Ribosomal Synthesis of Macrocyclic Peptides with β- and β-Homo-Amino Acids for the Development of Natural Product-Like Combinatorial Libraries.

The development of large, natural-product-like, combinatorial macrocyclic peptide libraries is essential in the quest to develop therapeutics for "undruggable" cellular targets. Herein we report the ribosomal synthesis of macrocyclic peptides containing one or more β-homo-amino acids (βhaa) to enable their incorporation into mRNA display-based selection libraries. We confirmed the compatibility of 14 β-homo-amino acids, ()- and ()-stereochemistry, for single incorporation into a macrocyclic peptide with low to high translation efficiency. Interestingly, N-methylation of the backbone amide of βhaa prevented the incorporation of this amino acid subclass by the ribosome. Additionally, we designed and incorporated several α,β-disubstituted β-homo-amino acids (βhaa) with different R-groups on the α- and β-carbons of the same amino acid. Incorporation of these βhaa enables increased diversity in a single position of a macrocyclic peptide without significantly increasing the overall molecular weight, which is an important consideration for passive cell permeability. We also successfully incorporated multiple ()-βhAla into a single macrocycle with other non-proteinogenic amino acids, confirming that this class of β-amino acid is suitable for development of large scale macrocyclic peptide libraries.