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

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Abstract
In an earlier study using Caco‐2 cells, an in vitro cell culture model of the 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, [Leu5]‐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 the cyclic prodrugs, we have determined the possible conformations of these cyclic 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 and B) in solution. Conformer A exhibited a type I β‐turn at Tyr1‐D‐Ala2‐Gly3‐Phe4. The presence of a turn was supported by ROE cross‐peaks between the 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 β‐turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus 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 have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well‐defined secondary structures in cyclic prodrugs 3–6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.