Dendrimers are branched synthetic macromolecules. This protocol describes the synthesis (1-2 weeks), functional screening (1.5 d) and decoding (2 d) of 'one-bead-one-compound' combinatorial libraries of dendrimers assembled from amino-acid building blocks by 'split-and-mix' solid phase peptide synthesis. The method resembles that for synthesizing linear peptides, except that a branching diamino acid is used at every third position to obtain the dendritic structure. Structural diversification by splitting is restricted to four amino acids per variable position, yielding libraries of approximately 60,000 sequences. In such libraries, the sequence of a dendrimer can be deduced uniquely from an amino-acid analysis of the solid support bead. This analysis is more reliable, faster and far less costly than Edman sequencing such that decoding multiple beads is affordable. The method is exemplified for the identification of catalytic peptide dendrimers catalyzing the hydrolysis of acyloxypyrene-trisulfonates with substrate binding (K(M) = 10-300 microM) and rate accelerations up to k(cat)/k(uncat) = 10(4) in aqueous buffer.
Exploring the structural diversity of peptide dendrimers as synthetic protein models: A 65 536‐membered combinatorial peptide‐dendrimer library was prepared by split‐and‐mix techniques on beads (see picture). The library was screened and revealed peptide dendrimers that catalyze fluorogenic ester hydrolysis and peptide dendrimers that bind to vitamin B12.
(3S)-4-(9-Fluorenylmethoxycarbonylamino)-3-methyl(allyloxycarbonyl)aminoethyloxyacetic acid (1) was prepared from (R)-3-aminopropane-1,2-diol and used as branching unit for the synthesis of second generation peptide dendrimers with six individually addressable variable amino acid positions. Three pairs of diastereomeric dendrimers were prepared bearing a common hydrophobic core and permutations of the catalytic triad amino acids aspartate, histidine and serine at the surface. Dendrimers with two surface histidine residues catalyzed the hydrolysis of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonates in aqueous buffer pH 6.0 with rate enhancement k cat /k uncat in the 10 3 range and Michaelis-Menten constants K M in the 10 À 4 M range. Substrate recognition involves electrostatic interactions, as shown by competitive inhibition of catalysis observed with pyrene-1,3,6,8-tetrasulfonate. The 4-fold to 7-fold lowering in K M between the butyryl and nonanoyl esters in the most active dendrimers provides evidence for a hydrophobic component in substrate binding, which is absent in a closely related, less active diastereomeric peptide dendrimer.
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.
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