Kinetex 5u evo c18 100a column

Analytical HPLC analysis was carried out on a SHIMADZU HPLC with Kinetex 5u EVO C18 100A column (100 mm × 4.60 mm, 5 μm, Phenomenex) monitoring at 215 nm and 280 nm. Solvents for analytical HPLC were water with 0.1% (v/v) trifluoroacetic acid (TFA) as solvent A and acetonitrile with 0.1% (v/v) TFA as solvent B. Compounds were analyzed at a flow rate of 0.5 mL/min.

Reagents were obtained from AlfaAesar and Sigma-Aldrich in at the highest purity available and used as supplied. ¹H NMR were performed on INOVA 400 spectrometers. Analytic HPLC analysis was carried out on a SHIMADZU LC with Kinetex 5u EVO C18 100A column (100 mm × 4.60 mm, 5 μm, Phenomenex) monitoring at 215 nm and 326 nm. Solvents for analytical HPLC were water with 0.1% trifluoroacetic acid (TFA) as solvent A and acetonitrile with 0.1% TFA as solvent B. Compounds were analyzed at a flow rate of 0.5 ml/min. LC-MS experiments were carried out on a Shimadzu HPLC LC20-AD and ThermoScientific LCQ Fleet with a Kinetex 5 μm EVO C18 100A column (30 × 2.1 mm, 5 μm, Phenomenex) monitoring at 215 and 260 nm with positive mode for detection of mass-to-charge ratio of ions. Solvents for LC-MS were water with 0.1% acetic acid (solvent A) and acetonitrile with 0.1% acetic acid (solvent B) at a flow rate of 0.3 ml/min.

Different concentrations (0.686, 2.058, 6.173, 18.519, 55.556, 166.667μM) of small molecule inhibitors were pre-incubated with 0.3 μM sirtuins (SIRT1, SIRT2, and SIRT3) and 1 mM NAD in buffer (20 mM Tris-HCI, pH 8.0, 1 mM DTT) at 37 °C for 10 min. Then 10 μM of acyl peptide (KQTARK(Ac)STGGWW)was added to initiate the reactions. The reactions were then incubated at 37°C in a total volume of 60 μl (5 min for SIRT1 and SIRT2, 20 min for SIRT3). The conversion of each reaction was less than 20%.The reactions were stopped by adding 60 μl of an aqueous solution of 50% methanol containing 200 mM HCI and 320 mM acetic acid. The reaction mixtures were then centrifuged at 17000g for 10 min and analyzed on an analytical HPLC with Kinetex 5u EVO C18 100A column (100 mm × 4.60 mm, 5.0 μm, Phenomenex). The gradient was: 0% B for 2 min, 0 to 30% B in 13 min, and then 30% to 100% B in 10 min at a flow rate of 0.5 ml/min. All reactions were done in duplicate. The product and the substrate peaks were quantified using HPLC UV absorption traces at 280 nm.