Due to their restricted conformational flexibility, cyclic peptides are of great interest in connection with structure-activity relationships, especially the elucidation of bioactive conformations. For linear peptides that do not contain turn structure-inducing amino acid residues, the cyclization reaction may be an inherently improbable or slow process, and side reactions, such as cyclodimerization and epimerization at the C-terminal residue, may dominate. A number of 1-hydroxy-7-azabenzotriazole-based onium salts were examined for cyclization of thymopentin-derived pentapeptides and the results compared with data from more conventional coupling reagents. The azabenzotriazol-derived coupling reagents stood out as being the most effective by far. The cyclizations proceed extremely rapidly, and in contrast to other coupling reagents, C-terminal epimerization was generally less than 10%. C-terminal D-amino acid residues favor the formation of monocyclic pentapeptide rings. A similar effect was observed for cyclization of linear N-methylamino acid-containing peptides, suggesting that reversible amide bond alkylation such as Hmb-modification should be useful in promoting the cyclization of pepitdes devoid of turn-inducing amino acid residues.

The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N-and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible ⑀-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC 50 : 100 -300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC 50 : 0.6 -50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.

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The differences in the conformational properties of ovine (o) and human (h) CRH in aqueous solution, structure‐inducing TFE and in the presence of detergent micelles and lipid vesicles have been investigated by circular dichroism, Fourier transform infrared spectroscopy, NMR and dynamic light scattering. o‐CRH was found to exist as a monomer with little regular structure in dilute aqueous solution. Association at concentrations higher than 10−3 mol/L results predominantly in dimers. The induction of a substantial amount of intermolecular β‐structure seems to be the result of interactions of the C‐terminal hexapeptide and the N‐terminal region 6‐12 of o‐CRH chains in antiparallel orientation. In contrast, h‐CRH exhibits a high tendency of association which is highly sensitive to the pH. The formation of tetramers at millimolar peptide concentration is related to a helical content of ca. 50%. The potentially helical, highly hydrophobic region 6‐20 enlarged by more hydrophobic residues in position 23 and 25 is proposed to stabilize the h‐CRH associates. In the presence of structure inducing TFE (<40%v) both CRH peptides exist as monomers. o‐CRH reveals about 72% helicity, in h‐CRH the formation of about 85% helix is observed. The differences in helicity of the two CRH molecules are located in the C‐terminal heptapeptide, as concluded on the basis of NMR studies. Both peptides bind to detergent micelles at pH 4 as well as 7.4 associated with an increase in the α‐helical content. Interaction of the two peptides with DMPC vesicles was found exclusively at pH 4. Above the phase transition temperature of DMPC the α‐helical content in h‐CRH increases slightly; however, o‐CRH reveals a substantial amount of β‐type structure. The intramolecular type of β‐structure is associated with a deeper insertion of the o‐CRH region 6‐12 into the hydrophobic region of the lipid bilayer, whereas the corresponding region of h‐CRH is kept in the bilayer surface. The higher helicity of h‐CRH might explain to some extent its higher affinity to the CRH receptor, CRH antibodies and the CRH binding protein. © Munksgaard 1996.