Interplay between excited-state intramolecular proton transfer and charge transfer in flavonols and their use as protein-binding-site fluorescence probes.

Abstract

A comparative study is presented of competitive fluorescences of three flavonols, 3-hydroxyflavone, 3,3',4',7-tetrahydroxyflavone (fisetin), and 4'-diethylamino-3-hydroxyflavone (DEIF). The normal fluorescence Si -* So (400-nm region) is largely replaced by the proton-transfer tautomer fluorescence S' --S' in the 550-nm region for all three of the flavonols in aprotic solvents at room temperature. For DHF in polar solvents the normal fluorescence becomes a charge-transfer fluorescence (460-500 nm) which competes strongly with the still dominant proton-transfer fluorescence (at 570 nm). In protic solvents, and at 77 K, the interference with intramolecular hydrogen bonding gives rise to greatly enhanced normal fluorescence, lowering the quantum yield of proton-transfer fluorescence. The utility of DHF as a discriminating fluorescence probe for protein binding sites is suggested by the strong dependence of the charge-transfer fluorescence on polarity of the environment and by various static and dynamic parameters of the charge-transfer and protontransfer fluorescence which can be determined.The purpose of this paper is to explore the application of molecular fluorescence as probes for protein binding sites. The competition between proton-transfer (PT) fluorescence (1, 2) (i.e., excited-state intramolecular proton transfer, ESIPT) and charge-transfer (CT) fluorescence, and the application of solvent polarity studies, is presented for three hydroxyflavones selected as fluorescence probes. The model compounds chosen are 3-hydroxyflavone (3-HF) and its derivatives 3,3',4',7-tetrahydroxyflavone (fisetin) and 4'-diethylamino-3-hydroxyflavone (DHF). In the first two, PT tautomer fluorescence (Amax 529 nm and 540 nm, respectively) is predominant in aprotic solvents at 298 K, the normal tautomer fluorescence being observed only very weakly. The third molecule (DHF) emits both a PT fluorescence at Amos 570 nm and a prominent CT fluorescence at Amass 460 nm.3-HF has been the object of extensive photophysical studies (1-33). Fisetin has not been studied extensively (29) and attracted our attention again recently because of its extraordinary performance as a lasing material (unpublished work). DHF is a newly synthesized molecule (34-36) which we explore as a protein fluorescence probe.The spectroscopic behavior of these three hydroxyflavones offers some contrasting properties which, for these cases, permit a discrimination between normal fluorescence, strong CT fluorescence, and PT fluorescence and indicate the utility of the latter two as fluorescence probe phenomena. MATERIALS AND METHODSSpectroscopic Measurements. Absorption spectra were measured on a Shimadzu UV-2100 spectrophotometer. The fluorescence spectra were recorded with a SPEX Fluoromax spectrofluorimeter (Spex Industries, Edison, NJ).Chemicals. 3-HF and fisetin were from Aldrich. tDHF and 4'-diethylamino-3-methoxyflavone were synthesized in the laboratory of Pi-Tai Chou (University of South Carolina, Columbia). All solvents were of spectrograde quality and...

Effects of polar protic solvents on dual emissions of 3-hydroxychromones

“…3,4 Due to these unique features, numerous 3HC dyes have been synthesized and studied, resulting in the development of new fluorescent probes for solvent polarity, 2,5,6 ion binding 7 and electric fields 8,9 with applications in the fields of polymers, 10 reverse micelles, 11,12 host-guest complexes 13 , lipid vesicles, 9,14-17 cellular membranes 18 and proteins. [19][20][21][22][23] Reactive derivatives of 3HCs have been also proposed for fluorescence labelling of biomolecules in order to study their conformations and intermolecular interactions. 20,21 In this respect, compounds 2 and 3 (chart 1) and their derivatives are very promising since they exhibit solvent-dependent dual emission and satisfactory fluorescence quantum yields in highly polar environment.…”

2-(benzimidazol-2-yl)-3-hydroxychromone derivatives: spectroscopic properties and a possibile alternative intramolecular proton phototransfer

“…Association with solvent molecules, especially important for highly polar proton donor or acceptor solvents, competes with the intramolecular H-bonding and affects the ESIPT [16,17]. Intermolecularly H-bonded species with a broken intramolecular H-bond generally lose the capacity for excited state proton phototransfer.…”

Energy transfer to a proton-transfer fluorescence probe: Tryptophan to a flavonol in human serum albumin

“…When joined in one molecular system, excited-state events can compete dynamically. An example of such competition is the mutual exclusion of intramolecular electron and PT in 4Ј-diethylamino-3-hydroxyflavone (16)(17)(18)(19). Proper pairing of excited-state transformations can be a source of structural͞dynamical information about the molecules possessing these transformations, the excited-state transformations themselves, and the medium where the coupling takes place.…”