hPEPT1 affinity and translocation of selected Gln-Sar and Glu-Sar dipeptide derivatives

The intestinal di- and tripeptide transporter hPEPT1 is considered responsible for the absorption of di- and tripeptides arising from digestion, along with several drugs and prodrugs. In order to gather information on the binding site of the protein, several structure-affinity relationships have been suggested. However, these are not necessarily predictive of compounds that are actually translocated by hPEPT1. More information on affinity to and translocation via hPEPT1 of side-chain-modified dipeptides may be gained by conducting a study of selected dipeptide derivatives with variety in size, hydrophobicity, and bond type. The aim of the present study was to synthesize new esters and amides based on L-Glu-Sar and investigate the effects that bond type and size of modification of the N-terminal side chain of sarcosine-containing dipeptides have on the affinity to and translocation via hPEPT1. The esters L-Glu(O-i-Bu)-Sar and L-Glu(OCH(2)Ada)-Sar and the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were synthesized, and affinity to and translocation via hPEPT1 were investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay using (14)C-labeled Gly-Sar. Translocation was measured as fluorescence ratios induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All compounds showed high affinity to hPEPT1, but only the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were translocated by hPEPT1. hPEPT1 is very susceptible to modifications of the N-terminal amino acid side chain of dipeptidomimetic substrates, in terms of achieving compounds with high affinity for the transporter. However, as affinity is not predictive of translocation, derivatization in this position must be performed with great caution since some of the compounds investigated turn out not to be translocated by the transporter.