Excitation-Dependent Multiple Fluorescence of a Substituted 2-(2'-Hydroxyphenyl)benzoxazole.

Excitation-dependent multiple fluorescence of a 2-(2'-hydroxyphenyl)benzoxazole (HBO) derivative (1) is described. Compound 1 contains the structure of a charge-transfer (CT) 4-hydroxyphenylvinylenebipy fluorophore and an excited-state intramolecular proton transfer capable (ESIPT-capable) HBO component that intersect at the hydroxyphenyl moiety. Therefore, both CT and ESIPT pathways, while spatially mostly separated, are available to the excited state of 1. The ESIPT process offers two emissive isomeric structures (enol and keto) of 1 in the excited state, while the susceptibility of 1 to a base adds another option to tune the composite emission color. In addition to the ground-state acid-base equilibrium that can be harnessed for the control of emission color by excitation energy, compound 1 exhibits excitation-dependent emission that is attributed to solvent-affected ground-state structural changes. Therefore, depending on the medium and excitation wavelength, the emission from the enol, keto, and anion forms could occur simultaneously, which are in the color ranges of blue, green, and orange/red, respectively. A composite color of white with CIE coordinates of (0.33, 0.33) can be materialized through judicious choices of medium and excitation wavelength.

A fluorescent indicator for imaging lysosomal zinc(II) with Förster resonance energy transfer (FRET)-enhanced photostability and a narrow band of emission.

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)-Enhanced Photostability and a Narrow Band of Emission.

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)-sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET-conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)-enhanced emission of 3 and 4 in lysosomes. It was further shown using two-color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino-functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.

Distinguishing Förster Resonance Energy Transfer and solvent-mediated charge-transfer relaxation dynamics in a zinc(II) indicator: a femtosecond time-resolved transient absorption spectroscopic study.

A bifluorophoric molecule (1) capable of intramolecular Förster Resonance Energy Transfer (FRET) is reported. The emission intensity of the FRET acceptor in 1 depends on the molar absorptivity of the donor, which is a function of zinc(II) complexation. The FRET dynamics of [Zn(1)](ClO4)2 is characterized by femtosecond time-resolved transient absorption spectroscopy. The solvent-mediated relaxation of the charge-transfer (CT) state of the isolated donor and the FRET process of the donor­acceptor conjugate are on similar time scales (40­50 ps in CH3CN), but distinguishable by the opposite solvent polarity dependency. As the solvent polarity increases, the efficiency of Columbic-based FRET is reduced, whereas CT relaxation is accelerated. In addition to revealing a method to distinguish CT and FRET dynamics, this work provides a photophysical foundation for developing indicators based on the FRET strategy.

Tricolor emission of a fluorescent heteroditopic ligand over a concentration gradient of zinc(II) ions.

The internal charge transfer (ICT) type fluoroionophore arylvinyl-bipy (bipy = 2,2'-bipyridyl) is covalently tethered to the spirolactam form of rhodamine to afford fluorescent heteroditopic ligand 4. Compound 4 can be excited in the visible region, the emission of which undergoes sequential bathochromic shifts over an increasing concentration gradient of Zn(ClO(4))(2) in acetonitrile. Coordination of Zn(2+) stabilizes the ICT excited state of the arylvinyl-bipy component of 4, leading to the first emission color shift from blue to green. At sufficiently high concentrations of Zn(ClO(4))(2), the nonfluorescent spirolactam component of 4 is transformed to the fluorescent rhodamine, which turns the emission color from green to orange via intramolecular fluorescence resonance energy transfer (FRET) from the Zn(2+)-bound arylvinyl-bipy fluorophore to rhodamine. While this work offers a new design of ratiometric chemosensors, in which sequential analyte-induced emission band shifts result in the sampling of multiple colors at different concentration ranges (i.e., from blue to green to orange as [Zn(2+)] increases in the current case), it also reveals the nuances of rhodamine spirolactam chemistry that have not been sufficiently addressed in the published literature. These issues include the ability of rhodamine spirolactam as a fluorescence quencher via electron transfer, and the slow kinetics of spirolactam ring-opening effected by Zn(2+) coordination under pH neutral aqueous conditions.

