The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine(6)-oxytocin and penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs.

Six Pen(6)]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, Mpa(1)]oxytocin, dPen(1)]oxytocin, 5-t-BuPro(7)]oxytocin, Mpa(1), 5-t-BuPro(7)]oxytocin and dPen(1), 5-t-BuPro(7)]oxytocin. When tested in the uterotonic test in vitro Pen(6)]oxytocin, Pen(6), 5-t-BuPro(7)]oxytocin, Mpa(1), Pen(6)]oxytocin and Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. dPen(1), Pen(6)]Oxytocin and dPen(1), Pen(6), 5-t-BuPro(7)]oxytocin were found to be pure antagonists with similarly high pA2 values of approximately 8.2. Replacement of proline by 5-tert-butylproline increased binding affinity by a factor of two in Pen(6)]oxytocin and had no influence on the binding affinity of Mpa(1), Pen(6)]oxytocin and dPen(1), Pen(6)]oxytocin. Assignment of the proton signals for prolyl amide cis- and trans-isomers by NMR experiments in water indicated that the Pen(6)-5-tert-BuPro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-tert-butylproline(7) analogs of Pen(6)]oxytocin, Mpa(1), Pen(6)]oxytocin and dPen(1), Pen(6)]oxytocin. This augmentation in cis-isomer population was correlated with a 21-fold reduction in the agonistic potency and 2-fold augmentation in antagonistic potency for Pen(6), 5-t-BuPro(7)]oxytocin relative to Pen(6)]oxytocin. Augmentation of cis-isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of Mpa(1), Pen(6)]oxytocin to Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. In the potent oxytocin antagonist, dPen(1), Pen(6)]oxytocin, substitution of 5-tert-butylproline for proline augmented the cis-isomer population without affecting antagonistic potency. The synthesis and evaluation of Pen(6)]oxytocin and Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.