Synthesis of [alpha-methyltyrosine-4]angiotensin II: studies of its conformation, pressor activity, and mode of enzymatic degradation.

Abstract

Modifications in angiotensin II and its antagonistic peptides that should have increased in vivo half-lives but not reduced biological activity were studied by determining the effect of a-methylation of the tyrosine in position 4. angiotensin II, synthesized by the solid-phase procedure, showed 92.6 ± 5.3% pressor activity of angiotensin H. Incubation with a-chymotrypsin for 1 hr indicated absence of degradation although, under the same conditions, angiotensin H was completely degraded to two components. Comparison of the 'H NMR spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [a-methyltyrosine-4]angiotensin II suggested that a methylation of the tyrosine residue in angiotensin HI does not lead to major changes in the overall solution conformation. These results are in contrast to those obtained with N-methylation in position 4, which drastically reduced the biological activity and produced remarkable changes in the peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize potent angiotensin II analogs that have greater resistance to enzymatic degradation by a-methylation in position 4 (or 5) and simultaneous suitable modification at the NH2 and COOH termini.The antagonists for the pressor hormone angiotensin II (AspArg-Val-Tyr-Ile-His-Pro-Phe; All) have proved useful in the study ofexperimental hypertension and as possible new clinical diagnostic tools (1). However, their long-term application has been limited because they have a short in vivo half-life and, due to degradation by peptidases, are not orally active. We therefore attempted to make these peptides resistant to this enzymatic degradation by replacing the natural amino acid residues with N-methylamino (2) or /3homoamino (3) acid residues. These modifications drastically reduced the biological activity ofthese analogs, and conformation studies suggest that N-methylation in positions 4 or 5 results in remarkable changes in the peptide backbone and a severe limitation in the rotational freedom of the side chains in tyrosine, isoleucine, and histidine residues (4). The replacement ofan a proton by an a methyl group, however, is thought to produce minimal changes in backbone and side-chain structures. The analogs thus obtained might mimic the parent hormone in recognizing and binding with the receptor on the cell membrane and, in addition, be stable to enzymatic degradation (4, 5). Based on this hypothesis, we have synthesized [a-methyltyrosine4]angiotensin II ([a-MeTyr4]AII).And, indeed, this peptide is resistant to chymotrypsin degradation and yet retains almost the full pressor activity of All. Conformation studies suggest minimum changes in backbone and side-chain structures. These results again suggest that, for full biological activity (agonist or antagonist), the backbone and side-chain structure of the analog should resemble that of the hormone, All (2, 4). These observations may be helpful in the design of new...

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