Limitations of the coupling of amino acid mixtures for the preparation of equimolar peptide libraries
Jean A. Boutin, Isabelle Gesson,
Jean-Michel Henlin,
Jean-Luc Fauchere
Department of Peptide and Combinatorial Chemistry, Institut de
Recherches SERVIER, 11 Rue des Moulineaux, F-92150 Suresnes, France
Sophie Bertin, Pierre-Herve Lambert, Jean-Paul Volland
Department of Analytical and Physical Chemistry, Institut de
Recherches SERVIER, 11 Rue des Moulineaux, F-92150 Suresnes, France
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 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.
Keywords
analysis of libraries, coupling of mixtures, incomplete libraries, peptide
libraries, positional scanning
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