Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
LetterExcited-state intramolecular proton transfer (ESIPT) and charge transfer (CT) fluorescence probe for model membranes
Introduction
The use of fluorescent molecules in probing the structural and dynamic changes in lipid bilayers is a well known technique [1]. This method relies on the exploitation of various environmentally sensitive photophysical parameters of fluorescent guest molecules in the host lipid matrix. In recent years, molecules showing excited-state intramolecular proton transfer (ESIPT) fluorescence have been proposed as probes for microenvironments of biological systems e.g. for the study of protein binding-sites [2], [3], [4]. To date, 3-hydroxyflavone (3HF), which is the basic structural moiety of naturally-occurring bioactive flavonols, is the most extensively studied molecule showing ESIPT and dual fluorescence behaviour [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. Earlier reports from our laboratory have demonstrated the potential applications of ESIPT and dual fluorescence parameters of 3HF in probing its microenvironments in different membrane-mimetic organized assemblies [16], [17]. Moreover, 3HF has been proposed as a novel fluorescence probe for studying the thermotropic phase transitions of liposomal membranes [18].
Recently, it has been demonstrated that substitution of strong electron-releasing groups such as N,N-dialkylamino groups on the para position of the 2-phenyl ring of 3HF, can dramatically alter the proton transfer dynamics [19], [20], [21]. The presence of a 4′-N,N-dialkylamino group results in a large excited-state dipole moment, particularly, in the normal form, giving rise to charge transfer (CT) character for the normal fluorescence [20]. Furthermore, it has been shown that switching between proton-transfer (PT) fluorescence and CT fluorescence of such 3HF derivatives depends on solvent polarity and intermolecular (solute–solvent) hydrogen-bonding perturbations of the internal hydrogen-bond of the molecule [20], [21]. In continuation of our studies on the spectroscopic properties of natural and synthetic flavonols exhibiting ESIPT in different membrane-mimetic organized assemblies [16], [17], [18], [22], [23], [24], [25], we report, in this letter, the fluorescence behaviour of 4′-N,N-dimethylamino-substituted 3-hydroxyflavone (DMA3HF, structure shown in Scheme 1) incorporated into small unilamellar liposomes made of the synthetic phospholipids dimyristoyl- and dipalmitoyl-phosphatidylcholine (DMPC and DPPC). The dual emission parameters of DMA3HF have been used to probe its microenvironments in the liposomes. Further, it is demonstrated that the temperature-dependence profiles of the steady-state fluorescence anisotropy of PT fluorescence of DMA3HF dramatically reveal the thermotropic phase transitions of phospholipids from gel to liquid-crystalline states. The estimated phase transition temperatures (Tm) are in good agreement with the previously reported values. We have also initiated fluorescence lifetime studies, and preliminary findings are included in this letter.
Section snippets
Materials and methods
DMA3HF was synthesized and purified as described in the literature [21]. Purity was checked by thin-layer chromatography and by comparing the fluorescence emission and excitation spectra in different solvents with the literature data. DMPC and DPPC were obtained from Sigma. Solvents used were of spectroscopic grade and were preliminarily checked for absence of absorbing and fluorescent impurities. Triple-distilled water was used for all liposome experiments. Small unilamellar liposomes of DMPC
Results and discussions
Fig. 1 shows a typical fluorescence emission spectrum of DMA3HF incorporated into liposomes (shown here for DPPC). The absorption and emission characteristics of DMA3HF in liposomal environments are listed in Table 1. In both DPPC and DMPC (spectrum not shown) liposomes, DMA3HF exhibits dual emission behaviour. The high energy band having emission maximum ≈508 nm can be assigned to the S1→S0 normal emission (CT fluorescence), whereas the large Stokes shifted green fluorescence band can be
Concluding remarks
In both DPPC and DMPC liposomes, DMA3HF exhibits dual fluorescence. The emission parameters, namely, position of emission maximum of CT fluorescence, the ratio between the intensities of PT and CT fluorescence and the fluorescence excitation profiles, reveal that DMA3HF molecules are solubilized in a predominantly aprotic environment, proximal to the polar moieties of the phospholipids. Observation of significant REES effects and high fluorescence anisotropy values indicate that the DMA3HF
Acknowledgements
We are grateful to Professor S. Basak and his coworkers of the Nuclear Chemistry Division of our institute for access to the nanosecond fluorescence lifetime setup and to our colleague Bidisa Sengupta for her kind assistance.
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