We have discovered a new platform for precision target identification (target ID) of proteins through the orchestration of small molecule-photocatalyst conjugates, blue light, and otherwise inert diazirines. Through a photophysical process known as Dexter energy transfer, we can, for the first time, effectively decouple the small molecule from the photoreactive group, which has been a mainstay of classical photoaffinity labelling for over half a century. Visible-light excitation of the small molecule-photocatalyst conjugate can sensitize diazirines within the immediate vicinity (0.1 nm), giving rise to a carbene with an extremely short solution half-life in water (~4 ns). Crucially, the catalytic generation of these reactive intermediates, at the precise location of the ligand binding site, gives rise to multiple labelling events. This unprecedented step, allows, for the first time, not only signal amplification at the protein level for target ID, but also at the peptide and single amino acid level, facilitating bind-site mapping. We've demonstrated the platform on a number of small molecules (including cyclic peptides) to label individual proteins and multi-protein complexes. Importantly, we have demonstrated that unlike traditional probe conjugates which require extensive SAR optimization, successful implementation of this platform requires no optimization of the catalyst or linker, saving critical time and expense. Moreover, the residue agnostic nature of carbene insertion, combined with the tight radius of activation, leads to precision mapping of the ligand binding site, which we have successfully highlighted on both single proteins and protein complexes.
