SPED-(styrene-polyethyleneglycol diacrylate-9-decen-1-ol): a novel resin for solid phase peptide synthesis; synthesis and characterization of biologically potent endothelin classes of peptides.

Here we present a novel beaded chemically stable, highly permeable hydrophobic/hydrophilic balanced support for solid phase peptide synthesis. The resin (SPED) was prepared by free radical suspension polymerization using monomers styrene and 9-decen-1-ol with amphiphilic cross-linking agent polyethyleneglycol diacrylate (Mn 258). Different cross-linking densities were prepared to check the extent of swelling in different polar and non-polar solvents. The SPED resin was characterized with IR, 13C NMR and surface by SEM. The chemical stability of the support in various peptide synthetic conditions was investigated and monitored by IR spectroscopy. To evaluate the applicability of the new resin in synthetic conditions more challenging peptide sequence of retro-ACP (74-65) was synthesized and compared to commercially available Merrifield resin. The efficiency of SPED was further confirmed by synthesizing biologically important endothelin family of peptides in high yield and purity. The purity of all peptides was checked by RP-HPLC and mass by MALDI-TOF MS.

Endothelin receptor antagonists: an overview of their synthesis and structure-activity relationship.

Endothelins (ETs) are potent vasoconstrictor peptides and are associated with several disease states like pulmonary hypertension, systemic hypertension and heart failure. Endothelin-1 (ET-1) is the first member of the family and it has the receptor subtypes known as ETA and ETB. The receptors ETA and ETB are attractive new therapeutic targets for diseases associated with elevated ET-1 levels. Several studies have thus led to the discovery of selective ETA receptor antagonists as well as non-selective ETA/ETB antagonists. The preclinical and clinical studies have clearly established that these antagonists are effective in the treatment of essential hypertension, pulmonary hypertension, heart failure and atherosclerosis. The advances in this area have resulted in the FDA approval of the orally active dual antagonist Bosentan for pulmonary hypertension in 2001. This review highlights the synthesis and structure-activity of the endothelin receptor antagonists and covers the literature in this area up to 2001.

Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620-derived photoprobe.

On the basis of the structure of IRL-1620, a specific agonist of the endothelin-B receptor subtype (ET(B)), a few photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing the photoreactive amino acid, p-benzoyl-l-phenylalanine in position 5 showed, as assessed with endothelin-A (ET(A)) and ET(B) receptor paradigms, pharmacological properties very similar to those of IRL-1620. The binding capacity of the probe was also evaluated on transfected Chinese hamster ovary (CHO) cells overexpressing the human ET(B) receptor. Data showed that binding of the radiolabeled peptide was inhibited by ET-1 and IRL-1620. Therefore, this photolabile probe was used to label the ET(B) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 49 kDa. An excess of ET-1 or IRL-1620 completely abolished the formation of the complex, showing the selectivity of the photoprobe. Digestions of the [Bpa(5),Tyr((125)I)(6)]IRL-1620-ET(B) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacts. Results showed that Endo Lys-C digestion gave a 3.8-kDa fragment corresponding to the Asp(274)-Lys(303) segment, whereas migration after V8 digestion revealed a fragment of 4.6 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp(275)-Asp(313) stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.6 kDa, corresponding to Gln(267)-Met(296). Thus, the combined cleavage data strongly suggested that the agonist binding domain of ET(B) includes a portion of the fifth transmembrane domain, between residues Trp(275) and Met(296).

Syntheses of the first endothelin-A- and -B-selective radioligands for positron emission tomography.

We have synthesized two potential positron emission tomography (PET) radioligands for the endothelin (ET) receptor. [11C]-PD156707 was produced by O-methylation of PD169390 using [11C]iodomethane. Radiochemical conversions of the order of 74 +/- 3.2% (n = 8) were obtained. The radiochemical purity of the isolated [11C]-PD156707 was 99% and the specific activity was 538 mCi/micromol. [18F]-BQ3020 was produced from [18F]fluoride in a total radiochemical yield of 2.7 +/- 0.4% (n = 10) in 238 +/- 5 min. The radiochemical purity was 95% and specific activities of the order of 670-930 mCi/micromol were obtained.

