Effect of modification of enkephalin C-terminal functions on affinity selection of opioid receptors

Enkephalin analogs with various C-terminal modifications have been synthesized to evaluate the corresponding structural elements in the opioid receptors. The carboxyl group of the C-terminal leucine5 or glycine5 was converted into the mercaptomethyl (-CH2SH) and hydroxymethyl (-CH2OH) groups, starting from leucinthiol or leucinol for Leu5-derivatives and from cysteamine or ethanolamine for Gly5-derivatives. Interactions of synthetic peptides with the opioid receptors were examined by the radioligand receptor binding assays using rat brain and tritiated enkephalin analogs. The data suggest that the C-terminal carboxyl group in enkephalins is important, but not electrostatically, for interaction with delta-opioid receptors. With leucinthiol-enkephalin in biological assays which examine its inhibitory activity for electrically stimulated contractions of isolated smooth muscle, it was found that the reactive thiol group exists in the mu receptors present in the guinea pig ileum. Leucinthiol-enkephalin became bound covalently to this receptor-thiol group via disulfide formation after prolonged incubation.