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Abstract

The standard method of peptide library synthesis involves coupling steps in which a single amino acid is reacted with a mixture of resin-bound amino acids. The more recently described positional scanning strategy (in which each position in the peptide sequence is occupied in turn by a single residue) is different since it involves the coupling of mixtures of amino acids to mixtures of resin-bound amino acids. In the present study, we analyze the compounds produced under these conditions measuring coupling rates and amounts of formed products, using mainly UV, HPLC, LC/MS and MS/MS techniques. Our data do not permit to conclude that the resulting libraries are complete. Indeed, our analytical data indicate that a large part of the di-, tri- and tetrapeptides synthesized with this method are not present in the final mixture. Although chemical compensation (in which poor coupling kinetics is compensated by a larger excess of the incoming amino acid) has been thought to counterbalance these biases, our experiments show that the compensation method does not take into account the crucial influence of the resin-bound amino acid and that even the dipeptide libraries obtained in this way are far from completeness. The present work provides strong evidence that the coupling of mixtures of amino acids to resin-bound residues, which is required by the positional scanning strategy, results in incomplete and/or non-equimolar libraries. It also clearly confirms that coupling rates in solid-phase peptide synthesis are dependent on the nature of both the incoming and the immobilized amino acid.

Assessment of Protein-tyrosine Phosphatase 1B Substrate Specificity Using “Inverse Alanine Scanning”

“…Unfortunately, classic combinatorial library methods do not readily lend themselves to detailed kinetic analyses, especially at the individual member level. We ruled out the one bead/one compound approach (15) as well as the positional scanning method (16,17) due to difficulties associated with quantitatively measuring kinetic constants in either the solid state or on nonequimolar mixtures of Tyr(P) peptides (18). Although these combinatorial methods cannot be used to assess the substrate efficacy of individual library members, both approaches generate a large number of peptides and therefore furnish a comprehensive appraisal of the diversity space associated with the substrate binding pocket on the target enzyme(s).…”

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

“…These mixtures were white to yellowish powders that could be handled in the assay, the same way as if they were discrete chemicals. They were analyzed using MS and MS/MS techniques (40,45,46) providing enough information to ensure (i) the lack of major by-products in the mixture and (ii) the presence of the accounted structures in the sublibraries. Furthermore, in each sublibrary, individual amino acids were indeed found to be present in roughly equal amounts by two-dimensional NMR (40).…”

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography–mass spectrometry and ion mobility mass spectrometry

“…As a matter of fact, extremely different pharmacokinetic (PK) data have been reported in all these studies and the reality is not clear. It is also uncertain what diosmetin metabolites are circulating in body fluids; a single work on identification of diosmetin conjugates in animal samples has been published [14], and another paper mentions, without methodological details, quantitative measurements of diosmetin-3- O -glucuronide (3- O -Gluc) in human plasma [13] (chemical formulas of the possible glucuronide metabolites can be seen in Fig. 1).…”