Synthesis and Opioid Activities of [D-Leu⁸]Dynorphin(1-8) Analogs Containing a Reduced Peptide Bond, Ψ(CH₂NH)

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[D-Leu⁸]Dynorphin(1-8)-NH₂ analogs, in which each peptide bond was systematically replaced with a ψ(CH₂NH) peptide bond, were synthesized by the solid-phase method. The ψ(CH₂NH) bond was introduced by the Boc-amino acid aldehyde/NaCNBH₃ method on a solid support. In the syntheses of the analogs, undesirable double alkylation took place at the sequences of Tyr¹ψ(CH₂NH)Gly² and Gly²ψ(CH₂NH)Gly³, possible due to the low steric hindrance of the glycine residue. To suppress the double alkylaiton, a minimum amount of aldehydes was employed. In the receptor binding assay, the ψ(CH₂NH) replacement of N-terminal peptide bonds which led to ¹ψ²- (2) and ²ψ³-analogs (3) resulted in a marked reduction in binding affinities for μ-, δ-, and κ-opioid receptors, while that of the other peptide bonds afforded analogs with a high κ-receptor affinity. A ³ψ⁴-analog (4) showed extremely high κ-receptor selectivity (μ/κ K_i ratio=339, δ/κ K_i ratio=24104). In the in vitro bioactivity assay (guinea pig ileum assay), 2 showed a very low IC₅₀ ratio (2.0) in the presence and absence of peptidase inhibitors whereas those of other analogs were >27, suggesting that the introduction of the CH₂NH isostere at Tyr¹-Gly² greatly enhanced the enzymatic stability of the parent peptide. Furthermore, analogs 2 and 3 showed a very low sensitivity to the inhibitory effect of NaCl plus 5'-guanylylimidodiphosphate of their binding at a κ-receptor site as compared with the other analogs and the parent peptide. These results suggest that the two analogs (2 and 3) have partial κ-antagonist properties.