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

Abstract

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...