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The cyclic tetrapeptide tentoxin and the conformationally related analog [Pro1] tentoxin have been synthesized by a new solution methodology. D, L-3-phenyl-serine was employed as a synthetic precursor to the Z-dehydrophenylalanine substituent. Increased yields of both compounds were obtained when a modified cyclization procedure was employed through ring closure from the N-terminal substituents of N-methylalanine and proline to a C-terminal glycine. Biological activities were determined in a seedling assay used to measure chlorosis induction. Both tentoxin and [Pro1] tentoxin exhibited similar chlorosis inducing activity. Whole leaf extracts of chlorotic, [Pro1] tentoxin-treated seedling leaves lacked active polyphenoloxidase when subjected to electrophoretic analysis. Coupling factor1 (CF1) ATPase isozymes were assayed for ATPase activity in 10μuM-100 μuM solutions of tentoxin and [Pro1] tentoxin. CF1 ATPase inhibition was observed for both tentoxin and [Pro1] tentoxin. Inhibition of a single ATPase isozyme by tentoxin was alleviated at or above 50μuM while [Pro1] tentoxin inhibited two CF1 ATPases at concentrations up to 110μuM. No alleviation of ATPase inhibition was noted for higher concentrations of [Pro1] tentoxin.

The peptide bond in the 4-5 position of the cyclic and linear enkephalin analogs H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] and H-Tyr-D-Ala-Gly-Phe-L(or D)-Leu-OH was replaced by a thiomethylene ether linkage. Each of the configurational isomers of the cyclic pseudopeptide H-Tyr-cyclo[-D-Lys-Gly-Phe psi [CH2S]L(or D)-Leu-] showed high potency in both the guinea pig ileum and the mouse vas deferens assay and, therefore, had no preference for either mu- or delta-opioid receptors, in contrast to the cyclic parent peptides H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] which are mu-receptor selective. The loss of selectivity observed with the cyclic pseudopeptides may be due to the greater flexibility of their 18-membered ring structures as a consequence of the peptide bond substitution. The linear pseudopeptide analogs were both less potent and less delta-receptor selective than their parent compounds. These results indicate that thiomethylene ether peptide bond replacements can have a pronounced effect on the activity profile of peptide hormones and neurotransmitters.

An isomeric series of four leucine-enkephalin analogs containing the thiomethylene ether unit as an amide bond replacement in all positions have been prepared by solid phase methods. The resulting pseudopeptides divulged widely differing retentive behaviors on reversed phase high performance liquid chromatography (HPLC). An analog containing the Pheψ[CH 2 S]Leu dipeptide replacement at the 4–5 position exhibited binding close to the parent, leucine enkephalin; its guinea pig ileum (GPI) activity was the highest of the analous tested. Another compound, Tyrψ[CH 2 S]Gly 1–2 ]-Leu-enkephalin, also displaced 3 H-etorphine well in the binding assay, but caused increased contractions in the GPI assay at low concentrations. The Pheψ[CH 2 S]Leu results are not compatible with the necessity of a β-turn structure for agonist activity in the GPI assay.