Ground and Singlet Excited State Hydrogen-Bonding Interactions between 1-Azacarbazole and Amides

Manuel Galán, Carmen Carmona, Pilar Guardado, María A. Muñoz and Manuel Balón.*

Departamento de Química Física, Facultad de Farmacia,

Universidad de Sevilla, 41012 Sevilla, Spain.

Abstract

In cyclohexane as the solvent, 1-azacarbazole (or a -carboline), AC, forms fluorescent ground state 1:1 hydrogen-bonded complexes with N,N-dimethylformamide, DMF, N,N-dimethylacetamide, DMA, and hexamethylphosphoramide, HMPA. The absorption and fluorescence spectra of the complexes are red shifted with respect to those of the no-bonded AC, and their association constants increase as the hydrogen-bonding acceptor properties of the amides increase. Fluorescence of the AC-DMF and AC-DMA solutions show mono- or biexponential decays depending on the monitored emission wavelength. To aid the interpretation of these results we have also studied the effect that triethylamine and methylethylcetone addition produces on the absorption and fluorescence spectra of AC. Triethylamine does not significantly affect the absorption spectrum of AC, but it dynamically quenchs its fluorescence. Methylethylcetone, conversely, behaves as amides do. On the basis of the above results, we assume that, in the ground state, the hydrogen bonding interaction takes place between the pyrrolic NH group of AC and the carbonyl group of the amide. Hydrogen bonded complexes and no-bonded AC behave in the singlet excited state as independent fluorophores. Singlet excited state of free AC is dynamically quenched by DMF and DMA. The quenching mechanism involves the hydrogen-bonding interaction of the pyrrolic NH group of AC and the lone electron pair of the amide. For HMPA, probably due to geometrical restrictions, this quenching process is absent.

Keywords: 1-azacarbazole, amides, hydrogen-bonding, ground state, excited state.

* Author for correspondence (E-mail: balon@fafar.us.es)

1. Introduction

1-Azacarbazole, or a -carboline, is a representative member of the carboline family, a class of drug-binding alkaloids possessing interesting photophysical and biological properties [1]. Because of its structural relation with 7-azaindole, AI, a molecule whose dimer has long been recognized as a model for the DNA base pairs, the photophysics of AC has received much attention. Similarly to AI, AC forms doubly hydrogen-bonded dimers that, upon photoexcitation, undergo a concerted intermolecular excited state double proton transfer reaction, ESDPT. Excited-state tautomerization reactions of AI and AC dimers have been intensively studied as model systems for understanding the photoinduced mutations of the DNA base pairs [2].

Owing to its bifunctional proton donor and acceptor properties, AC may form a variety of complexes with different hydrogen-bonding partners. Particularly, cyclic doubly hydrogen-bonded complexes of the sort illustrated in Fig. 1, have arisen great interest, since as the AC dimers, they are also able to undergo ESDPT reactions. While much work has been done on the photophysics of the cyclic complexes [3-12], single non-cyclic 1:1 hydrogen-bonded complexes involving only one of the hydrogen-bonding centres of the AC ring have received very scarce attention. However, the study of the nature of the last complexes and the dynamics of their hydrogen-bonded mediated proton transfer reactions is key for the full understanding of the photophysics and photochemistry of AC. Thus, it would be of interest to the photophysics of these hydrogen-bonded complexes of AC.

In this context, we have carried out a spectroscopic study (uv-vis, steady-state and time-resolved fluorescence) of the ground and singlet excited state hydrogen-bonding interactions of AC with different amides; namely, N,N-dimethylformamide, DMF, N,N-dimethylacetamide, DMA, and hexamethylphosphoramide, HMPA. These amides possess only proton accepting groups, hence they can only form hydrogen-bonds with the pyrrolic NH site of the AC. These studies have been conducted in cyclohexane, a solvent that due to its low polarity and its negligible hydrogen-bonding properties can be considered inert.

