Phe-Gly Dipeptidomimetics Designed for the Di-/Tripeptide Transporters PEPT1 and PEPT2:  Synthesis and Biological Investigations

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester <BO>1</BO>, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (<BO>2a</BO>), (R)- and (S)-hydroxyethylidene (<BO>3a</BO> and <BO>4a</BO>), and (R)- and (S)-hydroxyethylene (<BO>5a</BO> and <BO>6a</BO>) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere <BO>2a</BO> and the natural substrate Phe-Gly (K<INF>i</INF> values of 18.8 and 14.6 μM, respectively). Also the affinities for hPEPT1 are in the same range (K<INF>i</INF> values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C<INF>4</INF>-position for this class of compounds. Furthermore, the intracellular uptake of <BO>2a</BO>−<BO>4a</BO> in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of <BO>2a</BO> in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of <BO>3a</BO> and <BO>4a</BO> was observed. Transepithelial transport studies also indicated active transport of <BO>2a</BO> across Caco-2 monolayers.