Effect of linker length on the function of biotinylated OSW-1 probes.

Abstract

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 the probes possessed similar cytotoxicity compared to the parent natural product, showing that the linker moiety did not significantly affect 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 an affinity pulldown assay, suggesting that the cytotoxicity and pulldown efficiency did not correlate among the probes studied. The selectivity of affinity pulldown using the optimal probe was also verified 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 to be an optimal biotinylated probe for isolating OSW-1 binding proteins from cell lysate.