We previously applied the electrochromic modulation of excited-state intramolecular proton-transfer (ESIPT) reaction for the design of novel 3-hydroxyflavone (3-HF) derivatives as fluorescent probes for measuring the dipole potential, ΨD, in lipid bilayers (Klymchenko et al., Proc. Natl. Acad. Sci. USA, 2003, 100, 11219). In the present work, this method was revisited to take into account the influence of the bilayer hydration on the emission ratiometric response of 3-HF probes. For this reason, it was necessary to deconvolute the whole fluorescence spectra into three bands corresponding to the non H-bonded forms, normal N* and tautomer T* forms, both participating to the ESIPT reaction, and to the H-bonded H–N* form, excluded from this reaction. This allowed us to determine the pure N*/T* intensity ratio, without any contribution from the H–N* form emission depending essentially on the bilayer hydration. This new approach allowed us to confirm the correlation we obtained between the response of 3-HF probes on dipole potential modifications and the corresponding response of the reference fluorescent probe di-8-ANEPPS, thus further confirming the potency of 3-HF probes as excellent emission ratiometric probes to measure dipole potential in lipid membranes.
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ACKNOWLEDGMENTS
This work was supported by CNRS and Université Louis Pasteur. GM is a fellow from Agence Universitaire de la Francophonie. VVS was a student from Collège Doctoral Européen and was supported by the Région Alsace. ASK was a fellow from the European project TriOH.
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M`Baye, G., Shynkar, V.V., Klymchenko, A.S. et al. Membrane Dipole Potential as Measured by Ratiometric 3-Hydroxyflavone Fluorescence Probes: Accounting for Hydration Effects. J Fluoresc 16, 35–42 (2006). https://doi.org/10.1007/s10895-005-0022-3
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DOI: https://doi.org/10.1007/s10895-005-0022-3