Cyclopeptides have become the subject of much investigation as possible therapeutic leads, specifically in the inhibition of intracellular protein-protein interactions (PPIs) which have been challenging to target with typical small molecular inhibitors. We plan to identify a mutant-specific, high affinity macrocyclic peptide inhibitor for the KRas(G12V)−Sos PPI through the development of a one-bead one-compound (OBOC) tetrazine-cyclized peptide library for screening against KRas(G12V) and wtKRas. This study focuses on the development of a facile one-step reaction to prepare tetrazine macrocycle post-screen “hits” for tandem mass spectrometry and de novo sequencing, following the OBOC library screen against the target PPI. Tandem mass spectrometry (MS/MS) is a fast and reliable approach to peptide identification and sequencing; however, it is considerably more challenging when applied to cyclic peptides. Because cyclic peptides typically result in internal ions, the already complex task of interpreting the spectra is made even more complicated as peptides of length k are characterized by k distinct ion series. In order to address this, we developed a one-step reaction that allows for simultaneous peptide linearization and cleavage. This is done through the incorporation of Fmoc-(R, S)-3-amino-3-(2-nitrophenyl)propionic acid to the resin prior to synthesis, which acts as a photolabile linker which can be cleaved through irradiation of UVA light in a simple lab apparatus, which simultaneously linearizes the peptide at the photocleavable tetrazine staple. We have shown the applicability of this reaction to a panel of peptides with varying charges, hydrophobicities, and steric hindrances, which we have been able to identify and sequence through MS/MS and de novo sequencing in our collaboration with AbbVie.

Development, characterization, and cleavage of on-bead linked cyclic peptides for expedient sequencing

ACS Spring Meeting 2021

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