic excimer decay rate constant), and thus, the compound shows HTL behavior.14 In this case, the difference between a situation with and without preformed dimers will be reflected in the term ln{[ελ(E)Keq/ ηE + ελ(M-M)]/[ελ(M-M) + ελ(E)Keq]} (see eq S14 in the Supporting Information). The presence of an additive additional term, when compared with the situation where only dynamic excimers exist, always implies that a higher value of ln(IE/IM) will be obtained if ground state dimers are present, with respect to the situation where they are absent. Inspection of Figure 5 shows that in carboxymethylcellulose gel the excimer intensity is much lower than that in water and the most obvious explanation is that in the case of H6L2 preformed dimers exist in water, as pointed out in a previous paper.13 When the behavior of the two compounds is compared, the following question arises: why are ground state dimers favored in the gel in the case of H4L1 and not in the case of H6L2? The answer lies in the different structural motifs of compounds L1 and L2 and their implications on the process of ground state dimer formation. The arms bearing naphthalenes in the case of L2 possess amines, which can be protonated and undergo intramolecular hydrogen bonding, thus favoring conformation where the naphthalenes are in close contact (ground state dimers). L1, on the other hand, has all of the amines in the cyclic structure, while the arms bearing naphthalenes have no hydrogen bonding ability. Thus, in the case of the L1 dimer, formation is not dependent on H-bond interactions, while, in the case of L2, it is strongly dependent. Hydrogen bonding of the protonated amines of L2 with carboxymethylcellulose will compete with intramolecular hydrogen bonding, thus decreasing

Formation of intramolecular excimers in carboxymethylcellulose gels can change dramatically when compared with solution. The gel can favor ground state dimers, in the case where confinement effects overcome specific interactions, or, on the contrary, disrupt dimers that are observed in solution, when hydrogen bond interactions play a major role in dimer formation. The dynamic and static processes of excimer formation can be used as a tool to access microenvironmental properties of the gel in the vicinity of the fluorophore, if these contributions can be adequately separated. Polyamine chains connecting fluorophores which form intramolecular excimers have, differently from intramolecular excimers based on different chains, the advantage of allowing modulation between LTL and HTL regimes, solely based on structural design and/or pH change. This property results from the fact that unprotonated amines close to the naphthalene chromophores can quench the excimer and change the relation between the excimer dissociation rate constant and the excimer decay to the ground state. Supporting Information Available: Information on theory and the high- and low-temperature limits and figures showing spectral decomposition and fluorimetric titration. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Albelda, M. T.; Bernardo, M. A.; Dı´az, P.; Garcı´a-Espan˜a, E.; de Melo, J. S.; Pina, F.; Soriano, C.; Luis, S. V. Chem. Commun. 2001, 1520. (2) Melo, J. S.; Albelda, M. T.; Diaz, P.; Garcia-Espan˜a, E.; Lodeiro, C.; Alves, S.; Lima, J. C.; Pina, F.; Soriano, C. J. Chem. Soc., Perkin Trans. 2 2002, 991. (3) Pina, J.; de Melo, J. S.; Pina, F.; Lodeiro, C.; Lima, J. C.; Parola, A. J.; Soriano, C.; Clares, M. P.; Albelda, M. T.; Aucejo, R.; Garcia-Espana, E. Inorg. Chem. 2005, 44, 7449. (4) Clares, M. P.; Aguilar, J.; Aucejo, R.; Lodeiro, C.; Albelda, M. T.; Pina, F.; Lima, J. C.; Parola, A. J.; Pina, J.; de Melo, J. S.; Soriano, C.; Garcia-Espana, E. Inorg. Chem. 2004, 43, 6114. (5) Bernardo, M. A.; Alves, S.; Pina, F.; de Melo, J. S.; Albelda, M. T.; Garcia-Espan˜a, E.; Llinares, J. M.; Soriano, C.; Luis, S. V. Supramol. Chem. 2001, 13, 435. (6) Zachariasse, K. A.; Ktihnle, W.; Weller, A. Chem. Phys. Lett. 1980, 73, 6. (7) Snare, M. J.; Thistlethwaite, P. J.; Ghiggino, K. P. J. Am. Chem. Soc. 1983, 105, 3328. (8) Zachariasse, K. A.; Duveneck, G.; Busse, R. J. Am. Chem. Soc. 1984, 106, 1045. (9) Turley, W. D.; Offen, H. W. J. Phys. Chem. 1985, 89, 2933. (10) Hara, K.; Yano, H. J. Phys. Chem. 1986, 90, 4265. (11) Hara, K.; Yano, H. J. Am. Chem. Soc. 1988, 110, 1911. (12) de Melo, J. S.; Pina, J.; Pina, F.; Lodeiro, C.; Parola, A. J.; Lima, J. C.; Albelda, M. T.; Clares, M. P.; Garcia-Espana, E.; Soriano, C. J. Phys. Chem. A 2003, 107, 11307. (13) Alarco´n, J.; Albelda, M. T.; Belda, R.; Clares, M. P.; DelgadoPinar, E.; Frı´as, J. C.; Garcı´a-Espan˜a, E.; Gonza´lez, J.; Soriano, C. Dalton Trans. 2008, 6530. (14) Albelda, M. T.; Garcı´a-Espan˜a, E.; Gil, L.; Lima, J. C.; Lodeiro, C.; de Melo, S. J.; Melo, M. J.; Parola, A. J.; Pina, F.; Soriano, C. J. Phys. Chem. B 2003, 107, 6573. (15) de Melo, S. J.; Fernandes, P. F. J. Mol. Struct. 2001, 565, 69. (16) Striker, G.; Subramaniam, V.; Seidel, C. A. M.; Volkmer, A. J. Phys. Chem. B 1999, 103, 8612. (17) Stevens, B.; Ban, R. J. Trans. Faraday Soc. 1964, 60, 1515. (18) Birks, J. B. Photophysics of Aromatic Molecules; Wiley: London, 1970.

JP907490W