Conformational Studies on Tocinamide and Deaminotocinamide by 220 MHz Nuclear Magnetic Resonance Spectroscopy

Brewster, Anne I.; Glasel, Jay A.; Hruby, Victor J.

doi:10.1073/pnas.69.6.1470

Cited by 18 publications

(17 citation statements)

References 25 publications

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“…The assignments of almost all of the proton resonances of oxytocin in DMSO have been reported (Johnson et al, 1969;Brewster et al, 1972Brewster et al, ,1973b. In these assignments, however, some contradiction between authors was apparent in the chemical shifts of the backbone amide protons (Johnson et al, 1969;Walter et al, 1972).…”

Section: Introductionmentioning

confidence: 91%

Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide

Kato

Endo²,

Fujiwara

et al. 1993

J Biomol NMR

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With the combined use of various two-dimensional (2D) NMR techniques, a complete assignment of the 1H and 13C resonances of oxytocin, Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2, for two molecular states, protonated and unprotonated at the N-terminal group, was performed in dimethyl sulfoxide. A small but distinct change in the backbone conformation of the six-residue cyclic moiety, associated with the protonation, was first suggested from those NMR parameters relevant to conformation, such as change with temperature in the chemical shifts of the peptide amide protons and changes in chemical shifts and homonuclear as well as heteronuclear three-bond coupling constants. The solution structures of oxytocin for the protonated and unprotonated forms were then calculated using distance analysis in dihedral-angle space, based on a relaxation matrix evaluated from quantitative NOE intensities at different mixing times. Total amounts of 93 and 105 distances were determined for the protonated and the unprotonated forms, respectively. There were 25 interresidue distances relevant to the structure of the cyclic moiety for the protonated form of oxytocin and 43 for the unprotonated form. Overall structures with the lowest target penalty function were similar between the two forms, having a beta-turn structure at the endocyclic residues of the Tyr-Ile-Gln-Asn moiety. The local backbone conformations near the N-terminus, however, were significantly different between the two forms. This was found to be due to a change in the dihedral angle of the disulfide bridge (chi ss around C-S-S-C), which closes the ring in the cyclic peptide. The dihedral angle was about +90 degrees for the unprotonated form and an intermediate value of about +45 degrees for the protonated form.

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Section: Introductionmentioning

confidence: 91%

Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide

Kato

Endo²,

Fujiwara

et al. 1993

J Biomol NMR

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“…Conformations have also been proposed for deamino-oxytocin and for several analogs of oxytocin (1,4). Since it is not known whether oxytocin performs its biological functions in the body in an aqueous or a nonaqueous environment, it is also important to investigate its conformational possibilities when it is dissolved in water.…”

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confidence: 99%

300-MHz Nuclear Magnetic Resonance Study of Oxytocin in Aqueous Solution: Conformational Implications

Brewster¹,

Hruby

1973

Proc. Natl. Acad. Sci. U.S.A.

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The 300-MHz nuclear magnetic resonance spectrum of the peptide hormone, oxytocin, in water, was obtained. Extensive nuclear magnetic resonance spindecoupling experiments, including those involving irradiation of the a-proton resonances under the water peak, studies using partially deuterated hormone derivatives, and nuclear magnetic resonance studies of some precursor peptides to oxytocin, permitted us Current interest in the structure-activity relationships of the neurohypophyseal hormone, oxytocin, has led to the proposal of a number of possible energetically favorable conformations for this molecule in dimethylsulfoxide solution (1-3). Conformations have also been proposed for deamino-oxytocin and for several analogs of oxytocin (1,4). Since it is not known whether oxytocin performs its biological functions in the body in an aqueous or a nonaqueous environment, it is also important to investigate its conformational possibilities when it is dissolved in water.Oxytocin is a nonapeptide with the following sequence:H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 L 2 3 4 5 6 1 7 8 9The cyclic hexapeptide portion is closed by a disulfide bridge.Whereas the a-and (3-proton resonances can be seen and assigned in deuterium oxide (D20) solution, the amide proton resonances are exchanged with deuterium and do not appear. In water (H20), the solvent peak obscures the a-proton peaks, making assignment of the peptide NH peaks extremely difficult. Feeney et al. (5) under the water peak and to assign the peptide NH resonances from the resultant spin-decoupling. In this paper we present our assignments for oxytocin and for [4-leucine]oxytocin and the conformational implications deduced from the nuclear magnetic resonance (NMR) spectra of these two molecules.

