Part I. The use of small-molecule substrates as probes of the ubiquitination pathway Part II. Intramolecular 1,3-dipolar cycloaddition reaction for peptide 'stapling'

Posttranslational modifications are modifications which occur after a polypeptide or protein has been translated and are used for cell signaling, protein trafficking, localization and degradation among many other pathways. Ubiquitination is one example of a post-translational modification pathway, which is involved protein translocation within the context of the cell, in addition to being involved in protein degradation. We have demonstrated small-molecule carboxy-terminal derived small-molecule peptide fragments can serve as modest substrates in the ubiquitination cascade, in vitro. We further demonstrated that small-molecule substrates can be optimized to increase catalytic efficiency and desired structural characteristics.Peptide "stapling" involves covalent cross-linking of peptide side chains with an aim to stabilize peptide secondary structures. Herein we have reported the first use of a photoinduced 1,3-dipolar cycloaddition reaction in stapling peptide helices. We found this stapling chemistry resulted in minimal structural perturbation, and in one case reinforcement, of a model 310 helical peptide in trifluoroethanol. Fluorescent microscopy studies indicated that one stapled peptide was able to permeate the HeLa cell membrane. Given the encouraging data in our model system, we are extending our stapling chemistry to prepare biologically active stapled peptides targeting the protein-protein interactions implicated in cancers, such as the p53-Mdm2/Mdmx interactions. The results of 'peptide stapling' in PDI (peptide dual inhibitor) based compounds afforded excellent in vitro ELISA data and modest p53 dependent activation in vivo.