In position 7 l- and d-Tic-substituted oxytocin and deamino oxytocin: NMR study and conformational insights

Abstract

Incorporation of L- or D-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa(1)]OT) resulted in four analogues, [L-Tic(7)]OT (1), [D-Tic(7)]OT (2), [Mpa(1),L-Tic(7)]OT (3) and [Mpa(1),D-Tic(7)]OT (4). Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799-806, 2007). Their NMR study (NOESY, TOCSY, (1)H-(13)C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC(50) value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of D analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys(6)-D-Tic(7) bond is supported by observation of two sets of cross-peaks for (1)H and (13)C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in (1)H-(13)C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.

Revealing the Relationship between Electric Fields and the Conformation of Oxytocin Using Quasi-Static Amide-I Two-Dimensional Infrared Spectra

“…In conclusion, when the electric fields are antiparallel to the X-axis, the OT molecules have longer γ-turns including five amino acids and stronger hydrogen bonds between Tyr2 and Asn5, indicating the tighter secondary spatial structure of the OT molecules. The different residues in position 1 play an important role in the nature of the disulfide bond which is between Mpr1 and Cys6, 16,43 and its stability is studied under electric fields based on Mayer bond order density (BOD). 58,64 The nature of Mayer BOD corresponds to the delocalization index in the Hibert atomic space, which is more comprehensive for investigating the stability of covalent bonds, and the number of Mayer BOD is favorable to the stability of covalent bonds.…”

“…The initial geometry of an OT molecule is taken from the X-ray structure (PDB ID: 1xy2, 43 Figure 1). The replacement of Cys1 with Mpr1, which is a nonprotein residue and is less bulky than Cys1, has been shown to affect the chemical environment and consequently the nature of the disulfide bond linking residues 1 and 6, 16,22 and the backbone carboxyl groups of Cys6 and Pro7 are labeled with 13 C 18 O, which has about a 60 cm −1 red shift. 24,34−36 The geometry optimization and anharmonic frequency calculation of OT are carried out using the B3LYP (D3) functional and 6-31G (d, p) basis set as implemented in the Gaussian 16 program, 44−49 where the SMD continuum water model is included.…”

“…Then, they reported the dynamics of cis/trans isomerization of the Cys6-Pro7 peptide bonds of OT in aqueous and methanol solutions and found that no polar resonance structures were possible in the transition state and the rate of cis/trans interconversion was faster in nonaqueous solvents . Wittelsberger et al confirmed that cis conformations between Cys6-Pro7 of OT did not lead to antagonistic activity, and a cis/trans conformational change played an important role in OT receptor binding and activation. − There are several research studies focusing on the spectra of OT, too. Plinska et al reported and compared the terahertz time-domain spectroscopy investigation on crystalline forms of both OT and derivatives, and the results might be helpful to identify unique biologic signatures of OT .…”

“…The cis/trans conformations of OT have been widely investigated using 2D NMR spectroscopy. − Quasi-static amide-I two-dimensional infrared (2D IR) spectra can be viewed as an extended version of 2D NMR spectroscopy, , which contain lots of detailed information to elucidate the protein three-dimensional structures by the inherent sensitivity to the mechanisms of coupling and energy transfer in intermolecular and intramolecular aspects. − In proteins, the amide-I vibrational normal mode usually consists of a CO stretching vibration with weaker contributions from the amide N–H wag. The vibration has a large transition dipole moment, leading to strong IR activity, and is delocalized over many residues, which gives a detailed structural fingerprint of the protein backbone. − Quasi-static amide-I 2D IR spectra in combination with isotope labeling methods have been extensively used to identify misfolded proteins and get access to the mechanism of the process. ,− …”

“…Structure–activity studies concern the structural features leading to agonism or antagonism of the OT effect on uterus prompted based on the cis/trans conformation, ,− and it is reported that electric fields of specific intensity and frequency can excite certain vibrational modes of proteins, which alters their conformations. − Therefore, in this paper, we theoretically study the response of the OT conformation to electric fields using quasi-static amide-I 2D IR spectra in combination with isotope labeling methods. On one hand, we aim to study hydrogen bonds among different residues because OT has an obvious β-turn geometry. − ,− ,, On the other hand, based on the Cys6-Pro7 isomer, the cis/trans conformations of OT are detected using 2D IR spectra under different electric fields. − …”