Analogues of the competitive angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (sarmesin) in which the sarcosine-1, O-methyltyrosine-4, and phenylalanine-8 residues were modified have been synthesized by the solid-phase method. The agonist and antagonist potencies of the 23 peptides synthesized were determined in the rat isolated uterus assay. At position 1, replacement of Sar with Asp, Ala, or Pro gave inactive analogues, and deletion of the N-terminal amino acid produced inactive heptapeptides for all analogues investigated. At position 4, substitution of Tyr with Tyr(Et), D-Tyr, D-Phe, Ile, Thr, or Hyp resulted in inactive analogues, whereas substitution of Phe gave a potent competitive antagonist (pA2 = 7.9), which retained significant agonist activity (22%). For position 8, [Sar1,Tyr(Me)4,Ile8]ANG II and [Sar1,Phe4,Ile8]ANG II were weaker antagonists (pA2 = 6.6 and 6.7, respectively) than [Sar1,Ile8]ANG II (pA2 apparent = 8.1) and, moreover, were reversible competitive antagonists. These findings demonstrate that the structural requirements for receptor blockade by sarmesin are remarkably stringent--modifications at positions 1, 4, and 8 markedly reduce the antagonist activity of this peptide.

Analogues of angiotensin II and III (ANG II and ANG III) in which the tyrosine and/or phenylalanine residues were substituted have been synthesized by the solid-phase method and purified by (carboxymethyl)cellulose chromatography and reversed-phase HPLC. The antagonist and agonist potencies of these peptides were determined in the rat isolated uterus assay. [Sar1,Tyr(Me)4]ANG II, [Tyr(Me)3]ANG III, [Sar1,D-Trp4]ANG II, [D-Trp3]ANG III, [Sar1,D-Trp8]ANG II, [D-Trp7]ANG III, [Sar1,Tyr(Me)4,Ile8]ANG II, [Tyr(Me)3,Ile7]ANG III, [Sar1,D-Trp4,Ile8]ANG II, [D-Trp3,Ile7]ANG III, [Sar1,Tyr(Me)4,D-Trp8]ANG II, and [Tyr(Me)3,D-Trp7]ANG III had antagonist activities (pA2) respectively of 8.1, less than 6, less than 6, less than 6, (7.7), (6.7), 7.2, less than 6, less than 6, less than 6, 7.1, and less than 6. The agonist activity of each peptide was less than 0.1% of that of ANG II. Analogues in which only the Phe residue was substituted were not readily reversible in the bioassay, whereas analogues in which only the Tyr residue or both the Tyr and Phe residues were substituted were reversible antagonists. Peptides that were twice substituted had lower antagonist activities than peptides having a single aromatic residue substitution. Substitution of the Tyr residue in ANG II, but not ANG III, provides a new route for the synthesis of potent and competitive angiotensin antagonists. Differences in the biological properties of ANG II and ANG III analogues substituted at the Tyr residue suggest different binding/conformation requirements for the two endogenous ligands at angiotensin receptors in smooth muscle.

We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N, N, N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manud or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.

The desensitizing potencies of angiotensin II (ANG II) analogues modified at positions 1, 2, 4, 7, and 8 have been examined in the rat isolated uterus assay by determining the time of recovery of the half-maximal concentration (EC50) response to angiotensin II after treatment of the tissues with a high dose (10(-5) M) of each analogue for 2 min. The magnitude of the desensitization effect was substituent dependent in the following manner: position 1, sarcosine (Sar) greater than Asp greater than des-Asp; position 2, Arg greater than Sar; position 4, Tyr greater than Tyr(Me) approximately Phe; position 7, 3,4-dehydroproline (Dpr) greater than Pro greater than thioproline (Tpr) greater than Sar; position 8, Ile greater than D-Trp greater than Ala greater than Phe. The "additivity" rule applied to these structure-desensitization relationships and the most potent desensitizer, requiring 3 h for reestablishment of the EC50 response, was [Sar1, Dpr7, Ile8]-ANG II. The desensitizing potencies of these analogues did not correlate with agonist or antagonist activities and demonstrated that the angiotensin-mediated tissue desensitization process has unique structural determinants. Methylation or elimination of the tyrosine hydroxyl group of strong desensitizers virtually eliminated the desensitization effect, implicating the phenoxyl moiety in the mechanism of desensitization. The initial phase of recovery of angiotensin responsiveness after desensitization by several analogues appeared to obey first-order kinetics. The results are discussed in the contexts of both one- and two-site receptor models.

Analogues of the competitive angiotensin antagonist [Sar1,Tyr(ME)4]angiotensin II (sarmesin) with modifications at the N-terminus have been prepared by the solid-phase method and purified by reversed-phase HPLC. Substitution of the Sar1 residue of sarmesin with N,N-dimethyl-Gly, N-ethyl-Gly, aminoisobutyric, (methylamino)isobutyric, aminocaproic, and oxamic acids gave analogues that had the following respective antagonist activities (pA2) in the rat isolated uterus assay: less than 6, 6.9, 5.5, 6.0, less than 6, and 5.3. The additional substitution of Ile for Phe at the C-terminus of the latter four peptides gave pA2 values of 7.1, 5.1, less than 5, and 5. Substitution of the Arg2 residue of sarmesin with Nle or Sar abolished antagonist activity. These data emphasize the stringent and discriminating structural requirements in the N-terminal domain of sarmesin that endow this analogue with its antagonist properties and suggest the presence of defined steric constraints in this region of the molecule during receptor blockade.