SYNTHESIS AND CONFORMATIONAL PROPERTIES OF THE LANTHIONINE-BRIDGED OPIOID PEPTIDE [D-ALAL2,ALAL5]ENKEPHALIN AS DETERMINED BY NMR AND COMPUTER-SIMULATIONS

We report the synthesis and conformational analysis by means of NMR and computer simulations of a novel opioid peptide with the sequence [GRAPHICS] which we write as [GRAPHICS] abbreviated [D-Ala(L)2, L-Ala(L)5]EA, where Ala(L) denotes each of the lanthionine amino acid ends linked by a monosulfide bridge and EA indicates enkephalinamide. Data from 2D NMR (HOHAHA and ROESY) provide short-range NOEs that are used as constraints in molecular modeling; measurement of coupling constants shows that chi1 (D-Ala(L)2) is predominantly in either the t or g- conformation, and temperature coefficient data suggest the participation of the Ala(L)5 amide proton in an intramolecular hydrogen bond. The use of NOE and hydrogen-bond constraints in a distance-geometry program yields a large number of initial conformations compatible with the data. Energy minimization of these structures using CHARMM results in three families of backbone ring conformations, labeled Al, A2, and B. The torsion chi1 in D-Ala(L)2 remains close to trans for all three conformations. Molecular dynamics in vacuo at 300 K show that these three families of conformers interconvert, with concerted shifts in two of the three torsions psi(Phe), phi(Ala(L)5), and chi(Ala(L)5). The [D-Ala(L)2,L-Ala(L)5]EA is superactive in the guinea pig ileum (GPI) and mouse vas deferens (MVD) in vitro tests and also in the rat hot plate test in vivo. At the same time, this analog with a constrained 13-membered ring shows virtually no selectivity with a ratio IC50 (MVD)/IC50 (GPI) of 0.882.