Comparative studies of Nsc and Fmoc as N(alpha)-protecting groups for SPPS.

2-(4-Nitrophenyl)sulfonylethoxycarbonyl (Nsc) is an alternative base-labile N(alpha)-protecting group to 9-fluorenylmethoxycarbonyl (Fmoc) for amino acids. The UV spectrum of the Nsc group exhibits moderate absorption at 380 nm which is excellent for real-time monitoring of the deprotection process. It also decreases the rearrangement of X-Asp, which can be a serious problem in SPPS.

Solution-structure of a peptide designed to mimic the C-terminal hexapeptide of endothelin.

Ac-cyclo(Cys-His-Leu-Asp-Cys)-Ile-Trp-OH, has been designed by computer-aided molecular-modelling techniques to mimic the proposed alpha-helical conformation of the C-terminal hexapeptide of endothelin. Two-dimensional proton nuclear magnetic resonance spectra were acquired for the peptide dissolved in d6-DMSO or D2O-H2O and the distance and angle constraints incorporated into simulated annealing experiments. Conformers generated from the D2O-H2O data superposed on the corresponding main-chain atoms in the crystal structure of endothelin 1 and the solution structure of BQ-123 with root mean square co-ordinate differences of 0.9A and 0.77A, respectively. The peptide did not elicit antagonism of endothelin-induced in-vitro contractions of rabbit aorta (endothelin A receptor) or rabbit bronchus (endothelin B receptor) preparations. Because the peptide can adopt a conformer which closely matches the equivalent residues in the endothelin 1 crystal structure and in BQ-123, we suggest BQ-123 does not necessarily mimic the endothelin C-terminal region to achieve its antagonism, and that a helical conformation of the endothelin C-terminal hexapeptide does not favour its interaction at the endothelin B receptor.

[Studies on development of disulfide bond forming reaction and the application to regioselective disulfide formation].

An efficient method for the disulfide bond formation in peptides by the silylchloride-sulfoxide system is described. Methyltrichlorosilane in trifluoroacetic acid, in the presence of diphenylsulfoxide, is found to cleave various S-protecting groups of cysteine to form cystine directly within 10 to 30 min. No side reactions were observed with nucleophilic amino acids such as Met, His, or Tyr, except for Trp, under the reaction conditions of the silylchloride-sulfoxide treatment. A chlorination of the indole moiety of unprotected Trp, rather than the sulfur-sulfur bond formation, is a dominant reaction when the peptide containing unprotected Trp is treated with the chlorosilane-sulfoxide. However, the disulfide bond can be formed efficiently with no modification at the indole ring by the treatment of the peptide having formyl-protected Trp residue with the silylchloride-sulfoxide system. The formyl group is removed by a brief treatment at basic pH without affecting the disulfide bond formed by the silylchloride-sulfoxide treatment. Total synthesis of human insulin, a two chain peptide containing three disulfide bonds, was achieved unambiguously by sequential and selective formation of disulfide bonds in the protein for the first time. The key reaction in the synthesis is regioselective formation of three disulfide bonds separately using the silyl chloride method described above. Prior to the insulin synthesis, it was confirmed by the syntheses of double-disulfide peptides: b-hANP, unnatural parallel dimer of a-hANP, and human endothelin-1 that no disulfide exchange occurred during the silyl chloride treatment. Using three orthogonal thiol protecting groups, Trt, Acm, and But, three disulfide bonds of human insulin were efficiently constructed by the successive reactions using thiolysis, iodine oxidation, and the sily1 chloride method. Each reaction for the stepwise disulfide formation proceeded within 15 to 60 min with no polymeric product and no solubility problem. The synthetic human insulin had the correct structure and was indistinguishable from natural human insulin.

Receptor binding and biologic activity of synthetic ET-1 peptides in the retinal pericyte.

