Synthesis, molecular modelling, and NMR structure determination of four cyclic peptide antagonists of endothelin

Abstract

A combined distance geometry and molecular mechanics/dynamics (MM/MD) protocol was unable to predict the active conformation of the cyclic pentapeptide inhibitor of endothelin-1 receptor, BQ-123, and two analogues. However, the MM/MD method alone is sufficient to predict the solution conformation of a third analogue. In that one case, the combination of proline at residue 3 and an N α-methyl substitution at residue 5 provides enough internal constraints to eliminate conformational flexibility seen in the other three analogues. For this constrained analogue, the 50 lowest energy conformations (out of a set of 500 DGEOM-generated, MM/MD minimized conformations) differ by no more than 3.9 kcal/mol. Thirty three of these 50 conformations have backbone atom RMSDs of less than 0.33 Å, relative to the lowest energy conformation. The accuracy of this MM/MD model is verified by determining the solution structure of each of the four analogues with 2D NMR techniques. Each of the cyclic pentapeptides has a well defined solution conformation where a proline residue is clearly in a γ-turn, leaving the remaining residues in a loose β-turn. All four experimental NMR conformations agree closely with the MM/MD model. MM/MD molecular mechanics/molecular dynamics, DG distance geometry, RMS root mean square, RMSD root mean square deviation. NMR nuclear magnetic resonance, 2D NMR 2-dimensional NMR, ROESY 2-dimensional rotating frame nuclear Overhauser effect spectroscopy, COSY 2-dimensional J-correlated spectroscopy, PECOSY 2-dimensional exclusive correlation spectroscopy, TOCSY 2-dimensional total correlation spectroscopy, 3J Nα, NH-C αH vicinal proton coupling constant, 3J αβ C αH-C βH vicinal proton coupling constant. FMOC 9-(fluorenylmethoxy)carbonyl, PyBOP benzotriazole-1-yl-oxy-tris-(pyrrolidino)phosphonium hexafluorophosphate, PyBrOP, bromo-tris-(pyrrolidino)phosphonium hexafluorophosphate, HOBt hydroxybenzotriazole, DMSO dimethyl sulfoxide, DCM dichloromethane, DMF dimethyl formamide, DIEA diisopropylethanolamine, TFA trifluoroacetic acid, EtOH ethanol, HOAc acetic acid, TMS tetramethylsilane, DMAP dimethylaminopyridine, DIC diisopropylcarbodiimide. RP-HPLC reverse phase high pressure liquid chromotography, AAA amino acid analysis, FAB-MS fast atom bombardment mass spectral analysis. We use the designations ETR A and ETR B, rather than the convention ET A and ET B to ensure clarity that we are discussing the two receptor subtypes of the Endothelin family.