2822 zyxwvutsrqpon Biochemistry zyxwvu 1981, 20, 2822-2828 Conformational Study of the Potent Peptide Hormone Antagonist zyx [ 1 zyxwvutsr -Penicillamine,2-leucine]oxytocin in Aqueous Solution+ Henry I. Mosberg, Victor J. Hruby,* and Jean-Paul Meraldi ABSTRACT: [ 1 -Penicillamine,2-leucine] oxytocin is a confor- mationally restricted analogue of oxytoxin in which the half-cystine- 1 and tyrosine-2 residues of the native hormone are replaced by half-penicillamine @,B-dimethyl-half-cystine) and leucine, respectively. This analogue is a surprisingly potent oxytocin antagonist [Hruby, V. J., Deb, K. K., Yamamoto, D. M., Hadley, M. E., & Chan, W. Y. (1979) zyxwvut J. Med. Chem. 22, 71. Extensive proton magnetic resonance experimentswere performed to determine the conformational properties of this analogue in aqueous solution, and the results were compared with the previously published model for the conformation of [ 1-penicillamine]oxytocin. The results are consistent with a conformation similar to that of [ 1 -penicillamine]oxytocin except that, while [ 1-penicillamine]oxytocin in aqueous solu- tion possesses two l e 3 (C,) type turns involving the iso- leucine-3 peptide amide proton and the half-penicillamine- 1 A major goal in efforts to understand the chemical-physical basis for information transfer in biological messengers such as peptide hormones is to differentiate those conformational and dynamic features important to the interaction of the hormone with its receptor (the binding message) from those important to the biological response (the biological activity message). For this purpose, examination of the conformational and dynamic differences of peptide hormone agonists and antagonists should provide important insights (Meraldi et al., 1977; Hruby, 1980). This is because, while agonists such as the native hormone contain the necessary information to both bind to the receptor and transduce a biological effect (agonist activity), competitive inhibitors (antagonists) can bind to the receptor but lack some critical structural, conformational, and/or dynamic property necessary for biological activity. In previous investigations, we have examined and compared the conformational and dynamic properties in aqueous solution of the peptide hormone, oxytocin, H-Cys-Tyr-Ile-Gln-Asn- Cys-Pro-Leu-Gly-NH2, and related agonists, and the con- formationally restricted oxytocin inhibitor, [ l-penicill- amineloxytocin ([Pen'loxytocin), an analogue in which the half-cystine- 1 residue is replaced by a half-penicillamine (P,P-dimethylcysteine) residue.' Using high-resolution proton and carbon- 13 nuclear magnetic resonance (NMR) spec- I F + From the Departments of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721. zyxwvutsrqpo Received November 4, 1980. This research was supported by grants from the U S . Public Health Service (AM- 17420) and the National Science Foundation. Experiments per- formed on the Nicolet NT 360 N M R spectrometer were performed at the Purdue University Biochemical Magnetic Resonance Laboratory under the direction of Dr. J. Markley, which is supported by National Institutes of Health Grant RR-01077. We thank Dr. Markley for the use of this facility. Preliminary experiments on the Bruker HX-270 were performed at the Francis Bitter National Magnet Laboratory of the Massachusetts Institute of Technology which is supported by National Science Foundation Contract (2-670 and National Institutes of Health Grant RR-00995. carbonyl and the asparagine-5 peptide amide proton and the isoleucine-3 carbonyl, [ 1 -penicillamine,2-leucine]oxytocin has only the latter 1+-3 turn. This difference between the an- tagonists is reflected in the different 4 and zyx I ) angles in the three N-terminal residues of the two inhibitor analogues and in differences in the preferred side-chain conformations for several residues. One particular result of these conformational dif- ferences is that, whereas for [ 1 -penicillamine]oxytocin the tyrosine-2 side chain is unable to assume the rotamer for maximal binding to the uterine receptor, [ l-penicillamine,Z leucine]oxytoxin retains conformational and dynamic prop- erties at residues two and three which are more similar to those of oxytocin. It is postulated that these conformational and dynamic properties are consistent with the stronger binding and, hence, greater antagonist activity for this penicillamine analogue relative to [ 1 -penicillamine]oxytocin. troscopy (Meraldi et al., 1975, 1977; Hruby et al., 1979a), carbon-1 3 spin-lattice relaxation (T,) studies (Meraldi et al., 1977), and CD and laser Raman spectroscopy (Hruby et al., 1978) we showed that, while oxytocin had considerable con- formational and dynamic flexibility, [Pen']oxytocin was conformationally and dynamically more restricted. The spectroscopic data were consistent with a conformation for the 20-membered ring moiety of [Penl]oxytocin that possessed two 1+-3 intramoleculai hydrogen bonds (C, structures) (Meraldi et al., 1975, 1977; Hruby, 1980), a disulfide bond with a dihedral angle of 110-1 15O and right-handed chirality (Hruby et al., 1978), and a restricted Tyr2 side chain in which the rotamer that would place the aromatic side chain pointing toward the 20-membered disulfide ring was excluded. We suggested that the restricted dynamic and conformational properties were important for the antagonist properties of [Pen']oxytocin. As a further test of this model, we more recently prepared [Pen1,Leu2]oxytocin and found that it was a very potent oxytocin antagonist, much more potent than expected (Hruby et al., 1979b). Carbon-13 chemical shift and T1 measurements (Hruby et al., 1979b) and CD and laser Raman studies (Hruby et al., 1978) indicated that it had very similar conformational and dynamic properties to those of [Pen']oxytocin. In this paper, we further examine the con- formational properties of [Pen',Leu2]oxytocinby using proton NMR ('H NMR). The results are consistent with a con- formation in which [Pen1,Leu2]oxytocinretains a restricted conformation in the 20-membered disulfide ring similar to that previously proposed for [Pen']oxytocin, but with considerably more flexibility of the side chain at the 2 position and greater conformational restrictions at the 5 and 6 positions. These * Standard abbreviations and nomenclature for amino acids, peptides, and peptide derivatives are used throughout. All amino acids except glycine are of the L configuration. Other abbreviations used: NMR, nuclear magnetic resonance; CD, circular dichroism; 'H NMR, proton magnetic resonance; TI, spin-lattice (longitudinal) relaxation time; Pen, half-penicillamine. OOO6-2960/8 110420-2822$01.25/0 0 1981 American Chemical Society