The effect of beta-turn structure on the passive diffusion of peptides across Caco-2 cell monolayers.
OBJECTIVE
To investigate the relationships between the beta-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-XaaIle Val-NH2; Yaa = Tyr, Phe and Xaa = Gly, Ile: and Ac-PhePro-XaaIle-NH2; Xaa = Gly, Ile) peptides were synthesized and their effective permeability coefficients (Peff) were determined across Caco-2 cell monolayers. The lipophilicities of the peptides were estimated by measuring their partition coefficients (Po/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 beta-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 beta-turn structure in solution than the Ile-containing hydrophobic peptides. The Peff 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 pentapeptides within either the Asp or Asn series. The Peff values for the hydrophobic Gly-containing peptides were significantly greater than the Peff 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 beta-turn structure in solution are more lipophilic as measured by log Po/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 endopeptidase. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties of the peptides so as to alter their paracellular flux through unperturbed Caco-2 cell monolayers.
Knipp
G T
GT
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence 66047, USA.
Vander Velde
D G
DG
Siahaan
T J
TJ
Borchardt
R T
RT
D001692:Biological Transport / Q000502:physiology; D018938:Caco-2 Cells; D002942:Circular Dichroism; D004058:Diffusion; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010539:Permeability; D017433:Protein Structure, Secondary; D012996:Solutions; D013329:Structure-Activity Relationship
D009842:Oligopeptides; D012996:Solutions