Improved synthesis of DA364, an NIR fluorescence RGD probe targeting αvß3 integrin.

Optical imaging (OI) is gaining increasing attention in medicine as a non-invasive diagnostic imaging technology and as a useful tool for image-guided surgery. OI exploits the light emitted in the near-infrared region by fluorescent molecules able to penetrate living tissues. Cyanines are an important class of fluorescent molecules and by their conjugation to peptides it is possible to achieve optical imaging of tumours by selective targeting. We report here the improvements obtained in the synthesis of DA364, a small fluorescent probe (1.5 kDa) prepared by conjugation of pentamethine cyanine Cy5.5 to an RGD peptidomimetic, which can target tumour cells overexpressing integrin αvß3 receptors.

Bacterial transferase MraY inhibitors: synthesis and biological evaluation.

New inhibitors of the bacterial transferase MraY are described. Their structure is based on an aminoribosyl-O-uridine like scaffold, readily obtained in two key steps. The amino group can be coupled with proline or guanylated. Alternatively, these amino, prolinyl or guanidinyl groups can be introduced through a triazole linker. Biological evaluation of these compounds on MraY from Bacillus subtilis revealed interesting inhibitory activity for both amino compounds.

Combinatorial libraries of peptide dendrimers: design, synthesis, on-bead high-throughput screening, bead decoding and characterization.

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.

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.