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The delta-opioid antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP-OH) or its C-terminal amide analogue was systematically modified topologically by substitution of each amino acid residue by all stereoisomers of the corresponding beta-methyl amino acid. The potency and selectivity (delta- vs mu- and kappa-opioid receptor) were evaluated by radioreceptor binding assays. Agonist or antagonist potency were assayed in the mouse vas deferens and in the guinea pig ileum. In the TIPP analogues containing L-beta-methyl amino acids the influence on delta-receptor affinity and on delta-antagonist potency is limited, the [(2S,3R)-beta-MePhe3]TIPP-OH analogue being among the most potent delta-antagonists reported. In the D-beta-methyl amino acid series, the [D-beta-MeTic2] analogues are delta-selective antagonists whereas [D-Tic2]TIPP-NH2 is a delta-agonist. NMR studies did not indicate any influence of the beta-methyl substituent on the conformation of the Tic residue. The [(2R,3S)-beta-MePhe3]TIPP-NH2 is a potent delta-agonist, its C-terminal carboxylic acid analogue being more delta-selective but displaying partial agonism in both the delta- and mu-bioassay. These results constitute further examples of a profound influence of beta-methyl substitution on the potency, selectivity, and signal transduction properties of a peptide.

The shortened analogue of insulin, des-(B26 -B30)-pentapeptide insulin, has been characterized by twodimensional 'H NMR. The 'H resonance assignments and the secondary structure in water solution are discussed. The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2-Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1There is a long standing interest in insulin on account of its extreme importance as a drug for the treatment of diabetes [l]. In particular, several recent studies have been reported on insulins modified by site-directed mutagenesis, which have altered aggregation properties [2]. We have started a highresolution NMR study of insulin with the aim of elucidating the solution structure of the hormone and of its various modified forms. Here we report the 'H resonance assignments for des-(B26 -B30)-pentapeptide insulin (DPI) and discuss some structural aspects. This should provide the basis for a full structure determination of DPI in solution and should facilitate the interpretation of the NMR spectra and structural comparisons of modified insulins.Insulin consists of two polypeptide chains, called the A and B chains, which are linked by two disulfide bridges (see Fig. 1). The crystal structures of insulin [3--81 and of the monomeric des-(B26 -B30)-pentapeptide insulin [9 -1 I] are known. From X-ray crystallography [12,13] and CD measurements [14], it was found that the insulin monomer can adopt a number of different conformations. Careful analysis of the different X-ray crystal forms showed that these conformations can be grouped into two classes which are sometimes referred to as molecule 1 and molecule 2 (Chinese nomenclature) [6]. The major differences between the two forms are found at the end of the B chain (B25-B30) and the first part of the A The shortened analogue DPI obtained by removal of five C-terminal amino acids from the B chain has a relative molecular mass of about 5200 and differs from insulin in several aspects. Normal insulin can occur in various aggregation states in solution : monomeric, dimeric, hexameric and even polymeric states have been identified by a variety of methods [3, 13, 151. However, it is known that DPI has much less tendency to form such aggregates. A rationale for this comes from the X-ray structure which shows that the residues B26 ~ B30 of two insulin monomers form an antiparallel P-sheet. In the blood, insulin is present in very low concentrations which ensures that it circulates and brings about its biological effects as a monomer [3]. Monomeric DPI still has full biological activity provided that the carboxy-terminus of the B chain is amidated [17].The crystal structure of DPI has been solved at a resolution of 0.12 nm [9]. Furthermore, MD simulations of DPI in the crystal have been reported [18]. Several au...