CONFORMATIONALLY CONSTRAINED ANALOGUES OF THE NEUROHYPOPHYSEAL HORMONE OXYTOCIN.

Abstract

The synthesis of seventeen novel conformationally constrained analogues of the neurohypophyseal peptide hormone oxytocin is described. Synthesis of the peptides was accomplished using solid-phase synthesis techniques on either Merrifield or p-methyl-benzhydrylamine resin. Cleavage of peptides from the solid support and deprotection were carried out by either ammonolysis followed by treatment with sodium in liquid ammonia or anhydrous HF. Disulfide formation was accomplished by treatment of the deprotected peptide with aqueous potassium ferricyanide. Purification of the peptide analogues involved a combination of either partition and/or size exclusion chromatography followed by reverse-phase high-performance liquid chromatography. Several conformationally constrained unnatural amino acids were incorporated into the synthetic peptides. Two were prepared and incorporated as a mixture of isomers and the resulting peptides were separated and purified by HPLC. The types of analogues prepared fall into three categories: analogues incorporating conformational restrictions in positions 1 and 2; bicyclic oxytocin peptides; oxytocin antagonists with changes at the Asn⁵ residue. The peptides with conformational restrictions at position 1 or 2 are: [Tic²]OT, [DTic²]OT, [DTic²,Thr⁴]OT, [β-MePhe²]OT, [ΔPhe²]OT, [Cys(CH₂)₅¹,Phe²,Thr⁴,Orn⁸]OT and [Pen¹,DPhe²,Thr⁴,Orn⁸]OT. Bicyclic peptide analogues and their monocyclic precursors include: [Mpa¹,Lys⁴,Glu⁵]OT, [Mpa¹,Lys⁴,Glu⁵]OT, [Mpa¹,Glu⁴,Lys⁸]OT, and [Mpa¹,Glu⁴,Lys⁸]OT. Antagonists with changes in the Asn⁵ residue are: [Pen¹,DPhe²,Thr⁴,Thr⁵,Orn⁸]OT; [Pen¹,DPhe²,Thr⁴,Leu⁵,Orn⁸]OT; [Pen¹,DPhe²,Thr⁴,Asp⁵,Orn⁸]OT; and [Pen¹,DPhe²,Thr⁴,Tyr⁵,Orn⁸]OT. Biological assays of these analogues for oxytocic activity in the rat uterus model have shown one of the β-MePhe²-containing peptides, [L-threo-β-MePhe²]OT, to be a very potent agonist and one bicyclic, [Mpa¹,Glu⁴,Lys⁸]OT to be an extremely potent oxytocin antagonist. Initial biophysical investigations employing 250 MHz nuclear magnetic resonance spectroscopy were also undertaken in order to determine possible solution conformations of these peptide analogues.