Synthesis and Screening of an Indexed Motif-Library Containing Non-proteinogenic Amino Acids

Abstract

In an effort to increase the probability of finding novel peptides in resin-bound combinatorial libraries displaying affinity to various macromolecular targets, we increased the diversity of a solid-phase library considerably by synthesizing multiple structures on each bead - a motif-library - including 45 building blocks. The building blocks consist of L-aa, D-aa and eight hydrophobic non-proteinogenic alpha-amino acids. A library with the format O-Z0-1-O-Z0-1-O-XX-resin was synthesized giving the four motifs OOOXX, OZOOXX, OOZOXX, OZOZOXX corresponding to 364.500 different motifs (45(3) x 4 theoretical combinations). The positions O are defined amino acids while Z represents three mixtures pi, omega, phi, where pi is a mixture of polar and charged residues, omega is a mixture of aliphatic residues and phi is a mixture of aromatic residues. X represents a mixture of all 45 residues. The library was screened with the macromolecular target streptavidin which served as a model receptor. Binding peptides were sequenced by microsequencing. We included small amounts of norvaline and norleucine in the library, which served as index residues to be able to distinguish between LD-amino acids and other residues with the same retention time in the HPLC system. Beads that interact with the receptor were found, and the binding motifs that appeared had no homology to known binding motifs found in either L-aa or D-aa libraries, instead motifs with the non-proteinogenic residues L-phenylglycine, O-benzyl-L-hydroxyproline and O-benzyl-L-tyrosine dominated. The novel peptides inhibit binding of biotin to streptavidin but do not bind to avidin, and the affinity is higher than the peptides found in linear all L-aa peptide libraries.

Silymarin Decreases Connective Tissue Growth Factor to Improve Liver Fibrosis in Rats Treated with Carbon Tetrachloride

“…There is no doubt that silymarin has anti-oxidant-like activity, and this finding is consistent with previous reports (Lang et al, 1993;Kiruthiga et al, 2010). Hydroxyproline is a common non-proteinogenic amino acid (Ostergaard and Holm, 1997). It is found only in collagen and elastin in mammals but exists in a number of other proteins in plants.…”

“…Hydroxyproline is a common non‐proteinogenic amino acid (Ostergaard and Holm, ). It is found only in collagen and elastin in mammals but exists in a number of other proteins in plants.…”

Identification of NCAM‐binding peptides promoting neurite outgrowth via a heterotrimeric G‐protein‐coupled pathway

“…The fractions of acidic and basic amino acid residues are close to the average values for soluble proteins, whereas the fraction of aromatic residues is higher than for soluble protein and comparable with that of trans ‐membrane spanning sequences (Stevens and Arkin 2000). A high fraction of aromatic residues is common in binding peptides derived from combinatorial libraries (Østergaard and Holm 1997). Nevertheless, the amino acid composition of the 10 peptides is very different from that seen in the NCAM‐binding peptides identified in previous studies with combinatorial peptide libraries that contained a large fraction of basic residues and low fractions of acidic and aromatic residues (Rønn et al.…”