Long fluorescence lifetime triangulenium dyes in imaging and fluorescence polarization assay.

The development of new fluorescent dyes-new fluorochromes-has a large potential to improve the established methods in enzymology, by empowering both detection capability and the scope of the individual method. Unfortunately, there are huge barriers when adopting new improved fluorescent dyes in established methods. The dyes have to be generally available, protocols for labeling and analysis must be in place, and the field has to be aware how the new improved dye can enhance their method of choice. In this chapter, we will address these issues for the triangulenium dyes. A class of dyes that has a long fluorescence lifetime and emission in the red. A unique combination that opens up new possibilities for the study of protein rotational motion, when developing fluorescence polarization (FP) assays, and for all time-resolved imaging or analysis platforms. To make these dyes generally available, the features of the long fluorescence lifetime triangulenium dyes are described and an optimized labelling protocol are reported.

Extended Triangulenium Ions: Syntheses and Characterization of Benzo-Bridged Dioxa- and Diazatriangulenium Dyes.

The very limited class of fluorophores, with a long fluorescence lifetime (>10 ns) and fluorescence beyond 550 nm, has been expanded with two benzo-fused triangulenium derivatives and two cationic [5]-helicene salts. The syntheses of the benzo-bridged dioxa- and diazatriangulenium derivatives (BDOTA+ and BDATA+, respectively) required two different synthetic approaches, which reflect the structural and physiochemical impact on the reactivity of [5]-helicenium precursors. Spectroscopic investigations show that the introduction of the benzo bridge into the triangulenium chromophore significantly redshifts the absorption and emission while maintaining fluorescence lifetimes above 10 ns. The combination of a high quantum yield, long fluorescence lifetime, and emission above 600 nm is possible only if the structural aspects of the triangulenium framework are perfectly harmonized to secure a low rate of nonradiative deactivation. The new benzo bridge may be a general motif to obtain red-shifted derivatives of other dye classes.

Azadioxatriangulenium and Diazaoxatriangulenium: Quantum Yields and Fundamental Photophysical Properties.

Over the last decade, we have investigated and exploited the photophysical properties of triangulenium dyes. Azadioxatriangulenium (ADOTA) and diazaoxatriangulenium (DAOTA), in particular, have features that make them useful in various fluorescence-based technologies (e.g., bioimaging). Through our work with ADOTA and DAOTA, we became aware that the reported fluorescence quantum yields (ϕfl) for these dyes are lower than their actual values. We thus set out to further investigate the fundamental structure-property relationships in these unique conjugated cationic systems. The nonradiative processes in the systems were explored using transient absorption spectroscopy and time-resolved emission spectroscopy in combination with computational chemistry. The influence of molecular oxygen on the fluorescence properties was explored, and the singlet oxygen sensitization efficiencies of ADOTA and DAOTA were determined. We conclude that, for these dyes, the amount of nonradiative deactivation of the first excited singlet state (S1) of the azaoxa-triangulenium fluorophores is low, that the rate of such deactivation is slower than what is observed in common cationic dyes, that there are no observable radiative transitions occurring from the first excited triplet state (T1) of these dyes, and that the efficiency of sensitized singlet oxygen production is low (Ï•Δ ≤ 10%). These photophysical results provide a solid base upon which technological applications of these fluorescent dyes can be built.

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

The azaoxa-triangulenium dyes are characterised by emission in the red and a long fluorescence lifetime (up to 25 ns). These properties have been widely explored for the azadioxatrianguelnium (ADOTA) dye. Here, the syntheses of reactive maleimide and NHS-ester forms of the diazaoxatriangulenium (DAOTA) system are reported. The DAOTA fluorophore was conjugated to bovine serum albumin (BSA) and investigated in comparison to the corresponding ADOTA-BSA conjugate. It was found that the fluorescence of DAOTA experienced a significantly higher degree of solvent quenching if compared to ADOTA as non-conjugated dyes in aqueous solution, while the fluorescence quenching observed upon conjugation to BSA was significantly reduced for DAOTA when compared to ADOTA. The differences in observed quenching for the conjugates can be explained by the different electronic structures of the dyes, which renders DAOTA significantly less prone to reductive photoinduced electron transfer (PET) quenching from e.g. tryptophan. We conclude that DAOTA, with emission in the red and inherent resistance to PET quenching, is an ideal platform for the development of long fluorescence lifetime probes for time-resolved imaging and fluorescence polarisation assay.

Azadioxatriangulenium: Synthesis and Photophysical Properties of Reactive Dyes for Bioconjugation.

Azadioxatriangulenium (ADOTA) is a fluorescent triangulenium dye with a long fluorescence lifetime, highly polarized transitions and emission in the red part of the visible spectrum. These properties make the chromophore suited for application in fluorescence polarization/anisotropy assay. To be useful for these applications, reactive forms of the dyes must be available in significant quantities. Here, the synthesis and photophysical properties of amine-reactive NHS esters and a thiol-reactive maleimide derivate of ADOTA are reported. The synthesis involves two steps of nucleophilic bridge forming reactions starting from tris(2,6-dimethoxyphenyl) methylium tetrafluoroborate, which can readily be made on 100 gram scale. In the third and final step the reactive NHS or maleimide groups are formed. The beneficial photophysical properties of the ADOTA chromophore are maintained in these derivatives, and we conclude that these systems are ideal to study protein motion and protein-protein interactions for systems of up towards 1000 kDa.

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements.

Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r 0 = 0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕ fl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕ fl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.