Novel Techniques for the Synthesis and Coupling of Peptide Segments

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This research is being funded by the Organic Synthesis Chemistry Program. New techniques for the synthesis of naturally occurring medium or long chain peptides are being developed which will make them rapidly available in multi-gram quantities for testing purposes. New synthetic techniques based on base- and solvent-sensitive amino-protecting groups promise to make available quickly multi-gram quantities of shorter peptides and to markedly improve the synthesis of small quantities of longer peptides. Methods for the synthesis of long peptide chains of 100 or more amino acid units which involve the coupling of segments enjoy significant advantages over those involving stepwise approaches. Three methods for assembling the appropriate segments will be studied: (a) the FMOC/Polyamine solution technique, (b) the two polymer ("inverse merrifield") approach and (c) the standard Merrifield solid phase method. In all three approaches the applicability of newly-discovered FMOC amino acid fluorides as rapid-acting, inexpensive coupling agents will be studied. Such reagents are now available for all but four of the common amino acids. Synthesis of appropriate acid fluoride reagents for these four compounds (Arg, Asn, Gln, His) will be developed. To facilitate solid phase syntheses two special resin supports will be investigated: (a) tertiary alcohol resins which should completely inhibit loss of peptide from the resin due to diketopiperazine formation at the depeptide stage, especially in the case of C-terminal or penultimate proline or glycine and (b) resin supports bearing tertiary aminoalcohol functions which should serve to catalyse loading of the first amino acid and thereby promote complete on-line automated syntheses from the first to the last amino acid. Once rapid, routine methods for the synthesis of protected segments are available, both solution and solid phase methods of segment coupling will be examined. A novel method of effecting racemization-free segment coupling involving protection of the C-terminal amide bond by FM-bar or a related amide protectant will be pursued. The model chosen to study the application of these techniques is a 19-unit segment of a silkworm prothoracicotropic hormone, PTTH-II-(1-19).