2. Experimental

1-Azacarbazole was synthesized and purified as described in the literature [13]. The hydrogen-bonding acceptors DMF, DMA, and HMPA, triethylamine, methylethylcetone and the cyclohexane used as the solvent were commercial products (Sigma, Aldrich) of the best available quality, they were used without further purification and were stored on 4 Å molecular sieves.

The uv-visible absorption spectra were recorded on a Perkin-Elmer Lambda-5 spectrophotometer. Stationary fluorescence measurements were carried out in a Hitachi F-2500 spectrofluorimeter. Fluorescence lifetimes were measured with an Edinburgh Analytical Instruments CD-900 spectrofluorimeter employing the time correlated single photon counting technique. The source was a nanosecond flashlamp filled with H₂ (0.4 bar) opperating at 6.8 kV, with a repetition rate of 40.0 kHz. Fluorescence decays were acquired to 10⁴ counts in the peak. The decays were repeated at least twice and were fitted by reference deconvolution to a sum of exponentials:

with amplitudes, A_i, and lifetimes, t _i. Decay curves were both individually and globally analyzed by using single, double and triple exponentials. Goodness of the individual fits was judged by the magnitude of c ²_r and the shape of the autocorrelation function of the weighted residuals. The analysis of the lifetime data at different acceptor concentrations were performed with a global analysis program based on the Marquardt algorithm. These results were judged by the statistical fitting parameter c ²_g. Fluorescence measurements were carried out with nondegassed solutions under temperature controlled conditions (25±0.1 ºC).

3. Results and Discussion

As stated in the introductory section, AC is prone to dimerize in low polar solvents. To check this possibility, we previously studied the concentration dependent absorption and fluorescence spectra of AC in cyclohexane. In the AC concentration range used in this work, up to 10^-4 M, we did not observe changes in the absorption spectrum attributable to dimer formation and the absorbances fitted acceptably the Lambert-Beer law at different absorption wavelengths. Neither the fluorescence spectrum of AC showed indication of dimer formation. The dimer would be clearly detected by the emission of its photoinduced tautomer at 500 nm, which was absent in the fluorescence spectra of AC in cyclohexane under our experimental conditions.

Figure 2 shows the changes produced in the uv-visible and fluorescence spectra of AC in cyclohexane upon adding HMPA. Similar changes are observed for DMA and DMF. As this Figure shows, the uv-vis absorption spectrum of AC is shifted to the red as the amide concentration is increased. The presence of an isosbestic point in these spectra indicated the formation of AC-amide stoichiometric complexes. Owing to the nature of the interacting substrates we assume that these complexes are stabilized by hydrogen bonding interactions involving the pyrrolic NH group of the AC ring. Unfortunately, the small magnitude of the absorbance changes precluded the determination of the association constants of the complexes from the absorption spectra.As it is typically shown in Fig. 3 for the AC-HMPA system, the fluorescence spectra of the hydrogen bonded complexes of AC with DMA, DMF and HMPA are red shifted by 10-15 nm with respect to that of free AC. The fluorescence data were analyzed using the Benesi-Hildebrand equation for 1:1 stoichiometric binding,

where I₀ is the initial fluorescence intensity of free AC at the titration wavelength, I₁ the fluorescence intensity of the AC-amide complex and I the observed fluorescence intensity of the corresponding AC-amide mixtures. As the inset in Figure 3 shows, the linear dependence of 1/(I₀-I) on the reciprocal of amide concentration, confirms the assumed 1:1 stoichiometry of the complexes. The slope and the intercept of these plots allowed us to calculate the values of the ground state formation constants reported in Table 1. The sequence of these association constants DMF < DMA < HMPA closely follows that of b hydrogen-bonding acceptor parameters of the amides [14]. On the other hand, the red shifts observed in the absorption and fluorescence spectra upon the formation of the complexes indicate that the hydrogen-bonding interaction is reinforced in the excited state. This is in agreement with the charge density decrease experienced by the pyrrole nitrogen atom upon excitation of AC to its S₁ state [12].

