Gly pro amc

Vildagliptin was synthesized in our laboratory using the standard technique7 (link). Vildagliptin carboxylic acid metabolite (M20.7) was purchased from Santa Cruz Biotechnology (Delaware Avenue, CA, USA). Sitagliptin was obtained from LKT Laboratories (St. Paul, MN, USA). 7-Amino-4-methylcoumarin (AMC) was purchased from Setareh Biotech, LLC (Eugene, OR, USA). Gly-Pro-AMC was obtained from Bachem (Bubendorf, Switzerland). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primers were commercially synthesized at Life Technologies (Carlsbad, CA, USA). All other chemicals were of the highest grade available.

DPP-4 inhibitory activity was determined using a previously described method [36 (link)] where free AMC (7-amino-4-methyl-coumarin) is liberated from DPP-4 substrate Gly-Pro-AMC. Briefly, samples were prepared in PBS (pH 7.4) and assays were conducted in triplicate using a 96 well microtiter plate. Each well contained 20 µL of sample (3 mM, 1 mM and 0.3 mM), 20 µL of porcine DPP-4 (1 U/mL) (EMD Millipore, UK) and 30 µL of 1 mM Gly-Pro-AMC (Bachem, Switzerland). Plates were incubated (37 °C; 1 h) with gentle agitation. Reactions were stopped by addition of acetic acid (100 µL; 3 mM) and fluorescence (Ex 351 nm/Em 430 nm) measured (Safire II fluorometer, Tecan, Reading, UK). Berberine an isoquinoline alkaloid with DPP-4 inhibitory activity (IC50 13.3 µM) was used as a positive control in each experiment.