New Generation of Bioorthogonally Applicable Fluorogenic Dyes with Visible Excitations and Large Stokes Shifts

Synthesis of a set of new, azide bearing, biorthogonally applicable fluorogenic dyes with large Stokes shifts is presented herein. To assess the fluorogenic performance of these new dyes we have labeled a genetically modulated, cyclooctyne-bearing protein in lysate medium. Studies showed that the labels produce specific signal with minimal background fluorescence. We also provide theoretical insights into the design of such fluorogenic labels

2‑Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification

Photoaffinity labels are powerful tools for dissecting ligand–protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C–H/X–H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets <i>in vitro</i> based on mass spectrometry analysis. In the <i>in situ</i> target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery