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Endothelin-1 (ET), the most potent vasoconstrictor yet discovered, is a peptide containing 21 amino acids with two intrachain disulfide bridges. With the aim of obtaining two-chain derivatives, Et was submitted to chemical and enzymatic treatments. Reaction of ET with CNBr in 70% HCOOH gave, in addition to the expected [Hse7 lactone]-7,8-seco-ET and unreacted material, a by-product whose molecular weight was 25 m.u. greater than that of ET. When the reaction mixture, after lyophilisation, was immediately quenched with NH3-saturated dry MeOH, two products could be recovered in a 5:1 ratio, both obtained by nucleophilic attack of the homoserine lactone: the expected [Hse7-NH2]-7,8-seco-ET and [Hse7]ET, resulting from competitive intramolecular reaction of the deprotonated alpha-amino group of the Asp8 residue. The Lys9-Glu10 bond turned out to be very resistant to enzymatic attack both by Lys-C-endopeptidase and trypsin. The 9,10-seco-ET derivative could be obtained by treatment with Lys-C-endopeptidase only by using a high enzyme/ET ratio and after a prolonged incubation time. Cleavage of the Lys9-Glu10 bond could not be achieved by treatment with trypsin, even with a high enzyme/substrate ratio. The main product was 13,14-seco-ET, deriving from the action of chymotripsin (present as an impurity in the trypsin preparation) on Tyr13. The structure of these peptides was confirmed by amino-acid sequence analysis and fast atom bombardment mass spectrometry (FAB-MS). Nicking of the ET structure at different positions had different impact on the biological properties of the resulting derivatives.

The rotational relaxation times of the single tryptophan residues in endothelin-1, [Ala1,3,11,15]endothelin-1, human pro-endothelin-1, the linear hexapeptide Ac-His-Leu-Asp-Ile-Ile-Trp which corresponds to the C-terminal residues 16-21 in endothelin-1, the cyclic pentapeptide BQ123, and several di- and tri-peptides possessing C-terminal tryptophan residues have been determined from time-resolved fluorescence anisotropy decays obtained by phase/modulation techniques. Fluorescence lifetime distribution widths have also been examined as predictors of conformational heterogeneity/restriction. A significant contribution from a slow rotational component supports either the persistence, on the nano-second timescale at least, of a non-flexible alpha-helical structure for the C-terminal tail residues of endothelin-1 in water as solvent, as seen in the X-ray crystallographic structure, or the interaction of the C-terminal tail residues 16-21 with the constrained disulfide-bridged core residues 1-15. This slow rotational contribution is less evident in the linear, acyclic tetraalanine analogue but greatly increased in pro-endothelin-1. In BQ123 the fluorescence characteristics support the occurrence of a dominant rotameric form involving the indole sidechain of the D-tryptophan residue (C alpha-C beta torsion angle chi 1 of 60 degrees, as previously determined by NMR.

Disulfide bond formation in S-acetamidomethyl (Acm) cysteine-containing peptides by successive treatments with silver trifluoromethanesulfonate (AgOTf) and dimethyl sulfoxide (DMSO)/aqueous HCl is described. An S-Acm cysteine was found to be quantitatively converted into cysteine by deprotection of the Acm group with AgOTf followed by DMSO/aqueous HCl treatment. Under these reaction conditions, no significant side reactions were observed with oxidation-sensitive amino acids such as Met, Tyr and Trp. Oxytocin and a Trp-containing peptide, urotensin II, were prepared by this method. Furthermore, regioselective two disulfide bond formation was found to be feasible by the combination of air oxidation and the AgOTf-DMSO/HCl system. This strategy has been successfully applied to the syntheses of tachyplesin I and endothelin I, which have two disulfide bonds and a Trp residue in the molecule.

There are significant differences between the structures reported for members of the endothelin/sarafotoxin family of peptides, but also for the same peptides studied by different groups, raising the possibility that some of the differences are attributable to variation in solution conditions rather than intrinsic structural heterogeneity. We have shown, using circular dichroism spectroscopy and equilibrium sedimentation, that the secondary structures of these peptides are little affected by wide variations in pH, or by self-association. Although acetonitrile has a pronounced effect on the extent of peptide self-association it does not appear to alter the backbone structure of sarafotoxin SRTb, and has only minor effects on endothelin-1 and endothelin-3. The observed conformational variation thus appears largely to reflect sequence-dependent differences.

