Identification of Catalytic Peptide Dendrimers by "Off-Bead" in Silica High-Throughput Screening of Combinatorial Libraries

A combinatorial library of up to 65'536 peptide dendrimers ((AcXX7)-X-8)(8)(DapX(6)X(5))(4)(DapX(4)X(3))(2)DapX(2)X(1) (Dap = (S)-2,3-diaminopropionic acid branching point, X8-1 = groups of four proteinogenic L-amino acids) was prepared on a photocleavable tentagel resin. The library was assayed for catalytic hydrolysis of the fluorogenic substrate 1-butyryloxy-pyrene-2,7,8-trisulfonate 1 and analogs by a simple procedure involving (a) photocleavage from the support in the absence of solvent, (b) spreading of the solid Support beads on the Surface of a silicagel plate impregnated with an aqueous buffered substrate solution, and (c) identification of hits as beads surrounded by a fluorescent halo indicative of catalysis and sequence determination in hits and nonhits by amino acid analysis of the beads. The experiment provides direct access to structure-activity relationships in the library and delivers active esterase dendrimers. Anionic glutamate residues in the outer dendrimer branches were found to inhibit catalysis by histidine residues at the dendrimer core. The "off-bead" in silica assay is simple to implement and transferable to other library and reaction types.