THE EFFECT OF BETA-TURN STRUCTURE ON THE PASSIVE DIFFUSION OF PEPTIDES ACROSS CACO-2 CELL MONOLAYERS

Purpose. To investigate the relationships between the beta-turn struct ure of a peptide and its passive diffusion across Caco-2 cell monolaye rs, an in vitro model of the intestinal mucosa. Methods. Linear hydrop hilic peptides (Ac-TyrProXaaZaaVal-NH2; Xaa = Gly, lie and Zaa = Asp, Asn) and hydrophobic (Ac-Yaa-ProXaaIleVal-NH2; Yaa = Tyr, Phe and Xaa = Gly, lie: and Ac-PheProXaane-NH2; Xaa = Gly, lie) peptides were synt hesized and their effective permeability coefficients (P-eff) were det ermined across Caco-2 cell monolayers. The lipophilicities of the pept ides were estimated by measuring their partition coefficients (P-o/w b etween 1-octanol and HBSS. Two-dimensional NMR (2D-NMR) spectroscopy a nd circular dichroism (CD) spectroscopy was used to determine the solu tion structures of these model peptides. Results. Using 2D-NMR spectro scopy and CD spectroscopy, the hydrophilic Gly-containing peptides (Ac -TyrProGlyZaaVal-NH2; Zaa = Asp, Asn) were shown to exhibit a higher d egree of beta-turn structure in solution than the Ile-containing pepti des (Ac-TyrProIleZaaVal-NH2; Zaa = Asp, Asn). CD spectroscopy was used to show that the Gly-containing hydrophobic peptides (Ac-YaaProGlyIle Val-NH2; Yaa = Tyr; Phe: and Ac-PheProGlyIle-NH2) exhibited a higher d egree of beta-turn structure in solution than the lie-containing hydro phobic peptides. The P-eff values of all four hydrophilic peptides acr oss unperturbed Caco-2 cell monolayers were very low and no statistica lly significant differences were observed between the Gly-and lie-cont aining pentapeptides within either the Asp or Asn series. The P-eff va lues for the hydrophobic Gly-containing peptides were significantly gr eater than the P-eff values determined for their Lie-containing counte rparts. The Gly-containing penta- and tetrapeptides in the Phe series, which exhibited high permeation, were shown to be metabolically unsta ble. In contrast, the Gly-and lie-containing pentapeptides in the Tyr series and the lie-containing penta-and tetrapeptides in the Phe serie s, which exhibited low permeation, were metabolically stable. Conclusi ons. Hydrophobic peptides that exhibit significant beta-turn structure in solution are more lipophilic as measured by log P-o/w and more rea dily permeate Caco-2 cell monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. The ab ility of these peptides to permeate Caco-2 cell monolayers via the tra nscellular route also exposed them to metabolism, presumably by cytoso lic endopeptidases. Similar secondary structural features in hydrophil ic peptides do not appear to sufficiently alter the physicochemical pr operties of the peptides so as to alter their paracellular flux throug h unperturbed Caco-2 cell monolayers.