Synthesis and activity of histidine-containing catalytic peptide dendrimers

Peptide dendrimers built by iteration of the diamino acid dendron Dap-His-Ser ( His) histidine, Ser = Serine, Dap = diamino propionic acid) display a strong positive dendritic effect for the catalytic hydrolysis of 8-acyloxypyrene 1,3,6-trisulfonates, which proceeds with enzyme-like kinetics in aqueous medium ( Delort, E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2004, 126, 15642-3). Thirty-two mutants of the original third generation dendrimer A3 (( Ac-His-Ser)(8)( Dap-His-Ser)(4)( Dap-His-Ser)(2)Dap-His-Ser-NH2) were prepared by manual synthesis or by automated synthesis with use of a Chemspeed PSW1100 peptide synthesizer. Dendrimer catalysis was specific for 8-acyloxypyrene 1,3,6-trisulfonates, and there was no activity with other types of esters. While dendrimers with hydrophobic residues at the core and histidine residues at the surface only showed weak activity, exchanging serine residues in dendrimer A3 against alanine ( A3A), beta-alanine ( A3B), or threonine ( A3C) improved catalytic efficiency. Substrate binding was correlated with the total number of histidines per dendrimer, with an average of three histidines per substrate binding site. The catalytic rate constant k(cat) depended on the placement of histidines within the dendrimers and the nature of the other amino acid residues. The fastest catalyst was the threonine mutant A3C (( Ac-His-Thr)(8)( Dap-His-Thr)(4)( Dap-His-Thr)(2)Dap-His-Thr-NH2), with k(cat) = 1.3 min(-1), k(cat)/ k(uncat) = 90'000, K-M = 160 mu M for 8-bytyryloxypyrene 1,3,6-trisulfonate, corresponding to a rate acceleration of 18'000 per catalytic site and a 5-fold improvement over the original sequence A3.