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)]oxyt ocin. 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 antagonisti c properties. Their agonistic potency ranged from negligible (0.08 IU/mg) t o low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to ra nge from 6.6 to 7.9. [dPen(1), Pen(6)]Oxytocin and [dPen(1), Pen(6), 5-t-Bu Pro(7)]oxytocin were found to be pure antagonists with similarly high pA2 v alues of approximate to8.2. Replacement of proline by 5-tert-butylproline i ncreased 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- a nd trans-isomers by NMR experiments in water indicated that the Pen(6)-5-te rt-BuPro(7) peptide bond cis-isomer population was augmented relative to th e prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-te rt-butylproline(7) analogs of [Pen(6)]oxytocin, [Mpa(1), Pen(6)]oxytocin an d [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 au gmentation in antagonistic potency for [Pen(6), 5-t-BuPro(7)]oxytocin relat ive to [Pen(6)]oxytocin. Augmentation of cis-isomer population was also cor related to reduced agonist potency without effect on antagonism on conversi on of [Mpa(1), Pen(6)]oxytocin to [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. I n the potent oxytocin antagonist, [dPen(1), Pen(6)]oxytocin, substitution o f 5-tert-butylproline for proline augmented the cis-isomer population witho ut affecting antagonistic potency. The synthesis and evaluation of (Pen(6)] oxytocin and [Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that ster ic interactions influenced agonist and antagonist activity by modifying pep tide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained a ntagonistic potency and binding affinity and reduced agonistic potency.