Combinatorial synthesis, selection, and properties of esterase peptide dendrimers

A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.