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“…The restoration of the characteristic biological activities of oxytocin and deamino-oxytocin on addition of the tripeptide tail, Pro-LeuGly-NH2, to the ring structure might be due to the addition of residues at positions 7 and 8 which, together with residues at positions 3 and 4, provide binding sites for interactions with the receptors. However, the effect of the interaction of the complete natural acyclic tripeptide with the ring moiety has also to be considered (9,(34)(35)(36)(37) (38), the "cooperative model" (9,35) for the biologically active conformation of oxytocin can no longer include an absolute requirement for hydrogen bonding between the carboxamide carbonyl of asparagine and the peptide N-H of leucine because the oxytocin analogs described herein can assume the conformation needed to evoke the characteristic biological activities of oxytocin-albeit with lower potency-even though this hydrogen bond cannot form.…”

Section: Resultsmentioning

confidence: 99%

Biofunctional evaluation of a hydrogen bond linking the ring and tail beta-turns of oxytocin.

Roy¹,

Roy²,

Gazis³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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Deamino{8-N-methylleucinejoxytocin and deamino{-a-hydroxyisocaproic acidjoxytocin were synthesized to study the importance of hydrogen bonding between the carboxamide carbonyl of asparagine and the peptide N-H of leucine in stabilizing the biologically active conformation of oxytocin. The analogs were synthesized by coupling deaminotocinoic acid with Pro-Leu(Me)-Gly-NH2 and Pro-HyIc-Gly-NH2, respectively. (HyIc is a-hydroxyisocaproic acid.) Deamino-8- (7)-have had extremely low or negligible potency with respect to all of the biological activities characteristic of the parent hormone. The asparagine residue has been assigned an important, if not unique, role in the maintenance of the conformation of oxytocin in solution (8, 9); namely, it has been proposed that a hydrogen bond between the peptide N-H of asparagine and the C=O of tyrosine stabilizes a 1-turn (comprising the sequence -Tyr-Ile-Gln-Asn-) in the cyclic part of oxytocin and the C='O of the asparagine carboxamide may be hydrogen bonded to the peptide N-H of leucine, thus stabilizing the relationship of this 1-turn to a second COOH-terminal 1-turn in the molecule composed of the sequence -Cys-ProLeu-Gly-NH2. The "cooperative model"-i.e., the "biologically active" conformation of oxytocin (9)-introduced to rationalize the changes in biological activities accompanying structural modification of neurohypophyseal peptides, suggests that any replacement of asparagine at position 5 that fails to contribute to the stabilization of the ring (-Tyr-Ile-Gln-Asn-) 1-turn or to the stabilization of the relationship of the ring and tail 1-turns to each other would increase the conformational ambiguity of the resultant analog with a consequent deterioration of all biological activities. As far as the hydrogen bond in the cyclic moiety of oxytocin is concerned, there should not be, in all probability, an absolute requirement for an asparagine residue in position 5-i.e., at least some of the other amino acid residues In the first compound, the hydrogen of the peptide N-H of leucine is replaced by a methyl group, and the analog is thus incapable of forming any hydrogen bond involving the C=O of the asparagine carboxamide. In the second analog, there is also no possibility for the formation of the hydrogen bond under discussion because the residues in positions 7 and 8 are now linked through an "ester" rather than an "amide," the proline residue in position 7 supplying the carbonyl portion of the ester. Both of the analogs were synthesized by condensation of deaminotocinoic acid (10), containing the preformed disulfide bridge of deamino-oxytocin, with the appropriate tail fraction of the respective analogs. The starting point in the synthesis of the tail fraction, Pro-Leu(Me)-Gly-NH2 of deamino-[8-Nmethylleucineloxytocin was the preparation of N-methylleucine or one of its suitable derivatives. Benzyloxycarbonylleucine was N-methylated with sodium hydride and methyl iodide in tetrahydrofuran at room temperature by the method of McDermott and Benoiton (11). This me...

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Conformational Studies on Tocinamide and Deaminotocinamide by 220 MHz Nuclear Magnetic Resonance Spectroscopy

Cited by 18 publications

References 25 publications

Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide

Oxytocin solution structure changes upon protonation of the N-terminus in dimethyl sulfoxide

300-MHz Nuclear Magnetic Resonance Study of Oxytocin in Aqueous Solution: Conformational Implications

Biofunctional evaluation of a hydrogen bond linking the ring and tail beta-turns of oxytocin.

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