Combinatorial chemistry provides a powerful method for the rapid identification of high affinity ligands of macromolecular targets. In this work, we recognize three principles of combinatorial chemistry, which are the development of a combinatorial library, the screening of that library against biological targets, and the sequence determination of hits from those screenings. The majority of our work focuses on the sequence determination of support-bound “hit” peptides and peptidomimetics. Our work also focuses on the development of new peptidomimetic libraries, the efficiency of peptide cyclization, and the sequence determination of those novel groups of compounds.
We have developed a method to sequence support-bound peptides by partial Edman degradation and mass spectrometry (PED/MS). Briefly, support-bound peptides are subjected to a mixture of phenyl isothiocyanate (PITC) and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSU), which allows for the majority of the support-bound peptides to be degraded by Edman reaction chemistry but allows a small percentage of peptides to become capped by the Fmoc group. Following reaction with PITC and Fmoc-OSU, a trifluoroacetic acid (TFA) mediated cyclization occurs resulting in the cleavage of the N-terminal amino acid. However, since a small portion remains capped in each cycle of degradation, at the conclusion of all cycles there is a ladder of full-length peptide and capped truncation products on the resin. The Fmoc group is easily removed and the peptides are cleaved from the resin and analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). MS data shows a series of peaks with the mass difference of adjacent peaks corresponding to the amino acid sequence. This technology allows for greater than 95% sequencing success rate and costs approximately $1 US per peptide. We have also applied this technology to successfully sequence support-bound peptoid and peptide-peptoid hybrid libraries by making modifications to the sequencing conditions.
We have also successfully synthesized and sequenced support-bound cyclic peptoids, a novel class of compounds. By using a one-bead, two-compound approach, we have spatially segregated TentaGel microbeads (90 um) and synthesized a cyclic peptoid on the bead exterior to be used for biological screenings and a linear peptoid on the bead interior for sequencing by PED/MS. Cyclic peptoid libraries were used to investigate potential protein inhibitors and determine streptavidin ligands.
Our final work includes a comprehensive look at the factors affecting peptide cyclization via N to C macrolactamization. Work in this area has been limited due to a lack of technology to examine large numbers of combinatorially derived peptides. We synthesized five peptide libraries of varying number of random positions and monitored the efficiency of cyclization as affected by ring size and sequence composition. Utilizing a biotin-based screening assay, we were able to visualize fast and slow cyclizing peptides and sequence them using our PED/MS methodology to determine a general consensus for cyclization efficiency.