Synthesis and photophysical and cation-binding properties of mono- and tetranaphthylcalix[4]arenes as highly sensitive and selective fluorescent sensors for sodium.

The syntheses and properties of two calixarene-based fluorescent molecular sensors are reported. These comprise tert-butylcalix[4]arene either with one appended fluorophore and three ester groups (Calix-AMN1). or with four appended fluorophores (Calix-AMN4). The fluorophore is 6-acyl-2-methoxynaphthalene (AMN), which contains an electron-donating substituent (methoxy group) conjugated to an electron-withdrawing substituent (carbonyl group); this allows photoinduced charge transfer (PCT) to occur upon excitation. The investigated fluoroionophores thus belong to the family of PCT fluorescent molecular sensors. In addition to the expected red shifts of the absorption and emission spectra upon cation binding, a drastic enhancement of the fluorescence quantum yield-in an "off- on" fashion comparable to that seen in photoinduced electron transfer (PET) molecular sensors-was observed. For Calix-AMN1. it increases from 10(-3) for the free ligand to 0.68 for the complex with Ca2+. This exceptional behaviour can be interpreted in terms of the relative locations of the npi* and pipi* levels, which depend on the charge density of the bound cation. For Calix-AMN4. in addition to the photophysical effects observed for Calix-AMN1, interactions between the chromophores by complexation with some cations have been found in the ground state (hypochromic effect) and in the excited state (excimer formation). Steady-state fluorescence anisotropy measurements for the system Na+ is included in Calix-AMN4, show a depolarization effect due to energy transfer (homotransfer) between the fluorophores. Regarding the complexing properties, a high selectivity for Na+ over K+, Li+, Ca2+ and Mg2+ was observed in ethanol and ethanol-water mixtures. The selectivity (Na+/other cations) expressed as the ratio of the stability constants was found to be more than 400.

Voltage-Controlled Fluorometry of the Transfer of Nonfluorescent Ions across the 1,2-Dichloroethane/Water Interface Using Fluorescent Ionophores.

The voltage-driven transfer of alkali and alkaline-earth metal ions across the polarized 1,2-dichloroethane (DCE)/water interface has been measured fluorometrically by using two fluorescent ionophores, coumarin 153-linked monoaza-15-crown-5 (C153-crown(O(4))) and two-coumarin 153-linked diaza-18-crown-6 ((C153)(2)-K(22)). The 1:1 complex formation prevails for both monovalent and divalent ion transfer, when the concentration of C153-crown(O(4)) in DCE is at the micromolar level. In the Mg(2+) ion transfer, the formation of the 1:2 (metal-ligand) complex becomes significant at the millimolar level of C153-crown(O(4)). C153-crown(O(4)) seems to adsorb at the interface. The complexation of Ba(2+) with (C153)(2)-K(22) is significantly slower than the complexation with C153-crown(O(4)).

Recovery of lifetime distributions from frequency-domain fluorometry data by means of the quantified maximum entropy method.

The new quantified version of the maximum entropy method allows one to recover lifetime distributions with a precise statement of the accuracy of position, surface, and broadness of peaks in the distribution. Applications to real data (2,6-ANS in aqueous solutions of sodium dodecyl sulfate micelles of Β-cyclodextrin) are presented.

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

A new concept of photogeneration of cations: Evidence for photoejection of Ca(2+) and Li (+) from complexes with a crown-ether-linked merocyanine by picosecond spectroscopy.

Time-resolved transient absorption and gain spectra of DCM-crown and its complexes with Li(+) and Ca(2+) in acetonitrile are measured in the wavelength range 370-670 nm after subpicosecond excitation at 425 nm. The results give evidence for the fast formation of free DCM-crown from both excited complexes, with a faster rate for the Li(+) complex. A two-step mechanism is found for the initial decay (within 30 ps) of the free DCM-crown.

Coumarinic fluorescent chemosensors for the detection of transition metal ions.

Fluoroionophores consisting of coumarin-153 linked to mono- and diaza-crown ethers are able to complex transition metal cations (Ni(2+), Zn(2+), Cd(2+)) and Pb(2+), with concomitant large changes in photophysical properties. The stability constants in acetonitrile are higher than 3×10(6) M (-1) except for Ni(2+).

Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture.

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.

Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength.

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.

Changes in extrinsic fluorescence intensity of the electroplax membrane during electrical excitation.

The fluorescent dye 1-anilinonapthalene-8-sulfonic acid (ANS) has been used as a probe of changes in membrane conformation accompanying excitation of the electroplax ofElectrophorus electricus. ANS binds reversibly to the excitable membrane at rest. During generation of an action potential, an increase in ANS-fluorescence intensity is observed which resembles but does not strictly follow the membrane potential. Experiments using the current-clamp technique have demonstrated a linear relationship between the change in membrane potential and the change in ANS fluorescence intensity. The change in fluorescence intensity is not a consequence of binding to membrane sites of increased affinity nor of an electrophoretic concentration of ANS molecules at the membrane surface.It is not known whether the change in fluorescence intensity is due to a change in quantum yield of bound ANS or to an increase in the amount of bound ANS. In either case, the change in fluorescence intensity may be interpreted as a change in membrane conformation.