OSW-1 is a highly potent anticancer saponin with an unknown mechanism of action that is distinct from the currently used chemotherapeutic agents. Toward investigation of its binding proteins, we designed and synthesized a clickable photoaffinity probe from OSW-1 and characterized its photochemical reactivity. A mild, two-step procedure involving a Me₂SnCl₂-mediated acylation reaction was developed to site-selectively derivatize OSW-1 with a linker that bears a photoreactive group and an alkyne tag. The OSW-1-based photoaffinity probe retained potent anticancer activity similar to that of the parent natural product, thereby providing a cell-permeable analogue of OSW-1. Photoaffinity labeling studies demonstrated that the probe enabled crosslinking of a model sterol-binding protein in an affinity-dependent fashion, which could be efficiently detected by conjugating a fluorophore or biotin by click chemistry.

A set of lactose-based photoaffinity probes bearing either alkyl diazirine or trifluoromethylphenyl diazirine (TPD) groups were designed and synthesized to directly compare their efficiency in photocrosslinking a carbohydrate-binding protein. The crosslinking efficiency of the TPD probes was higher than the alkyl diazirine probes when reacted with a single binding protein. However, the alkyl diazirine probe with a small alkyne tag achieved more selective photoaffinity labeling of a binding protein in cell lysate than the corresponding TPD probe.

Carbohydrate-protein interactions mediate cellular signals, which are crucial in a diverse array of biological and pathological processes. Despite their importance, many carbohydrate-protein interactions remain unknown due to inherent difficulties in studying them. With the aim to provide the first step in elucidating the biological roles of carbohydrates, photoaffinity labeling has been used as a promising chemical strategy for the detection and identification of carbohydrate-binding proteins in their native environment. Recent efforts in the design of carbohydrate-based photoaffinity probes include the use of selective photoreactive groups, development of biotinylated photoreactive groups and linkers, multivalent carbohydrate ligands, affinity-based protein profiling probes, and photoreactive monosaccharide precursors for metabolic glycan labeling. These methods offer new tools to address challenges associated in capturing the often elusive carbohydrate-protein interactions.

A judicious choice of photoreactive group is critical in successful photoaffinity labeling studies of small molecule–protein interactions. A set of carbohydrate-based photoaffinity probes was prepared to compare the effects of three major photoreactive groups on the efficiency and selectivity of crosslinking a binding protein with low affinity. We showed that, despite the low crosslinking yield, the diazirine probe displayed the high ligand-dependent reactivity consistent with the ideal mechanism of photoaffinity labeling. Moreover, we demonstrated that, among the three photoreactive groups, only the diazirine probe achieved highly selective crosslinking of a low-affinity binding protein in cell lysate.