Aza-amino acid scanning of secondary structure suited for solid-phase peptide synthesis with Fmoc chemistry and aza-amino acids with heteroatomic side chains

Aza-peptides, peptide analogues in which the (x-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit a propensity for adopting P-turn conformations. A general Fmoc-protection protocol for the stepwise solid-phase synthesis of aza-peptides has now been developed based on the activation of N'-alkyl fluoren-9-ylmethyl carbazates with phosgene for coupling the aza-amino acid residues. This method has proven effective for introducing aza-amino acid residues with aliphatic (Ala, Leu, Val, and Gly) and aromatic (Phe, Tyr, and Trp) side chains. Acid promoted loss of aromatic side chains was noted with aza-Trp and aza-Tyr residues during peptide cleavage and suppressed by temperature control in the case of the latter. In addition, aza-peptides with heteroatomic side chain residues (Lys, Orn, Arg, and Asp) were conveniently synthesized using this protocol. Partial aza-amino acid scans were performed on three biologically active peptides: the potent tetrapeptide melanocortin receptor agonist, Ac-HiS-D-Phe-Arg-Trp-NH2; the growth hormone secretagogue hexapeptide, GHRP-6, HiS-D-Trp-Ala-Trp-D-Phe-Lys-NH2; and the human calcitonin gene-related peptide (hCGRP) antagonist, FVPTDVGPFAF-NH2. This practical procedure for aza-amino acid scanning using Fmoc-based solid-phase synthesis should find general utility for probing the existence and importance of U-turn conformations in bioactive peptides.