Design and Synthesis of DNA-Encoded Libraries of Unnatural Peptides to Discover Specific Chemical Probes

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Project SummaryVast libraries of encoded peptides, including cyclic systems, can be synthesised using biological methods, such as ribosomal expression systems, and can be screened by affinity selection to produce high affinity protein ligands. However, the scope of the aforementioned biological methods is usually limited to proteinogenic amino acids and their close analogues. The incorporation of unnatural moieties would allow for the more specific targeting of selected proteins classes, potentially leading to compounds with greater proteolytic stability and cell permeability. The aim of the proposed project is to develop synthetic approaches to realise this goal by developing chemical, rather than biological, techniques for peptide library synthesis. These techniques lack the requirement of identification by biological machinery and can, therefore, incorporate a far wider range of building blocks. In a second strand, hybrid methodology will be developed which allows the incorporation of unnatural warheads for selected protein classes into biologically displayed peptide libraries.Chemical Synthesis of Encoded Peptide LibrariesDELs are a technology for the synthesis and screening of a large number of molecules. Each DEL is a collection of these molecules, which are comprised of an organic moiety linked to a DNA oligonucleotide. The "genotype" (the DNA tag) serves as an amplifiable identification bar code for the displayed "phenotype" (the small organic molecule).Peptoids are oligomers of N-alkylated glycines; a peptoid monomer is an amino acid with the side chain connected to the nitrogen of the backbone, rather than the alpha carbon. Peptoids offer certain advantages to peptides as drugs, incl. resistance to proteolytic degradation due to the tertiary amide, improved cell-permeability relative to peptides due to the absence of the amide NH for H-bonding, and side chain diversity as they can be theoretically synthesised from any given primary amine.Peptoids are most often synthesised via a solid-supported submonomer method, using bromoacetic acid and then a primary amine. This is an issue, as an ?-halo carbonyl is a powerful electrophile, so will alkylate the DNA, so this method is incompatible with DEL synthesis.Initial work will focus on the off-DNA synthesis of peptoid monomers and the optimisation of multiple routes towards these building blocks. Consequently, optimised conditions will be applied to a variety of amines (to afford a range of peptoid monomers) and the scope of the reactions elucidated. Concurrently, the on-DNA coupling of peptoid monomers will be established using micellar conditions. This will be developed to realise the scope of the couplings by testing the coupling of the aforementioned synthesised peptoid monomers on-DNA.This will develop, in further work, to the synthesis of a range of peptoid-containing libraries, beginning with a small linear peptoid library, comprised of a range of the synthesised monomers. Extension of this will lead to the incorporation of building blocks that allow for the control of peptoid conformation, developing into the inclusion of conformational locks and cyclic structures.Hybrid Biological and Chemical SynthesisBiological techniques allow for the synthesis of even larger libraries than DELs, pushing the limit of library size to near 1013. The associated work in this strand will revolve around the incorporation of unnatural motifs into cyclic peptide libraries from ribosomal expression. These unnatural motifs, such as a single bromophenylalanine residue, will allow for the coupling of partners, e.g. boronates for Suzuku coupling, with known motifs that bind to certain target proteins. The work will involve the synthesis of said peptide libraries, the coupling of the specific binding motifs, and the subsequent testing of the diverse cyclic peptide libraries.