Photogenerated reagents and light-directed parallel synthesis of peptide microarrays

Peptides are biological compounds of considerable importance. In the forthcoming proteomics era, miniaturized, spatially addressable peptide microarrays will be of tremendous value for high-throughput peptide-protein interaction analysis. However, such peptide microarrays, especially peptide microchips of cm2 sizes, are currently beyond reach.; In this work, a novel chemistry for the parallel synthesis of peptides using photogenerated reagents and digital photolithography was investigated. Our in situ synthesis approach is based on Solid Phase Peptide Synthesis with in-solution removal of standard protecting groups using photogenerated reagent precursors that are activated by irradiation with light. These compounds are used as optical switches allowing for parallel reactions to occur only upon irradiation.; The synthesis and characterization of photogenerated acid, base, and hydrazine precursors was performed. These reagents were used for the deprotection of the protecting groups t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, dimethoxytrityl, and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl. The efficiency of the reactions was investigated on conventional solid phase supports as well as on glass plates. The synthesis of peptide microarrays containing 2304 sequences demonstrated that this approach is highly efficient and compatible with conventional amino acid monomers. Synthesis cycles of less than 20 minutes were used and stepwise yields of 98% were achieved.; The success of our light-directed parallel synthesis strategy was validated by showing that a library of variant peptides and peptidomimetics from an epitope of the human p53 protein could be synthesized on a microarray and show strong and specific affinities for the p53 antibody PAb240. Our microarray results, unambiguously confirmed by comparison to those of phage display, identified the known RHSVV epitope sequence. Through mutational analysis and the introduction of non-natural amino acids, peptidomimetic antibody-binding sequences were found and unique properties of the binding site were uncovered.