Structural requirements for the biosynthesis of backbone cyclic peptide libraries

Background: Combinatorial methods for the production of molecular libraries are an important source of ligand diversity for chemical biology. Syntheti c methods focus on the production of small molecules that must traverse the cell membrane to elicit a response. Genetic methods enable intracellular l igand production, but products must typically be large molecules in order t o withstand cellular catabolism. Here we describe an intein-based approach to biosynthesis of backbone cyclic peptide libraries that combines the stre ngths of synthetic and genetic methods. Results: Through site-directed mutagenesis we show that the DnaE intein fro m Synechoeystis sp. PCC6803 is very promiscuous with respect to peptide sub strate composition, and can generate cyclic products ranging from four to n ine amino acids. Libraries with five variable amino acids and either one or four fixed residues bwere prepared, yielding between 10(7) and 10(8) trans formants. The majority of randomly selected clones from each library gave c yclic products. Conclusions: We have developed a versatile method for producing intracellul ar libraries of small, stable cyclic peptides. Genetic encoding enables fac ile manipulation of vast numbers of compounds, while low molecular weight e nsures ready pharmacophore identification. The demonstrated flexibility of the method towards both peptide length and composition makes it a valuable addition to existing methods for generating ligand diversity. (C) 2001 Else vier Science Ltd. All rights reserved.