Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, raman, CD spectroscopy and molecular modeling

Abstract

The synthesis and biological evaluation of penicillamine 6 -5-tert-butylproline 7 -oxytocin analogs and comparison with their proline 7 -oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen 1 ,Pen 6 ]-oxytocin (1, pA 2 = 8.22, EC 50 = 6.0 nM) and [dPen 1 ,Pen 6 ,5-tBuPro 7 ]-oxytocin (2, pA 2 = 8.19, EC 50 = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using 1 H NMR, laser Raman and CD techniques. In H 2 O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln 4 and Pen 6 in the major trans-conformer of 1 as well as for Gln 4 in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 ± 0.2 kcal/mol) for isomerization of the amide N -terminal to 5-tBuPro 7 relative to OT was calculated from measuring the coalescence temperature of the Gly 9 backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C β -S-S-C β dihedral angles of +110-115°and ±90°, respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a χ CS−SC dihedral angle value close to 115°was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a ±90°angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a ±90°χ CS−SC dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114°and +116°. Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104°. In sum, the conformation about the disulfide in [dPen 1 ,Pen 6 ]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen 1 ,Pen 6 ,5-tBuPro 7 ]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro 7 (5R)-tert-butyl group, the Leu 8 isopropyl sidechain and the Pen 6 β-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110°may be a prerequisite for their binding at the receptor.

A Procedure for Oxytocin Measurement in Hair of Pig: Analytical Validation and a Pilot Application

“…It is important to point out that these results are only valid for our experimental conditions and specific assay and should be validated with other types of sample extraction or assays. Methanol has been used as a solvent for oxytocin with the advantage of not producing any conformational changes in this analyte [16] and have also been used for studies of oxytocin extraction from milk [17]. Although acetonitrile has been used for oxytocin extraction in some samples, such as saliva [3], previous studies do not recommend acetonitrile as extractor medium for selected drugs because of the low efficiency of extraction [14].…”

Conformations of a model cyclic hexapeptide, CYIQNC:1H-NMR and molecular dynamics studies

“…OT hormone has been known to be of the topic of immense investigation in lieu of its biological functions such as the maintenance of labor and milk ejection in mammals (Gimpl & Fahrenholz, 2001), facilitating the sexual maturation (Parent et al, 2008), stimulating the pre-ovulatory luteinizing hormone surge (Rettori, Canteros, Renoso, Gimeno, & McCann, 1997), and also its biomedical application such as in the treatment of autism (Newschaffer et al, 2007). Toward understanding the biological activities of OT, various works have been reported to be carried out for studying the interactions of OT with neurophysin (Blumenstein, Hruby, Viswanatha, & Chaturvedi, 1984;Eubanks, Lu, Peyton, & Breslow, 1999;Live, Cowburn, & Breslow, 1987;Rose, Wu, Hsiao, Breslow, & Wang, 1996) and with receptor (Bélec, Blankenship, & Lubell, 2005;Budesínsky, Procházka, & Slaninová, 2005;Carnazzi et al, 2001;Lebl et al, 1990;Lubecka, Sikorska, Marcinkowska, & Ciarkowski, 2014;Sikorska & Kwiatkowska, 2013;Turner, Matsoukas, & Moore, 1990).…”