Pseudoproline-containing analogues of morphiceptin and endomorphin-2: Evidence for a cis Tyr-Pro amide bond in the bioactive conformation

Analogues of the opioid peptides [D-Phe(3)]morphiceptin (H-Tyr-Pro-D-Phe-Pr o-NH2) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) containing the pseudoproli ne (Psi Pro) (4R)-thiazolidine-4-carboxylic acid (CyS[Psi (R1,R2) pro]) or (4S)-oxazolidine-4-carboxylic acid (Ser[Psi (R1,R2)pro]) in place of Pro(2) were synthesized. The pseudoproline ring in these compounds was either uns ubstituted (R-1, R-2 = H) or dimethylated (R-1, R-2 = CH3) at the 2-C posit ion. 2-C-dimethylated pseudoprolines are known to be quantitative or nearly quantitative inducers of the Cis conformation around the Xaa(i-1)-Xaa(i)[P si (CH3,CH3)pro] imide bond. All dihydropseudoproline-containing analogues (R1, R2 = H) showed goody opioid agonist potency in the guinea pig ileum (G PI) assay, high mu receptor binding affinity in the rat brain membrane bind ing assay, and, like their parent peptides, excellent mu receptor binding s electivity. H-1 NMR spectroscopic analysis of the Cys[Psi (H,H)pro](2)- and Ser[Psi (H,H)pro](2)-containing analogues in DMSO-d(6) revealed that they existed in a conformational equilibrium around the Tyr-Xaa[Psi (H,H)pro] pe ptide bond with cis/trans ratios of 40:60 and 45:55, respectively. The dime thylated thiazolidine- and oxazolidine-containing [D-Phe3]morphiceptin- and endomorphin-2 analogues (R-1, R-2 = CH3) all retained full mu agonist pote ncy in the GPI assay and displayed mu receptor binding affinities in the na nomolar range and high mu receptor selectivity. As expected, no conformers of the latter analogues with a trans conformation around the Tyr-Xaa[Psi (C H3,CH3)pro] imide bond were detected by H-1 NMR spectral analysis, indicati ng that in these compounds the cis conformation is highly predominant (> 98 %). These results represent the most direct evidence obtained so far to ind icate that morphiceptin and endomorphin-2 have the cis conformation around the Tyr-Pro peptide bond in their bioactive conformations.