Synthesis and biophysical characterization of stabilized α-helices of BCL-2 domains

Rational design of compounds to mimic the functional domains of BCL-2 family proteins requires chemical reproduction of the biologic complexity afforded by the relatively large and folded surfaces of BCL-2 homology (BH) domain peptide a-helices. Because the intermolecular handshakes of BCL-2 proteins are so critical to controlling cellular fate, we undertook the development of a toolbox of peptidic ligands that harness the natural potency and specificity of BH a-helices to interrogate and potentially medicate the deregulated apoptotic pathways of human disease. To overcome the classic deficiencies of peptide reagents, including loss of bioactive structure in solution, rapid proteolytic degradation in vivo, and cellular impermeability, we developed a new class of compounds based on hydrocarbon stapling of BH3 death domain peptides. Here we describe the chemical synthesis of Stabilized Alpha-Helices of BCL-2 domains or SAHBs, and the analytical methods used to characterize their secondary structure, proteotytic stability, and cellular penetrance.