The endothelins (ETs) are a family of bicyclic 21-amino acid peptides that are potent and prolonged vasoconstrictors. It has been shown that highly potent combined ETA/ETB receptor antagonists can be developed from the C-terminal hexapeptide of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21), such as Ac-(D)Dip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) and Ac-DBhg16-Leu-Asp-Ile-Ile-Trp21 (PD 145065). However, these compounds are relatively unstable to enzymatic proteolysis as determined in an in vitro rat intestinal perfusate assay. This instability is thought to be due to carboxypeptidase activity. In fact, incubation of PD 145065 with carboxypeptidase inhibitors greatly increased its half-life in rat intestinal perfusate. By performing a reduced amide bond and N-methyl amino acid scan, it was discovered that N-methylation of Ile-20 resulted in a compound (Ac-DBhg16-Leu-Asp-Ile-[NMe]Ile-Trp21, PD 156252) that retained full receptor affinity at both endothelin receptor subtypes along with enhanced proteolytic stability and cellular permeability. Interestingly, N-methylation of this bond allows the cis configuration to be readily accessible which greatly alters the preferred structure of the entire molecule and may be responsible for the observed enhanced metabolic stability.

As new methods for volatilizing and ionizing biological molecules for mass spectrometry (MS) analysis are developed, the conformation of gas-phase biomolecule ions and how it relates to solution-phase reactivity and structure has been generating considerable interest. 1 The conformations of gas-phase ions of large polypeptides formed by electrospray ionization (ESI) have been probed by a variety of means, including ion-molecule reactions, 2 collision cross-section measurements, 3 and hydrogendeuterium-exchange (H/D). 4 The number of exchangeable protons reflects the openness of the protein conformation. Ion mobility measurements have resolved gas-phase structural isomers for a number of proteins. 5 The charge state distribution produced by ESI as a function of pH, 6 solvent content, 7 and metal-binding properties 8 has also been related to the protein conformation in solution. Another recent application of ESI-MS includes the study of noncovalently bound complexes, 9,10 for which MS has unique advantages in determining complex stoichiometry of the binding partners. Characteristics of the native solution state of proteins and oligonucleotides and their interactions with other biomolecules can be also captured by MS measurements.Tandem mass spectrometry (MS/MS) with collisionally activated dissociation (CAD) has been used also to explore the reactivities of large ions. Mass-analyzed kinetic energy (MIKE) spectrometry was used to study the stability of secondary structure in the absence of solvent for the peptide melittin. 11 The CAD mass spectra of peptide-metal ion complexes show that relatively selective bond cleavages can be induced, as specific complexation with metal ions can direct specific dissociation pathways. 12 Smith and co-workers' CAD study of 12 kDa cytochrome c proteins suggests that the fragmentation pattern could be influenced by the protein's higher order structure. 13 However, from these studies, it is not clear whether these three-dimensional structures in a solvent-less environment bear any resemblance to the conformation in the solution phase. We present an example in which the gas-phase MS/MS-based measurements of a polypeptide are consistent with its known solution-phase structure.RES-701-1 (I), isolated from Streptomyces sp. RE-701, is a potent and selective endothelin ET B receptor antagonist. 14 The hexadecapeptide possesses a unique linkage between the -carbonyl carbon of Asp-9 and the R-amino group of Gly-1, forming a 9-residue cyclic "head" structure and a 7-residue "tail" (see Scheme 1). However, a synthetic version of the peptide (II) is characterized by an affinity for the ET B receptor reduced by nearly 3 orders of magnitude (IC 50 of 10 nM for the natural peptide and 6.5 µM for the synthetic peptide). 15,16 High-resolution, exact mass measurements with ESI show the two peptides to be within (0.004 mass units ((2 ppm) of the expected value (measured monoisotopic mass of I was 2041.8520, and a mass of 2041.8492 for II; expected mass from sequence is 2041.8536). 17 Dramatic differ...

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Factor Xa (FXa) has materialized as a key enzyme for the intervention of the blood coagulation cascade and for the development of new antithrombotic agents. FXa is the lone enzyme responsible for the production of thrombin and therefore is an attractive target for the control of thrombus formation. We have designed and synthesized a unique series of quinoxalinone FXa inhibitors. This series resulted in 3-[4-[5-((2S,6R)-2,6-dimethylpiperidin-1-yl)pentyl]-3-oxo-3,4-dihydroquinoxolin-2-yl]benzamidine (35) with 0.83 nM activity against FXa and excellent selectivity over similar serine proteases. An X-ray crystal structure of compound 35 bound to trypsin along with molecular modeling has led to a predicted binding conformation of compound 35 in FXa. Compound 35 has also been proven to be efficacious in vivo in both the rabbit veno-venous shunt and dog electrolytic injury models. In addition, it was shown that compound 35 did not significantly increase bleeding times in a rabbit model except at the highest doses and plasma concentrations were elevated in a dose dependent manner following a bolus dose and continuous intravenous infusion.

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Communications to the Editor Design of a Functional Hexapeptide Antagonist of Endothelin Endothelin-1 (ET-1, Figure 1), a bicyclic 21-amino acid peptide, is a potent constrictor of vascular smooth muscle.1 2"3 Since the isolation of ET-1 from the supernatant of cultured porcine endothelial aortic cells, human genomic analysis has identified two structurally and functionally related isopeptides (ET-2 and ET-3).4 56Previous structure-activity analyses have shown the importance of the C-terminal L-tryptophan indole ring, its carboxylate, and the two cystine bridges (1-15 and 3-11

Synopsis Ac-[Nle4,~-Phe7,~-Trps]-a-MSH4.,,-NH2was 3 X more potent than a-MSH in the frog skin bioassay. Proton nmr studies in aqueous solution revealed a marked preservation of the backbone conformation of these linear analogs. Chemical-shift variations due to the through-space anisotropic influence of the core aromatic amino acid residues permitted evaluation of side-chain topology. The observed topology was consistent with nonhydrogen-bonded B-like structure (# = -139O, + = +135" for L--O acids; 6 = +139", + = -135O for D-amino acids) as the predominant solution conformation. The biological and conformational data suggest that high melanotropic potency requires a close spatial arrangement of the His6, Phe7, and Args side chains.