Bright ideas for chemical biology

Abstract

Small-molecule fluorescent probes embody an essential facet of chemical biology. Although numerous compounds are known, the ensemble of fluorescent probes is based on a modest collection of modular "core" dyes. The elaboration of these dyes with diverse chemical moieties is enabling the precise interrogation of biochemical and biological systems. The importance of fluorescence-based technologies in chemical biology elicits a necessity to understand the major classes of small-molecule fluorophores. Here, we examine the chemical and photophysical properties of oft-used fluorophores and highlight classic and contemporary examples in which utility has been built upon these scaffolds.

Figure 1

Photophysical concepts (a, b) and biological applications (c–f) of small-molecule fluorophores. a) Jabłoński diagram. i) Absorption of a photon gives an excited state. ii) Internal conversion to S₁. iii) Fluorescence. iv) Non-radiative decay. v) Intersystem crossing to T₁. vi) Phosphorescence. vii) Non-radiative decay. b) Generic absorption and emission spectra. c) Site-specific labeling of a biomolecule by an orthogonal reaction between two functional groups (red). d) Enzyme substrates. i) Enzyme-catalyzed removal of a blocking group (red) elicits a change in fluorescence. ii) Enzyme catalyzes the cleavage of a labeled biomolecule (red) and concomitant decrease in FRET. e) Environmental indicators. i) Binding of an analyte (red) elicits a change in fluorescence. ii) Protonation of a fluorophore elicits a change in fluorescence. f) Staining of subcellular domains by distinct fluorophores.

Figure 2

Plot of fluorophore brightness (ε × Φ) versus the wavelength of maximum absorption (λ_max) for the major classes of fluorophores. The color of the structure indicates its wavelength of maximum emission (λ_em). For clarity, only the fluorophoric moiety of some molecules is shown.