The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides

In an earlier study using Caco-2 cells, an in vitro cell culture model of t he intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu(5)]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala -Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of th e cyclic prodrugs, we have determined the possible conformations of these c yclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A an d B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-G ly3-Phe4. The presence of a turn was supported by ROE cross-peaks between t he NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at t he same positions. The type II turn was stabilized by hydrogen bonding, thu s forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs ha ve solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic pr odrugs 3-6, but not in the linear peptides 1 and 2, could both contribute t o the enhanced ability of these prodrugs to permeate membranes.