Table 1. Ground state association constants, K_G, and dynamic quenching constants, k_q,

for the hydrogen-bonding interactions of AC with amides in cyclohexane at 298 K.

^(a) Hydrogen-bonding acceptor descriptor values according Y. Marcus [14].

In order to gain some insight into the dynamics of the hydrogen-bonding interaction in the singlet excited state, we carried out time-resolved fluorescence measurements of the AC-amide solutions in cyclohexane. We will discuss firstly the results obtained for the AC-DMF and AC-DMA systems, since they are somewhat different from those of the AC-HMPA system. The fluorescence decays of the AC-DMF and AC-DMA mixtures depended on the emission wavelength. As it is illustrated in Table 2 for the AC-DMA mixture, at 340 nm the fluorescence decays could be fairly well described by a linear combination of two exponentials:

where A₁ and A₂ stand for the contribution at zero time of the two components with lifetimes t ₁ and t ₂.

Table 2. Analysis of the biexponential decays of the AC-DMA system in cyclohexane at 298 K

( l _exc=320 nm, l _em =340 nm). The figures into parenthesis are the pre-exponential factors, A_i.

The shorter lifetimes, t ₁, decreased with the amide concentration, while the longer lifetime component, t ₂, was independent of the amide concentration and remained practically constant around 7.4 ns.As Fig. 4 shows, the plots of the reciprocal of the lifetimes, k₁=1/t ₁, against amide concentration according to equation

are linears with intercept values, k₀, very close to that of the reciprocal of the lifetime of no-bonded AC in cyclohexane, t ₀=6.4 ns. The slopes of these plots, k_q, are reported in Table 1. At the red end of the emission spectra, this short lifetime component dissapears and the fluorescence decays could reasonably be fitted to monoexponential functions with lifetimes whose values, within the experimental error, are equal to those of t ₂.

The above results show that, in the excited state, the equilibrium between the hydrogen bonded complexes and the free AC is not established during the life span of these species. If this were the case, these species would be coupled in the excited state and, therefore, the fluorescence decays would be always biexponential, irrespective of the monitored emission wavelength. On the other hand, in the case of irreversible formation of the hydrogen-bonded complexes in the excited state, we would expect biexponential decays at the longest wavelengths and the appearance of a rising time, i.e. a negative preexponential factor. Since such behavior was not observed, it is quite unlikely that free AC is the excited state precursor of the hydrogen-bonded complexes. Therefore we conclude that, as depicted in Scheme 1, the hydrogen-bonded complexes and the no-bonded AC behave as independent fluorophores. To account for the dynamic quenching of the no-bonded AC species by DMF and DMA, we will assume that, the interaction in the excited state is different than in the ground state.

In this sense, it is interesting to realize that DMF and DMA molecules have two potential acceptor centres for hydrogen-bonding interactions, the oxygen atom of the carbonyl group and the lone electron pair on the nitrogen atom. Therefore, it is conceivable that AC could interact independently with each one of these centres in the amide molecule.

Thus, to obtain information on this point, we have studied the interactions of AC with triethylamine and methylethylcetone as model compounds. Addition of triethylamine had not significant effects on the absorption spectrum of AC, but, as Figure 5 shows, it quenchs the fluorescence intensity and decreases the fluorescence lifetime of AC.These results show the dynamical nature of the quenching process; i.e. the quenching is due to the excited state interaction of AC with the amine. The mean value of the quenching constant obtained from Stern-Volmer plots of the steady-state and time-resolved fluorescence data, 5x10¹⁰ M^-1 s^-1, is similar to those of the quenching constants, k_q, reported in Table 1 for the AC-DMF and AC-DMA systems. These quenching constants are, on the other hand, of the order of magnitude expected for a difussion controlled process in cyclohexane. Conversely, the changes observed in the absorption and fluorescence spectra of AC in cyclohexane upon the addition of methylethylcetone entirely resemble those produced by the amides.