Colorimetric detection of copper ions in sub-micromolar concentrations using a triarylamine-linked resin bead.

The triarylamine derivative ETPA reacts with Cu(2+) to give deeply colored, stable radical cations in acetonitrile solution. ETPA was immobilized on to a tentagel resin bead which was then used for the fabrication of a simple device capable of the colorimetric detection of submicromolar concentrations of Cu(2+) ions in water. The naked eye detection limit reported here for Cu(2+) is one of the lowest ever reported for small molecule sensors.

Structurally diverse copper(II) complexes of polyaza ligands containing 1,2,3-triazoles: site selectivity and magnetic properties.

Copper(II) acetate mediated coupling reactions between 2,6-bis(azidomethyl)pyridine or 2-picolylazide and two terminal alkynes afford 1,2,3-triazolyl-containing ligands L(1)-L(6). These ligands contain various nitrogen-based Lewis basic sites including two different pyridyls, two nitrogen atoms on a 1,2,3-triazolyl ring, and the azido group. A rich structural diversity, which includes mononuclear and dinuclear complexes as well as one-dimensional polymers, was observed in the copper(II) complexes of L(1)-L(6). The preference of copper(II) to two common bidentate 1,2,3-triazolyl-containing coordination sites was investigated using isothermal titration calorimetry and, using zinc(II) as a surrogate, in (1)H NMR titration experiments. The magnetic interactions between the copper(II) centers in three dinuclear complexes were analyzed via temperature-dependent magnetic susceptibility measurements and high-frequency electron paramagnetic resonance spectroscopy. The observed magnetic superexchange is strongly dependent on the orientation of magnetic orbitals of the copper(II) ions and can be completely turned off if these orbitals are arranged orthogonal to each other. This work demonstrates the versatility of 1,2,3-triazolyl-containing polyaza ligands in forming metal coordination complexes of a rich structural diversity and interesting magnetic properties.

Generation of triarylamine radical cations through reaction of triarylamines with Cu(II) in acetonitrile. A kinetic investigation of the electron-transfer reaction.

Triphenylamine derivatives react with Cu(2+) in acetonitrile to give radical cations, which subsequently undergo dimerization to provide tetraphenylbenzidine derivatives. Kinetic aspects of radical cation formation were examined by stopped-flow spectrophotometry. A broad range of triphenylamine derivatives were studied, and the driving force for the electron-transfer reaction ranged from +3.67 to -8.56 kcal M(-1) with rate constants varying from 1.09 x 10(2) to 2.15 x 10(5) M(-1) s(-1) for these systems. Reorganization energy for the electron-transfer reaction was estimated using experimentally determined activation parameters. Fitting of the rate data to the Marcus equation using different values of the electronic coupling matrix element H(el) provided a good fit with H(el) = 100 cm(-1) suggesting that electron transfer in the TPA/Cu(2+) system conforms to the Marcus-type electron transfer. Furthermore, the high reorganization obtained from these studies is consistent with significant bond cleavage in the transition state, and a mechanism consistent with the experimental data is proposed.

Absorption and Emission Sensitivity of 2-(2'-Hydroxyphenyl)benzoxazole to Solvents and Impurities.

2-(2'-Hydroxyphenyl)benzoxazole (HBO) is known for undergoing intramolecular proton transfer in the excited state to result in the emission of its tautomer. A minor long-wavelength absorption band in the range 370-420 nm has been reported in highly polar solvents such as dimethylsulfoxide (DMSO). However, the nature of this species has not been entirely clarified. In this work, we provide evidence that this long-wavelength absorption band might have been caused by base or metal salt impurities that are introduced into the spectral sample during solvent transport using glass Pasteur pipettes. The contamination by base or metal salt could be avoided by using borosilicate glass syringes or nonglass pipettes in sample handling. Quantum chemical calculations conclude that solvent-mediated deprotonation is too energetically costly to occur without the aid of a base of an adequate strength. In the presence of such a base, the deprotonation of HBO and its effect on emission are investigated in dichloromethane and DMSO, the latter of which facilitates deprotonation much more readily than the former. Finally, the absorption and emission spectra of HBO in 13 solvents are reported, from which it is concluded that ESIPT is hindered in polar solvents that are also strong hydrogen bond acceptors.

