Limitations of the coupling of amino acid mixtures for the preparation of equimolar peptide libraries.

The standard method of peptide library synthesisinvolves coupling steps in which a single amino acidis reacted with a mixture of resin-bound amino acids.The more recently described positional scanningstrategy (in which each position in the peptide sequence is occupied in turn by a single residue) isdifferent since it involves the coupling of mixturesof amino acids to mixtures of resin-bound amino acids.In the present study, we analyze the compoundsproduced under these conditions measuring couplingrates and amounts of formed products, using mainly UV,HPLC, LC/MS and MS/MS techniques. Our data do notpermit to conclude that the resulting libraries arecomplete. Indeed, our analytical data indicate that alarge part of the di-, tri- and tetrapeptidessynthesized with this method are not present in thefinal mixture. Although chemical compensation (inwhich poor coupling kinetics is compensated by alarger excess of the incoming amino acid) has beenthought to counterbalance these biases, ourexperiments show that the compensation method does nottake into account the crucial influence of theresin-bound amino acid and that even the dipeptidelibraries obtained in this way are far fromcompleteness. The present work provides strong evidence that the coupling of mixtures of amino acidsto resin-bound residues, which is required by thepositional scanning strategy, results in incompleteand/or non-equimolar libraries. It also clearlyconfirms that coupling rates in solid-phase peptidesynthesis are dependent on the nature of both theincoming and the immobilized amino acid.