1260 infinity series 2 hplc

Stocks of 1 and 2 (250 mM in MeCN with 0.1% formic acid) were diluted to 1 mM using 10 mM ammonium bicarbonate buffer with 10% MeCN. Samples of 1 were incubated with purified RrNIT enzyme (90 µg). The final volume of each reaction was 500 µL. Reactions were incubated at 37 ºC for 1–24 h, and enzymes were precipitated via addition of 500 µL cold MeOH wih 1% formic acid. Reactions were filtered and the filtrate was further analyzed. Reactions incubated for 24 h were treated with an additional 90 µg RrNIT and incubated for an additional 1 h at 37 ºC. Synthetic standards for 1 and 2 were prepared in 50:50 H₂O:MeCN with 0.1% formic acid. The standards and reaction samples were analyzed using an Agilent Technologies 1260 Infinity Series II HPLC equipped with a diode array detector. Separations were performed using an Agilent Poroshell C18 column (eluting with 50–100% MeCN in H₂O containing 0.1% formic acid over 20 min). The flow rate was set at 0.2 mL/min. Raw traces were exported as .CSV files and analyzed in GraphPad Prism 6. Data were min-max normalized prior to plotting.