Concerning the photophysics of fluorophores towards tailored bioimaging compounds: a case study involving S100A9 inflammation markers

Abstract
A full understanding concerning the photophysical properties of a fluorescent label is crucial for a reliable and predictable
performance in biolabelling applications. This holds true not only for the choice of a fluorophore in general, but also for the
correct interpretation of data, considering the complexity of biological environments. In the frame of a case study involving
inflammation imaging, we report the photophysical characterization of four fluorescent S100A9-targeting compounds
in terms of UV–vis absorption and photoluminescence spectroscopy, fluorescence quantum yields (ΦF) and excited state
lifetimes (τ) as well as the evaluation of the radiative and non-radiative rate constants (kr and knr, respectively). The probes
were synthesized based on a 2-amino benzimidazole-based lead structure in combination with commercially available dyes,
covering a broad color range from green (6-FAM) over orange (BODIPY-TMR) to red (BODIPY-TR) and near-infrared
(Cy5.5) emission. The effect of conjugation with the targeting structure was addressed by comparison of the probes with their
corresponding dye-azide precursors. Additionally, the 6-FAM and Cy5.5 probes were measured in the presence of murine
S100A9 to determine whether protein binding influences their photophysical properties. An interesting rise in ΦF upon binding
of 6-FAM-SST177 to murine S100A9 enabled the determination of its dissociation equilibrium constant, reaching up
to KD = 324 nM. This result gives an outlook for potential applications of our compounds in S100A9 inflammation imaging
and fluorescence assay developments. With respect to the other dyes, this study demonstrates how diverse microenvironmental
factors can severely impair their performance while rendering them poor performers in biological media, showing
that a preliminary photophysical screening is key to assess the suitability of a particular luminophore.