This study investigated the diastereoselective synthesis of three dipeptide templates 1, 2 and 3, which may be regarded as conformationally restricted analogs of H-Gly-Xaa-OH, in which Xaa constitutes an aromatic amino acid. Bond formation between alpha-C of Gly and the aromatic moiety was achieved by proton-catalyzed intramolecular electrophilic aromatic substitution. The absolute configuration of the dipeptide templates was determined by single-crystal X-ray crystallography or by nuclear Overhauser enhancement measurements. A protective group strategy was elaborated to allow their incorporation into peptide sequences by liquid phase as well as by solid-phase peptide synthesis. The templates were used to generate an enkephalin analog 15, a modified peptidic neurokinin antagonist 20 and two dermorphin derivatives (24 and 33). Molecular dynamic simulations with 15 and 20 revealed the preference for a turn-like motif for 15. The biological activity, as investigated by respective receptor binding and functional assays, was strongly diminished with all four derivatives, indicating that their receptor-relevant molecular geometries lie outside the examined conformational space.

A discontinuous 17-amino acid peptide analog of neuropeptide Y (NPY), NPY 1-4-Ahx-25-36 containing 6-aminohexanoic acid instead of the residues 5 to 24, was found to bind preferentially to Y2 subtypes of NPY receptors. In order to further characterize the binding site, three different types of cyclic analogs were synthesized. Firstly lactamisation between residues 2 and 30 led to the most selective Y2-agonist, secondly lactamisation between the N-terminus and residue 31 reduced binding significantly. Thirdly, any cyclization including the C-terminus led to an inactive compound. Circular dichroism revealed different conformations for the three analogs with reduced alpha-helical content in comparison to the linear ana-log. The different conformation of the peptides has been confirmed by molecular dynamics simulations. A model for peptide-receptor interaction is suggested.

C-terminal analogues of neuropeptide Y have been synthesized. The influence of chain length, single-amino-acid substitutions and segment substitutions on receptor binding, biological activity and conformational properties has been investigated. Receptor binding and in vivo assays revealed biological activity already for amino acids 28-36 of neuropeptide Y [neuropeptide Y-(Ac-28-36)-peptide] which increased with increasing chain length. Replacement of Arg25 in neuropeptide Y-(Ac-25-36)-peptide had no influence on binding, whereas Arg33 and Arg35 cannot be replaced by lysine or ornithine without considerable decrease in receptor binding. The introduction of conformational constraints by the 2-aminoisobutyric acid residue (Aib) in position 30 and replacing the amino acids 28-32 by Ala-Aib-Ala-Aib-Ala decreased receptor binding. However, the corresponding Aib-Ala-Aib-Ala-Aib-substituted analogue and a more flexible analogue with Gly5 at position 28-32 exhibited considerable affinity for the receptor. All these substitutions led to a decrease in postsynaptic activity. Strong agonistic activities could be detected in a series of 10 discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was neuropeptide Y amino acids 1-4 linked to amino acids 25-36 through aminohexanoic acid (Ahx) [neuropeptide Y-(1-4-Ahx-25-36)-peptide].

Analogues of neuropeptide Y (NPY) containing small N- and C-terminal segments linked via flexible spacer arms were found to exhibit receptor binding affinity constants almost as high as NPY as well as post- and presynaptic NPY-agonistic activities. One of the most active analogues contains N-terminal NPY segment 1-4 linked via epsilon-aminocaproic acid (Aca) to the C-terminal partially alpha-helical peptide amide segment 25-36. NPY 1-4-Aca-25-36 is the first highly potent NPY agonist, which is of considerably reduced size in comparison to the native hormone. The analogues are accessible by solid-phase synthesis using Fmoc strategy.