The biotinylated probes based on anticancer saponin OSW-1 with varied linker lengths were synthesized and their cell growth inhibitory activity and affinity pulldown efficiency were evaluated. All probes demonstrated comparable cytotoxicity to the parent natural product, highlighting that the linker moiety had a minimal impact on cell uptake or target engagement. In contrast, when evaluated against the known target proteins, OSBP and ORP4, the biotinylated probe 3 with PEG5 linker enabled most effective enrichment of target proteins in the affinity pulldown assay, suggesting that the cytotoxicity and pulldown efficiency did not correlate among the probes studied. Our data provided the first evidence that OSW-1 specifically binds to endogenously expressed OSBP and ORP4. The selectivity of affinity pulldown using probe 3 was also validated by facile identification of the enriched protein by silver staining and LC/MS analysis. Therefore, probe 3 with PEG5 linker comprising of 25 atoms (28 Å) was found as an optimal biotinylated probe for isolating OSW-1 binding proteins from cell lysate.
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