A strategy to site-selectively monoacylate an antitumor saponin OSW-1 was developed using an organotin reagent to rapidly access its derivatives that are useful as chemical probes. 4″-O-Acylated OSW-1 derivatives bearing a fluorophore, an alkyne tag, or biotin were prepared in good yields and were shown to maintain highly cytotoxic activity.

OSW-1 is ah ighly potent anticancer saponin with an unknown mechanism of action that is distinct from the currently used chemotherapeutic agents. To ward investigation of its binding proteins, we designed and synthesized ac lickable photoaffinity probe from OSW-1 and characterized its photochemical reactivity.Amild, twostep procedure involving aM e 2 SnCl 2 -mediated acylation reaction was developed to site-selectively derivatize OSW-1w ith al inker that bears ap hotoreactive group and an alkyne tag. The OSW-1-basedp hotoaffinity probe retained potent anticancer activity similar to that of the parent natural product, thereby providing ac ell-permeable analogue of OSW-1. Photoaffinity labeling studies demonstrated that the probe enabled crosslinking of am odel sterolbinding protein in an affinity-dependent fashion,w hich could be efficiently detected by conjugating afluorophore or biotin by click chemistry.

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A novel fluorescent photoaffinity probe of OSW-1 was prepared in two steps from a naturally occurring inactive congener by a sequential site-selective acylation strategy using MeSnCl. It displayed highly potent anticancer activity and a similar intracellular localization property to that of a fluorescently-tagged OSW-1, thereby demonstrating its potential utility in live cell studies.

The structural basis for the intracellular delivery of OSW-1 is investigated using fluorescent derivatives of OSW-1 and its closely related congeners. Despite the large differences in activity, all the fluorescent probes are found to translocate across the plasma membrane to the ER and Golgi apparatus. This observation suggests that the glycosylated cholestane moiety plays an important role in the cell internalization and intracellular localization property of OSW-1.