Long-Lived Bright Red Emitting Azaoxa-Triangulenium Fluorophores

Abstract

The fluorescence lifetimes of most red emitting organic probes are under 4 nanoseconds, which is a limiting factor in studying interactions and conformational dynamics of macromolecules. In addition, the nanosecond background autofluorescence is a significant interference during fluorescence measurements in cellular environment. Therefore, red fluorophores with longer lifetimes will be immensely helpful.Azaoxa-triangulenium fluorophores ADOTA and DAOTA are red emitting small organic molecules with high quantum yield, long fluorescence lifetime and high limiting anisotropy. In aqueous environment, ADOTA and DAOTA absorption and emission maxima are respectively 540 nm and 556 nm, and 556 nm and 589 nm. Their emission extends beyond 700 nm. Both probes have the limiting anisotropy between 0.36–0.38 at their absorption peak. In both protic and aprotic solvents, their lifetimes are around 20 ns, making them among the longest-lived red emitting organic fluorophores. Upon labeling of avidin, streptavidin and immunoglobulin their absorption and fluorescence are red-shifted. Unlike in free form, the protein-conjugated probes have heterogeneous fluorescence decays, with the presence of both significantly quenched and unquenched populations. Despite the presence of significant local motions due to a flexible trimethylene linker, we successfully measured both intermediate nanosecond intra-protein motions and slower rotational correlation times approaching 100 ns. Their long lifetimes are unaffected by the cell membrane (hexadecyl-ADOTA) and the intra-cellular (DAOTA-Arginine) localization. Their long lifetimes also enabled successful time-gating of the cellular autofluorescence resulting in background-free fluorescence lifetime based images.ADOTA and DAOTA retain a long fluorescence lifetime when free, as protein conjugate, in membranes and inside the cell. Our successful measurements of intermediate nanosecond internal motions and long correlations times of large proteins suggest that these probes will be highly useful to study slower intra-molecular motions and interactions among macromolecules. The fluorescence lifetime facilitated gating of cellular nanosecond autofluorescence should be of considerable help in in vitro and in vivo applications.

Steady state and time resolved fluorescence studies of azadioxatriangulenium (ADOTA) fluorophore in silica and PVA thin films

“…Polyvinyl alcohol (130, 000 MW) was purchased from Sigma Aldrich (Sigma Aldrich, St Louis, MO USA). N -(ω-butanoic acid)-azatriangulenium tetrafluoroborate (ADOTA) was prepared as previously described [27,31]. …”

Diazaoxatriangulenium: synthesis of reactive derivatives and conjugation to bovine serum albumin

“…Subsequent reaction of the DMQA compounds in molten pyridinium chloride followed by basic hydrolysis of the intermediate amide yielded the desired N -(3-carboxypropyl)- N ′-methyl-DAOTA tetrafluoroborate ( 5a , Scheme 1) as a red powder in a good yield. 49 The synthesis of the 4-aminophenyl DAOTA derivative ( 5d ) was also performed via the route through the DMQA derivative ( 3d , Scheme 1). This yielded 3d in a high yield, while the ring closure reaction for conversion of 3d into 5d yielded 5d in a low yield.…”

“…46, 47 The spectra displayed in figure 1 show that DAOTA ( 5c ) has more disable absorption and emission wavelengths in the red region of the visible spectrum for measurements on biological systems as compared to ADOTA ( 4c ). 49, 53, 65 Table 1 summarises the photophysical properties of 4c and 5c in acetonitrile, dimethyl sulfoxide (DMSO), and phosphate buffered saline at pH = 7.4 (PBS). The differences between 4c and 5c in organic solvents are closely related to the oscillator strengths of the lowest energy transition of the two parent chromophores.…”

Sandwich type plasmonic platform for MEF using silver fractals

“…The water used for all solutions and washings was Millipore grade with > 18.2 MΩ resistivity. Me-ADOTA.Cl was prepared as previously described [15] while ADOTA-NHS (butyric acid derivative) was synthesised accordingly to established procedures[16]. …”