Macrocyclic peptides (MCPs) hold great promise as therapeutics due to their ability to target protein-protein interactions and other challenging biological targets with high affinity and selectivity. Despite their potential, a major limitation to their broader use in drug discovery is the lack of large and diverse MCP libraries suitable for high-throughput screening (HTS). At the same time, with thousands of unique commercially available building blocks, the number of synthetically accessible macrocyclic hexapeptides is greater than 10^18 – a number far too large to comprehensively screen in functional assays. We developed BRiTeCycle to enable the sparse synthetic sampling of this 10^18 space, followed by the rapid exploration of the chemical space around any primary hits. BRiTeCycle uses a novel spatially addressed, microfluidics-based synthesis approach that has broad chemical compatibility and high stepwise efficiencies. To assess BRiTeCycle, we synthesized a 2,304-membered library of cyclic hexapeptides, in quadruplicate. Sixty amino acid building blocks were incorporated into the library. The purity of 116 samples (5% of the total) was assessed from 220 nm UPLC chromatograms. Between replicates, crude LC/MS traces are highly reproducible, suggesting reproducible robustness in our synthetic approach. In addition to the desired cyclic hexapeptide, a common side-product was the corresponding cyclic dodecapeptide that results from the cyclic dimerization of two linear hexapeptide precursors. Linear product was rarely observed. To further assess the membrane permeability of these MCPs, we applied Parallel Artificial Membrane Permeability Assay (PAMPA) to a subset of the library, which supported the membrane permeable nature of our MCPs. This work demonstrates that the BRiTeCycle platform can efficiently synthesize large libraries of diverse, cyclic hexapeptides, enabling high-throughput screening and characterization for novel drug candidates.