While incorporation of penicillamine residues (Pen; beta,beta-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the beta-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys-Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1-15, 3-11 to the unnatural 1-11, 3-15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys-Pen disulfides. Random oxidation (air, aqueous NH4OH) of the tetrathiols of [Pen1,11, Nle7]-ET-1 or [Pen3,15, Nle7]-ET-1 produced the correct 1-15, 3-11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen1,15, Nle7]-ET-1 favored the unnatural 1-11, 3-15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen1,11, Nle7]-ET-1 was a partial agonist at the ETA receptor (EC50 = 7.5 nM in rabbit carotid artery rings; Kd = 4.5 nM in rat A10 cell membranes) while [Pen3,15, Nle7]-ET-1 (EC50 = 0.9 nM; Kd = 0.7 nM) was a full agonist with similar potency to ET-1.

Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin-1 (ET-1), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A), and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical (+/- 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET-1 and ETD8A.

A peptide hydropathically complementary to Big Endothelin [Big ET] residues 16-29 has been synthesized in a multimeric form starting from an octadentate polylysine core, essentially in a way similar to the procedure used for the production of multiple antigenic peptides [MAP's]. Interaction between the multimeric complementary peptide [8 delta ET] and the Big ET fragment 16-32 containing the target complementary region, also synthesized in a multimeric form [8ET], was evaluated by analytical high performance affinity chromatography and solid phase binding assays. While the binding interaction between the monomerics peptide pair was in the micromolar range, the recognition between the corresponding multimeric form was characterized by enhanced binding affinity of at least two orders of magnitude. In solution, complex formation between multimeric complementary peptide and target Big ET sequence in the monomeric and multimeric form was accompanied by precipitation at concentrations higher than 0.5 mg/mL and 0.1 mg/mL, respectively. Polyclonal antibodies raised against the multimeric target sequence recognized multimeric and monomeric ET target sequences with binding affinities similar to binding affinities exhibited by the multimeric complementary peptide. Multimerization of hydropathically complementary peptides could provide an improved opportunity to measure and thus probe quantitative binding properties of complementary peptides.

Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH2-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.

Syntheses are described for the putative human and porcine biosynthetic precursors (hET-38 and pET-39) of endothelin, with the sequence previously deduced from human- and porcine-cDNA coding for preproendothelin. The Boc based solid phase synthetic method was applied, followed by weak hard acid, trimethylsilyl bromide, cleavage. The peptide removal from the resin was optimally accomplished with hydrogen fluoride. Disulfide bridges were formed by air-oxidation, and the linkage modes determined by enzymic (Endoproteinase Asp-N) digestion and HPLC. Five additional C-terminally elongated endothelin homologs were also synthesized. For alternative synthesis of pET-39, the use of trimethylsilyl trifluoromethanesulfonate for the removal of peptide from the resin generated a major side product, which was characterized. hET-38 was found to be less effective in vitro, when compared to endothelin. The vasoconstrictor activity in vitro of other related peptides was comparable to that of hET-38.

[Nle7]-endothelin was synthesized and studied by 1H NMR and distance geometry calculations. The NMR study was performed first in DMSO-d6 and then in 50% acetonitrile/water since this peptide aggregates in pure water. In both cases, all spin systems were identified and assigned with the aid of two-dimensional spectroscopy (2D): COSY (for scalar couplings) and NOESY (for dipolar couplings). On the basis of the acetonitrile/water NMR parameters, and using the DISGEO program, a three-dimensional structure of [Nle7]-endothelin is proposed and discussed.

The solution conformation of Endothelin-1, a recently discovered bicyclic, 21 amino acid peptide, has been examined by 1H NMR in deuterated dimethylsulphoxide and circular dichroism in aqueous and organic solvents. A total of 158 NOEs were detected, which were used as distance constraints in the distance geometry program DISGEO. Two families of structures were obtained, both characterized by a helix-like region extending from Lys9 to Cys15, but with opposite "handedness". Circular dichroism studies of the peptide in both aqueous and trifluoroethanol solutions show a negative shoulder at 224 nm, characteristic of right-handed helices. Molecular dynamics and energy minimization yielded a solution structure for this new peptide compatible with all experimental observations.

A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilyl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nin-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.

A model is proposed for the 3-dimensional structure of endothelin, a potent vasoconstrictor and pressor peptide from vascular endothelium. The model is derived through protein structure prediction and circular dichroism studies, and is based on the atomic coordinates for the bee-venom peptide apamin. The model derived shows the same turn-helix motif as observed for apamin and mast-cell degranulating peptide. On the basis of this model we suggest possible strategies for endothelin antagonist design, and note that this motif may be common in a number of peptides acting on channel proteins.