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

Solution conformations of β-methyl-para-nitrophenylalanine⁴ analogues of the potent δ-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 ³J_HNCαH coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of [β-Me-pNO²Phe⁴]DPDPE in DMSO were compared with low energy conformer 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 homo-nuclear (³J_HαHβ) and heteronuclear (³J_HαCγ) coupling constants and ¹³C chemical shifts, show that the β-methyl substituent eliminates one of the three staggered rotamers of the torsion angle X¹for each stereoisomer of the β-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 δ-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 [β-Me-pNO₂Phe⁴]DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [ β-Me-p-NO₂Phe⁴]DPDPE in the δ-receptor-bound state may assume a conformation different from their favorable conformations in DMSO.