Zn(II)-coordination modulated ligand photophysical processes - the development of fluorescent indicators for imaging biological Zn(II) ions.

Molecular photophysics and metal coordination chemistry are the two fundamental pillars that support the development of fluorescent cation indicators. In this article, we describe how Zn(II)-coordination alters various ligand-centered photophysical processes that are pertinent to developing Zn(II) indicators. The main aim is to show how small organic Zn(II) indicators work under the constraints of specific requirements, including Zn(II) detection range, photophysical requirements such as excitation energy and emission color, temporal and spatial resolutions in a heterogeneous intracellular environment, and fluorescence response selectivity between similar cations such as Zn(II) and Cd(II). In the last section, the biological questions that fluorescent Zn(II) indicators help to answer are described, which have been motivating and challenging this field of research.

Cu(II) mediated generation and spectroscopic study of the tris(4-anisyl)amine radical cation and dication. Unusually shielded chemical shifts in the dication.

The reaction of tris(4-anisyl)amine (TAA) with Cu(2+) ion leading to formation of the TAA radical cation and dication is described. Spectroscopic studies confirm the formation of the radical cation and dication. (1)H and (13)C NMR spectral studies reveal interesting structural features of the dication.

A FRET-based indicator for imaging mitochondrial zinc ions.

A strategy based on fluorescence resonance energy transfer (FRET) to transform a red-emitting fluorophore into a ratiometric indicator for mitochondrial Zn(II) is demonstrated.

Phenothiazine attached Ru(bpy)(3)2+ derivative as highly selective "turn-ON" luminescence chemodosimeter for Cu2+.

The design of a highly selective "turn-ON" luminescence chemodosimeter for Cu(2+) is reported. The design strategy made use of the ability of Cu(2+) ions to oxidize aromatic amines in acetonitrile solution. The aromatic amine employed here is a phenothiazine moiety which is covalently linked to one of the bipyridine units of Ru(bpy)(3)(2+). Excitation of the Ru(bpy)(3)(2+) leads to electron transfer from the phenothiazine moiety to the MLCT excited state of Ru(bpy)(3)(2+) which resulted in efficient quenching of the luminescence. In the presence of excess Cu(2+), phenothiazine moiety is oxidized to a stable entity which is incapable of electron donation to the MLCT excited state of Ru(bpy)(3)(2+). The emission of the Ru(bpy)(3)(2+) moiety is thus restored and we show that this strategy can be used as the basis for sensing micromolar amounts of Cu(2+). Only Cu(2+) is capable of this reaction, making this an interesting, hitherto unexplored strategy for the selective detection of micromolar amounts of Cu(2+).

Cu(II)-mediated generation of triarylamine radical cations and their dimerization. an easy route to tetraarylbenzidines.

Triphenylamine (TPA) derivatives react with Cu2+ in acetonitrile to give TPA radical cations which undergo dimerization and deprotonation reactions to yield tetraphenylbenzidines (TPB). Synthetic utility of this reaction is demonstrated using several triphenylamine derivatives, and yields in excess of 80% are obtained in most cases. Involvement of the amine radical cations in these reactions was confirmed by ESR and absorption spectroscopic studies. A mechanism consistent with all observations is proposed. This study also revealed a very good correlation between the free energy change for radical cation formation and product yields.

Understanding reactivity patterns of the dialkylaniline radical cation.

N,N-Dimethylaniline and N,N-diethylaniline react with Cu2+ to form the corresponding amine radical cations. The radical cations were characterized by their absorption spectra. In the absence of any nucleophiles, the radical cations dimerize to give tetraalkylbenzidines, and this reaction can be monitored by absorption spectroscopy. In the presence of nucleophiles such as Cl[negative in circle], Br[negative in circle], or SCN[negative in circle], the radical cations undergo nucleophilic substitution to give para-substituted dialkylanilines in good yields.