Conformational analysis of beta-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations.

Abstract

Solution conformations of beta-methyl-para-nitrophenylalanine4 analogues of the potent delta-opioid peptide cyclo[D-Pen2, D-Pen5]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and 3JHNC alpha H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides (ECEPP/2) force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (3JH alpha H beta) and heteronuclear (3JH alpha C gamma) coupling constants and 13C chemical shifts, show that the beta-methyl substituent eliminates one of the three staggered rotamers of the torsion angle chi 1 for each stereoisomer of the beta-Me-p-NO2Phe4. Similar solution conformations were suggested for the L-Phe4-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe4-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main delta-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [beta-Me-p-NO2Phe4]DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [beta-Me-p-NO2Phe4]DPDPE in the delta-receptor-bound state may assume a conformation different from their favorable conformations in DMSO.