Most of our drugs derive from natural products, many more natural products possess biological activity but our inability to synthesise novel analogues hampers our ability to use them either as tools or medicines. Cyclic peptides are common structural motifs in natural products and medicines (vancomycin, gramicidin). They are widely recognised to constitute a promising and still underexploited class of molecule for novel therapeutics; specifically an important role for cyclic peptides in the inhibition of protein-protein interactions has been demonstrated. We will harness the power of the recently identified macrocyclases from the ribosomally-derived cyanobactin superfamily to prepare diverse modified cyclic peptides. These enzymes exhibit the remarkable ability to macrocyclise unactivated peptide substrates. Different members of this family of macrocyclases process peptides into macrocycles containing from six up to twenty residues. We have characterised and re-engineered one member of the family (PatG) which makes eight residue macrocycles. We will determine the structural and biochemical features of the macrocyclases that are known to lead to six or to twenty residue macrocycles. We will use these insights to put these enzymes to work in novel chemical reactions. We will combine macrocyclases with other enzymes from the cyanobactin biosynthetic pathways (whose structures and mechanism we have largely determined) and work on solid phase peptide substrates. By bringing together the power of solid phase methods (split and pool) and the novel chemistry enabled by the enzymes, we will generate highly diverse macrocyclic scaffolds containing amino acids, enzymatically modified amino acids, non-natural amino acids and non-amino acid building blocks. Successful completion of the project will revolutionise the design of cyclic peptide-inspired libraries with diverse backbone scaffolds for applications in target identification, drug discovery and tool screening.