CONFORMATIONAL DETERMINANTS OF AGONIST VERSUS ANTAGONIST PROPERTIES OF [D-PEN(2),D-PEN(5)]ENKEPHALIN (DPDPE) ANALOGS AT OPIOID RECEPTORS - COMPARISON OF X-RAY CRYSTALLOGRAPHIC STRUCTURE, SOLUTION H-1-NMR DATA,AND MOLECULAR DYNAMIC SIMULATIONS OF [L-ALA(3)]DPDPE AND [D-ALA(3)]DPDPE

c-[D-Pen(2),D-Pen(5)]enkephalin (DPDPE, 1) is a cyclic, constrained, h ighly potent, delta opioid receptor selective peptide agonist. Substit ution of Gly(3) with L-Ala in DPDPE to give [L-Ala(3)]DPDPE (2) has be en shown to produce a peptide with much greater delta receptor binding selectivity than DPDPE itself. However [L-Ala(3)]DPDPE is only a part ial agonist in in vivo antinociception and actually was found to poten tly antagonize the antinociceptive effects of DPDPE at delta receptors in the brain. In comparison, [D-Ala(3)]DPDPE (3) is a weak and poorly selective delta agonist. In an effort to correlate the biological pro files of these peptides with secondary structure, [L-Ala(3)]DPDPE and [D-Ala(3)]DPDPE were studied by X-ray crystallography and H-1 and C-13 NMR in DMSO solution. Crystals of both peptides were obtained using v apor diffusion techniques. [L-Ala(3)]DPDPE crystallizes in the monocli nic space group C2 with cell dimensions a = 36.35(1) Angstrom, b = 19. 737(4) Angstrom, c = 28.16(1) Angstrom, beta = 129.07(2)degrees, and V = 15688(9) Angstrom(3). The asymmetric unit contains four peptide mol ecules and approximately 20 water molecules, giving a calculated densi ty of 1.274 g cm(-3). The conformation of all four independent [L-Ala( 3)]DPDPE molecules is essentially the same. [D-Ala(3)]DPDPE crystalliz es in the monoclinic space group P2(1) with cell dimensions a = 12.271 (2) Angstrom, b = 9.600(a) Angstrom, c = 18.750(4) Angstrom, beta = 10 3.56(2)degrees, and V = 2147.2(7) Angstrom(3). The asymmetric unit con tains one peptide molecule and 10 molecules of water, giving a calcula ted density of 1.298 g cm(-3). Comparison of these X-ray structures wi th the crystal structure previously reported for DPDPE indicates that there are differences in the disulfide bond region for all three pepti des. ROEs determined about the disulfide regions of 1-3 in solution ar e indicative of a high degree of conformational interconversion, while heteronuclear coupling constants between the D-Pen(2.5) Ha and C gamm a,gamma' carbons indicate a Strong preference for a gauche (+) chi(1) angle in 2. The backbone conformations of DPDPE and [D-Ala(3)]DPDPE in the X-ray structures are virtually identical, while in [L-Ala(3)]DPDP E, there is a rotation of approximately 160 degrees about both psi(2) and phi(3) compared to DPDPE which has the effect of rotating this ami de group approximately 180 degrees relative to DPDPE. The solution NMR data for the peptide backbone conformations of 2 and 3 are mainly con sistent with their X-ray structures. However, MD simulation of all thr ee compounds, starting with the geometries of their X-ray structures, indicates that by comparison of observed and predicted ROE intensities an equilibrium between these conformations is likely in solution. The ''DPDPE-like'' conformation for [L-Ala(3)]DPDPE is however significan tly higher in energy than the X-ray structure reported here and, thus, is predicted to be less populated in solution and in receptor binding . It is concluded that the X-ray structure of DPDPE represents an agon ist conformation for this peptide at the delta opioid receptor and tha t the corresponding X-ray structure of [L-Ala(3)]DPDPE represents an a ntagonist conformation due to the differences in conformation between positions 2 and 3. Considerations on the structural implications of th is conformational difference on receptor binding are discussed.