PURPOSE: The purpose of this study was to examine the effect of synthetic endothelin (ET)-1 peptides with antigenic potential for binding and biologic activity using an in vitro model of microvascular pericytes. METHODS: All possible sequential hexapeptide fragments of endothelins -1, -2, and -3 were synthesized on polyethylene rods and tested for reactivity with antibodies for ET-1 and ET-3. The most highly antigenic peptide ET-1[3-8] and the least antigenic ET-1[1-6], which gave low reactivity in the enzyme-linked immunosorbent assay (ELISA), were synthesized. The C-terminal hexapeptide ET-1[16-21], which has been reported as an ETB receptor agonist but which gave no reactivity in the ELISA, also was investigated. The synthesized ET analogues were tested for receptor binding, inositol phosphate generation, and mitogenesis in bovine retinal pericytes. RESULTS: ET-1[16-21] partially inhibited both [125I]-ET-3 binding at high concentrations, as did ET-1[1-6]. In contrast, ET-1[3-8] displaced labeled ET-1 binding but not labeled ET-3 binding. None of the peptides had any significant mitogenic or second-messenger responses when compared to those elicited by the full ET-1 molecule. CONCLUSIONS: The result of the current work shows that the sequence, ET[3-8], is involved in isopeptide-specific binding to ETA receptors, but it suggests that other regions of the molecule are necessary for full bioactivity in microvascular pericytes.

Structure-activity relationships of the potent combined endothelin-A/endothelin-B receptor antagonist Ac-DDip16-Leu-Asp-Ile-Ile-Trp21: development of endothelin-B receptor selective antagonists.

The endothelins (ETs) are a family of bicyclic 21-amino acid-containing peptides that are highly potent and prolonged vasoconstrictors. The discovery of potent ET antagonists will facilitate the understanding of the physiological and/or pathophysiological role of ET. Structure-activity studies have revealed the importance of the C-terminal hexapeptide (residues 16-21) of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21) to the development of potent antagonists at both receptor subtypes (ETA and ETB). In particular, it has been shown that Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (Dip = 3,3-diphenylalanine) has low nanomolar affinity for the two endothelin receptor subtypes and is a functional antagonist of ET activity, both in vitro and in vivo at both receptors. Herein, we will describe the structure-activity relationships of Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) with a particular emphasis on modifications that lead to enhanced receptor affinity and/or individual receptor subtype selectivity. In particular, we will demonstrate how we utilized PD 142893 to develop ETB receptor selective ligands and the pharmacological differences that exist between species ETB receptors with respect to their affinity for C-terminal hexapeptide antagonists.

Disulfide bond-forming reaction using a dimethyl sulfoxide/aqueous HCl system and its application to regioselective two disulfide bond formation.

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.

Endothelin-1 analogues substituted at both position 18 and 19: highly potent endothelin antagonists with no selectivity for either receptor subtype ETA or ETB.

Novel endothelin-1 (ET-1) analogues which are highly potent endothelin antagonists at both receptor subtype ETA and ETB are reported. The replacement of Asp18 with the Thr18 and of Ile19 with a hydrophobic amino acid whose side-chain branches on the gamma-carbon such as Leu, cyclohexylalanine, and gamma-methylleucine (gamma-MeLeu) resulted in loss of or significantly decreased the biological activity of ET-1, while high affinity for the ETA (IC50 = 0.42-0.70 nM) and ETB (IC50 = 0.17-0.43 nM) receptor was retained. These compounds were shown to have high antagonist activities in ET-1-induced vasoconstriction of porcine coronary artery (pA2 7.4-7.7) and in Sarafotoxin S6c-induced vasoconstriction of rabbit pulmonary artery ([Thr18, gamma-MeLeu19]ET-1: pA2 8.4). Among these compounds, [Thr18, gamma-MeLeu19]ET-1 has the desirable characteristic of possessing no agonist activity at either receptor subtype.

Control of peptide disulfide regioisomer formation by mixed cysteine-penicillamine bridges. Application to endothelin-1.

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.

Solid-phase synthesis of [Ala1,3,11,15]-endothelin-1 by a modified double-coupling procedure.

[Ala1,3,11,15]-Endothelin-1, a linear analogue of endothelin-1 in which alanines replace the cystine residues, has been prepared by solid-phase synthesis in approximately 17% yield. Fmoc amino acids were coupled using an economical modified double-coupling (symmetrical anhydride followed by O-benzotriazolyl ester) procedure under fully automated conditions. Conditions for synthesis and purification were closely monitored and should be applicable to other endothelin analogues.

