Letter
Excited-state intramolecular proton transfer (ESIPT) and charge transfer (CT) fluorescence probe for model membranes

https://doi.org/10.1016/S1386-1425(99)00013-XGet rights and content

Abstract

Motivated by the burgeoning interest in designing and characterizing fluorescence probes with superior spectroscopic properties for exploring the structural and dynamic aspects of biological systems, we have investigated the photophysical behaviour of a synthetic flavonol, namely 4′-N,N-dimethylamino-3-hydroxyflavone (DMA3HF) in model membranes consisting of small unilamellar liposomes of the phospholipids DPPC and DMPC. The DMA3HF fluorophore is remarkable in the fact that it combines both excited-state proton transfer (PT) and charge transfer (CT) fluorescence emissions in a single molecular system. The nature and relevant parameters of the dual emission profiles, fluorescence excitation spectra, along with steady-state fluorescence anisotropy (r) and red edge excitation shift (REES) data have been used to probe the local environments of DMA3HF molecules in liposomes. Furthermore, r versus temperature (T) profiles are shown to provide estimates of thermotropic gel to liquid-crystalline state phase transition temperatures of the phospholipids, in excellent agreement with the existing literature data.

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.

References (33)

  • P.K Sengupta et al.

    Chem. Phys. Lett.

    (1979)
  • N.P Ernsting et al.

    J. Chem. Phys.

    (1989)
  • M Sarkar et al.

    Chem. Phys. Lett.

    (1991)
  • M Sarkar et al.

    Spectrochim. Acta

    (1996)
  • J Guharay et al.

    Spectrochim. Acta

    (1997)
  • J Guharay et al.

    Spectrochim.

    Acta

    (1997)
  • J Guharay et al.

    Chem. Phys. Lett.

    (1994)
  • O.P Bondar et al.

    Biochim. Biophys. Acta

    (1998)
  • J. Yguerabide, M.C. Foster, in: E. Grell (Ed.), Molecular Biology, Biochemistry and Biophysics, vol. 31, Membrane...
  • A Sytnik et al.

    Proc. Natl. Acad. Sci. USA

    (1994)
  • A Sytnik et al.

    Proc. Natl. Acad. Sci. USA

    (1994)
  • A Sytnik et al.

    Proc. Natl. Acad. Sci. USA

    (1996)
  • D McMorrow et al.

    J. Phys. Chem.

    (1984)
  • M Kasha

    J. Chem. Soc. Faraday Trans. II

    (1986)
  • M Itoh et al.

    J. Am. Chem. Soc.

    (1982)
  • A.J.G Strandjord et al.

    J.Phys. Chem.

    (1983)
  • Cited by (86)

    • Detection of hydrogen sulphide based on a novel G-quadruplex selective fluorescent probe

      2018, Sensors and Actuators, B: Chemical
      Citation Excerpt :

      Therefore, G4 has already been used to develop various sensors for protein [23], nucleic acids [24], metal ions [25] and small molecules [26]. 3-hydroxychromone (3HC) derivatives with excellent sensitivity to the polarity of microenvironment [27], which are important excited-state intramolecular proton transfer (ESIPT) based molecules, have been used to investigate micelles [28], phosphor lipid vesicles [29], metals [30] and proteins [31]. According to our previous reports, hydrophobic microenvironment provided by low-polar solvent [32], high α-helical level proteins [32,33], and intracellular environment [33], could enhance the fluorescence intensity of ESIPT molecules.

    • Ground and excited state dipole moments of some flavones using solvatochromic methods: An experimental and theoretical study

      2018, Journal of Molecular Structure
      Citation Excerpt :

      In addition to their marked biological activities such as anti-inflammatory, anti-cancerous, and anti-allergic [1–4], flavones show promising photo physical and photochemical properties [5,6]. This class of compounds is widely used as dye lasers, indicators of biophysical processes and optical brighteners [7–9]. Flavones have also been used as fluorimetric reagents for determination of metal ions [10].

    View all citing articles on Scopus
    View full text