COMBINED USE OF STEREOSPECIFIC DEUTERATION, NMR, DISTANCE GEOMETRY, AND ENERGY MINIMIZATION FOR THE CONFORMATIONAL-ANALYSIS OF THE HIGHLY DELTA-OPIOID RECEPTOR SELECTIVE PEPTIDE [D-PEN2,D-PEN5]ENKEPHALIN

Comparison of 1H and 13C NMR parameters for the cyclic, conformationally restricted, δ opioid receptor selective enkephalin analogue Tyr-D-Pen-Gly-Phe-D-Pen ([d-Pen2D-Pen5]enkephalin, DPDPE) in aqueous versus dimethyl sulfoxide (DMSO) solution indicates that this peptide adopts similar conformations in these solvents. This suggestion that the conformation of DPDPE is relatively environment independent allows conclusions regarding the receptor-bound conformation of this peptide to be drawn from studies performed on experimentally convenient DMSO solutions of the peptide, alone. Accordingly, 2D NOESY experiments were conducted on DPDPE in DMSO, and the observed interproton interactions were utilized for the quantitative calculation of the appropriate interproton distances. A commonly encountered limitation, the general inability to stereos pecifically assign diastereotopic and enantiotopic hydrogens within the amino acid residues, which results in increased error limits for calculated distances involving such hydrogens, was overcome by the synthesis of stereos pecifically deuterated amino acids (2S,3R)-[3-2H]tyrosine, (2S,3S)-[4,4,4-2H3]penicillamine, (R)-[2-2H]glycine, and (2S,3R)-[3-2H] phenylalanine and their incorporation into DPDPE. As a result all resonances in the 1H NMR spectrum of DPDPE were assignable, and more stringent interproton distances were calculated from the observed NOE interactions. These interproton distances were employed as distance constraints for distance geometry calculations of conformations consistent with the experimental data. Energy minimization of conformers generated by distance geometry calculations was performed by using the amber force field, and the resulting low-energy conformers were reexamined for agreement with distance constraints and other conformation- dependent NMR parameters. From these studies a conformer was identified that displayed significantly lower energy than all others found while maintaining good agreement with experimental data. Details of this model conformer and comparisons with recently proposed conformations for DPDPE are discussed. © 1990, American Chemical Society. All rights reserved.