Site-specific biotinylation. A novel approach and its application to endothelin-1 analogs and PTH-analog.

We have developed an expeditious method for the incorporation of the biotinylaminocaproyl moiety on the epsilon-amino group of a lysine residue within a peptide chain in a site-specific manner. Using t-Boc chemistry for the solid phase synthesis approach and a base labile, acid stable protecting group (Fmoc-) for the epsilon-amino group of the target lysine, we prepared fully protected resin bound peptides which are site-specifically biotinylated. Following HF cleavage, the uniquely biotinylated peptides were obtained in a high degree of purity. Using this approach, a number of biotinylaminocaproyllysyl derivatives of a monocyclic Endothelin-1 analog were prepared. Synthesis of selected bicyclic analogs of high affinity monocycles led to the preparation of the bicyclic [Nle7]ET-1 analog containing epsilon-biotinylaminocaproyllysine at position-9. This peptide, with Kd = 0.08 nM, has 1000-fold higher affinity for the ETA receptor than the commercially available N alpha-biotinylated Endothelin-1. The general utility of this biotinylation methodology was demonstrated by the synthesis of a site-specifically biotinylated PTH analog which contained several side chain functionalized amino acid residues in its sequence. The synthetic method reported here is convergent in that it allows the facile variation of the length of the spacer and also offers the potential to introduce in a site specific manner other groups such as affinity labels and fluorescent tags.

An endothelin B receptor-selective antagonist: IRL 1038, [Cys11-Cys15]-endothelin-1(11-21)

In the inhibition of specific binding of [125I]endothelins (ETs) to membrane from various tissues of rats, guinea pigs, pigs and humans, [Cys11-Cys15]-ET-1(11-21), IRL 1038, has a much higher affinity for ETB receptors (Ki = 6-11 nM) than for ETA receptors (Ki = 0.4-0.7 microM). In contraction assays, with ET-3 as a stimulant, 3 microM IRL 1038 antagonized the ETB receptor-mediated contraction of guinea pig ileal and tracheal smooth muscle without any significant agonistic activity, but did not effect the ETA receptor-mediated contraction of rat aortic smooth muscle. IRL 1038 is therefore, considered to be the first antagonist selective to the ETB receptor.

FMOC solid phase synthesis of an endothelin converting enzyme substrate. Use of allyl ester as the third orthogonal protecting group.

The synthesis of a substrate for the endothelin converting enzyme using the continuous flow FMOC solid phase method has been accomplished. The allyl group, employed to protect Glu side chain, was cleanly removed during the synthesis in the presence of tert.-butyl based protecting groups to enable the introduction of the Edans moiety. The substrate was obtained in 31% yield after reverse phase hplc purification.

Crystallization and preliminary X-ray analysis of human endothelin.

Endothelin, a potent regulator of vasoconstriction and hypertension, is a naturally produced peptide of 21 amino acids containing two disulfide bonds. We have crystallized endothelin from humans using the vapor-diffusion technique, characterized the crystals by X-ray diffraction analysis, and have collected the X-ray intensities to a resolution of 1.8 A. The crystals, which demonstrate physical properties similar to most protein crystals and have a comparable solvent content, are hexagonal prisms that frequently grow to lengths of 400 microns and widths of 150 microns. The space group of the crystals is P6(1)22 (or P6(5)22), with a = 27.4, c = 79.6 A. There is one molecule of endothelin in the asymmetric unit of the crystals.

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.

Continuous flow synthesis of peptides using a polyacrylamide gel resin (Expansin).

The continuous flow syntheses of endothelin 1, proendothelin 2, ATP binding site of the CDC2 kinase 3, and fragment 18-30 of an actin 4, have been performed by using a polyacrylamide gel resin Expansin (about 0.6 mmol NH2/g) with the glycolamidic ester handle as labile anchorage. In addition, we report here a method of air oxidation which reduces the formation of side-products related to the formation of intermolecular disulfide bridges.