The Effect of β-Turn Structure on the Passive Diffusion of Peptides Across Caco-2 Cell Monolayers

Abstract

Purpose. To investigate the relationships between the β-turn structure of a peptide and its passive diffusion across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa.

Methods. Linear hydrophilic peptides (Ac-TyrProXaaZaaVal-NH₂; Xaa = Gly, Ile and Zaa = Asp, Asn) and hydrophobic (Ac-YaaPro-XaaIleVal-NH₂; Yaa = Tyr, Phe and Xaa = Gly, Ile: and Ac-PhePro-XaaIle-NH₂; Xaa = Gly, He) peptides were synthesized and their effective permeability coefficients (P_eff) were determined across Caco-2 cell monolayers. The lipophilicities of the peptides were estimated by measuring their partition coefficients (P_o/w) between 1-octanol and HBSS. Two-dimensional NMR (2D-NMR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the solution structures of these model peptides.

Results. Using 2D-NMR spectroscopy and CD spectroscopy, the hydrophilic Gly-containing peptides (Ac-TyrProGlyZaaVal-NH₂; Zaa = Asp, Asn) were shown to exhibit a higher degree of β-turn structure in solution than the Ile-containing peptides (Ac-TyrProIleZaaVal-NH₂; Zaa = Asp, Asn). CD spectroscopy was used to show that the Gly-containing hydrophobic peptides (Ac-YaaProGlyIleVal-NH₂; Yaa = Tyr, Phe: and Ac-PheProGlyIle-NH₂) exhibited a higher degree of β-turn structure in solution than the Ile-containing hydrophobic peptides. The P_eff values of all four hydrophilic peptides across unperturbed Caco-2 cell monolayers were very low and no statistically significant differences were observed between the Gly- and Ile-containing penta-peptides within either the Asp or Asn series. The P_eff values for the hydrophobic Gly-containing peptides were significantly greater than the P_eff values determined for their Ile-containing counterparts. The Gly-containing penta- and tetrapeptides in the Phe series, which exhibited high permeation, were shown to be metabolically unstable. In contrast, the Gly- and Ile-containing pentapeptides in the Tyr series and the Ile-containing penta- and tetrapeptides in the Phe series, which exhibited low permeation, were metabolically stable.

Conclusions. Hydrophobic peptides that exhibit significant β-turn structure in solution are more lipophilic as measured by log P_o/w and more readily permeate Caco-2 cell monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. The ability of these peptides to permeate Caco-2 cell monolayers via the transcellular route also exposed them to metabolism, presumably by cytosolic endopeptidases. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties fo the peptides so as to alter their paracellular flux through unperturbed Caco-2 cell monolayers.