On the basis of the above results, we conclude that, in the ground state, the pyrrolic NH group of AC interacts preferently with the carbonyl groups of the amides, but, in the excited state, the hydrogen-bonding interactions take place through the lone electron pair of the nitrogen atoms of the amides. This excited state hydrogen bonding interaction efficiently quenchs the AC fluorescence. Possibly, this interaction involves the transfer of the pyrrolic proton to the amide and the formation of AC anions which are very weakly fluorescent.

Turning now to the AC-HMPA system, time-resolved fluorescence measurements showed that, independently of the emission wavelength, the fluorescence of the cyclohexane solutions of AC in the presence of HMPA decayed monoexponentially. The lifetimes of the decays were very close to that of no bonded AC, 6.4 ns, and slightly increased as the acceptor concentration increased. Double exponential analyses were also attempted, but neither c _r² nor c _g² improved. Furthermore, the second component of these analyses revealed at random and, occasionally, physically inexplicable negative contributions to the overall decay. We think that this conflicting result is an artifact of the fitting procedure which cannot satisfactorily resolve lifetimes whose values differ less than 1 ns [15]. Thus, the fluorescence decays of AC-HMPA solutions in cyclohexane could also be reasonably interpreted according to Scheme 1, assuming a system formed by a mixture of two independently absorbing and emitting species, the complex and the no-bonded AC, with very close fluorescence lifetime. Interestingly, HMPA, possibly due to geometrical constrains, does not appreciably quench the fluorescence of free AC.

Finally, it is interesting to note that the AC-amide hydrogen-bonded complexes have slightly larger fluorescence quantum yields, f , and longer lifetimes, t , than free AC. Therefore, because f = k_r/(k_r+k_nr) and t =1/(k_r+k_nr), complexation apparently hinders the non-radiative process, k_nr. In the singlet manifold of the AC monomer, there exist two low-lying singlet excited states that are analogous to the ¹L_a and ¹L_b states of indole and other polyacenes [6]. In indoles, the ¹L_b state remains lower in the gas phase and non polar solvents, while the more polar ¹L_a state becomes the lowest energy excited singlet state in polar solvents [16-18]. The fluorescence from ¹L_a is weaker than that from ¹L_b. Coupling of both states leads to an efficient nonradiative process involving the dissociation of the NH pyrrolic group. Therefore environmental changes that induce decoupling of these states would enhance both the quantum yield and fluorescence lifetime.

Since the close structural relation between AC and indole, it seems rather likely that the ¹L_a and ¹L_b states of AC behave in a similar way. Thus, we can tentatively suggest that the hydrogen-bonding interaction between AC and the amides decouples the ¹L_a and ¹L_b states of AC enhancing the quantum yield and fluorescence lifetime.

In contrast to that, hydrogen-bonding interactions of AC with alcohols and water are known to decrease the quantum yield and the fluorescence lifetime of AC [8]. Waluk and coworkers have attributed this behaviour to the enhancement of the internal conversion process caused by these hydrogen-bonding interactions [8]. Alcohols and water are bifunctional donor-acceptor hydrogen-bonding molecules that can interact with both the pyrrolic and the pyridinic nitrogen atoms of AC. Therefore, we suspect that the interaction through the pyridinic nitrogen atom is responsible of such a behaviour. In this case, a different mechanism which deactivates the S₁ state of AC could take place. In fact, Waluk and coworkers have also considered the possibility of a hydrogen-bonding mediated charge transfer process. We think that a systematic study of the hydrogen bonding interactions of the pyridinic nitrogen atom of AC with selected hydrogen-bonded donors could help to clarify this question. This study is now in progress in our laboratory and it will be the subject of a forthcoming publication.

4. Acknowledgements

We gratefully thank financial support from Dirección General de Investigación Científica y Técnica (PB98-1160) and Junta de Andalucía.

5. References