The reporter group is thus introduced after covalent bond formation between the probe and target protein. Following covalent labeling of protein targets via a (latent) chemically reactive moiety within the probe, probe-modified proteins are conjugated to the azide-bearing reporter under click chemistry conditions ( Figure 1A, (IV)). However, this arrangement has been shown to produce higher background labeling of proteins ( Speers and Cravatt, 2004). Alternatively, the azide can be incorporated into the probe and the alkyne incorporated into the reporter. The terminal alkyne is typically present in the small-molecule probe, while the azide is present in a fluorescent or biotinylated reporter group. During the click reaction, Cu(I) catalyzes a highly selective, bio-orthogonal 1,3 dipolar cycloaddition reaction between a terminal alkyne group and an azide group to form a stable triazole product ( Figure 1A, (III), (IV)). (B) Structures of the natural product HUN7293 ( 1), photo-affinity probe ( 2), and the photo-stable control compound ( 3).Ĭu(I)-catalyzed click chemistry is an important method for bio-conjugation of probe-labeled proteins with reporter groups ( Best, 2009). The adduct is then detected by conjugation with an azide-containing reporter group under click chemistry conditions (IV). UV irradiation of the diazirine generates a carbene intermediate (II) that covalently crosslinks to the protein target (III). (A) Generalized scheme for photo-affinity labeling and detection using a diazirine and alkyne-containing photo-affinity probe (I). Heterobifunctional amine-reactive alkyl diazirine crosslinkers, as well as alkyl diazirine-containing amino acid analogs are commercially available (Pierce, Thermo Scientific). Several improved methods for the synthesis of alkyl diazirines starting from alkyl ketone precursors have been recently reported ( Bond et al., 2009 MacKinnon et al., 2007). The alkyl diazirine is stable toward acidic and basic conditions and toward ambient light encountered during routine chemical synthesis. When not poised for insertion into bonds of the macromolecular target, the alkyl carbene intermediate undergoes rapid quenching by solvent or internal rearrangement to a stable olefin side-product ( Ford et al., 1998). Second, the carbene intermediate formed upon photo-activation of the diazirine ( Figure 1A, (II)) rapidly inserts into X-H bonds (X = N, S, O), as well as C-H bonds, to form stable covalent insertion products ( Brunner, 1993). This allows installation of the diazirine at positions of a small-molecule that would not tolerate larger, aryl-based photo-reactive groups. First, it is compact in size, being nearly isosteric to a methyl group, and is accessed synthetically via an alkyl ketone. However, the alkyl diazirine holds unique advantages. Like most useful photo-affinity groups, the alkyl diazirine ( Figure 1A, (I)) is activated at a wavelength of light (~355 nm) that is not damaging to protein(s). benzophenone, trifluoromethyl phenyl diazirine, aryl azide). There are several photo-reactive functional groups frequently used in PAL (e.g. The challenges are compounded with small-molecules that target integral membrane proteins, which often show decreased function after solubilization with detergents, a prerequisite for affinity purification. Affinity purification of protein targets is often difficult with non-covalently bound small-molecules, especially those with low to moderate binding affinity for the target. Covalent bond formation between the probe and targets enable the subsequent purification and identification of the targets using techniques such as SDS-PAGE, immunoprecipitation, biotin-streptavidin affinity purification and mass spectrometry. fluorophore, biotin, or radioactive label). Photo-crosslinked protein targets are then visualized by the reporter group (e.g. carbene, nitrene, or radical) that covalently crosslinks the photo-affinity probe to its macromolecular binding partner(s). Irradiation of the photo-reactive group generates a highly reactive chemical species (e.g. During PAL, the photo-affinity probe is incubated with a protein mixture and irradiated with UV light. The photo-affinity probe is designed and synthesized based on SAR (structure-activity relationships) of a parent small-molecule having known biological activity. PAL uses an analog of a biologically active small-molecule, known as a photo-affinity probe, that bears photo-reactive and reporter functional groups, to identify macromolecular binding partners. Photo-affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. Target identification of biologically active small-molecules is often the rate-determining step in forward chemical genetics.