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The conformationally constrained cyclic enkephalin analogs, [ 2- D - penicillamine , 5- L - cysteine ]- and [2- D - penicillamine , 5- D - cysteine]enkephalinamide were synthesized and their biological activities investigated. Both analogs effectively induced thermal analgesia as measured by the in vivo hot plate test. Both analogs were effective in inhibiting muscle contractions in the guinea pig ileum and mouse vas deferens assay systems and were shown to displace both [3H]naloxone and [3H] [ D - Ala 2 , D - Leu 5 ] enkephalin from rat brain receptor preparations. The analogs exhibited a significant preference for δ-receptors over μ-receptors, an unusual finding for enkephalinamide derivatives. In addition the 5- L - cysteine containing analog was more potent than the 5- D - cysteine analog in all the in vitro assays with the exception of the guinea pig ileum system. These uncommon results are attributed to the conformational constraints imposed by the cyclization via a disulfide and by the rigidizing effect of the penicillamine.

The rabbit reticulocyte heme-regulated eIF-2α kinase (HRI) utilizes adenosine-5′-0-(3-thiotriphosphate) (ATP-γ-S) as a substrate for its autophosphorylation and activation, and for the phosphorylation of eIF-2. The phosphorothioated binary complex [ eIF-2(α-[ 35 S]P)·GDP ], interacted with the reticulocyte reversing factor (RF) in in vitro assays, and inhibited the ability of RF to catalyze GDP exchange from (eIF-2·[3H]GDP) complexes. The phosphorothioate residue in the binary complex was resistant to phosphatase action under protein synthesis conditions. eIF-2(α-[ 35 S]P)·GDP inhibited protein synthesis in hemin-supplemented lysates with biphasic kinetics, but had no effect on protein synthesis in heme-deficient lysates. The data reported here indicate that phosphorylation of eIF-2·GDP alone, through the ability of eIF-2(α-P)·GDP to bind and sequester RF, is sufficient to inhibit protein chain initiation in the reticulocyte lysate.