Luna 5 c18 2 100 a

Strictosidine synthase was purified as in 48 (link). Strictosidine was generated enzymatically by incubation of 4 mM tryptamine (Sigma), 2 mM purified secologanin (Sigma) and 2 µM strictosidine synthase in 50 mM phosphate buffer pH 7.4 buffer, in a total volume of 50 ml, overnight at 37 °C. This reaction was monitored for strictosidine production and consumption of secologanin by LC-MS. The solution was purified by solid phase extraction, eluted in 100% MeOH, and dried under vacuum. The dried product was resuspended in H₂O, filtered with 2 µm filters and purified by preparative HPLC in 2 ml aliquots. The HPLC used was a Dionex Ultimate 3000 pump, with a variable wavelength UV detector. The column used for chromatographic separation was a Phenomenex Luna 5µ C18(2) 100 A (250 x 30 mm), with the binary solvent system consisting of 0.1% TFA and acetonitrile. The elution program was the following: 1 min isocratic at 10% ACN, 8 min gradient up to 30% ACN, 4 min gradient up to 100% ACN, 6 min isocratic at 100% ACN, 1 min gradient down to 10% ACN, and 6 min isocratic at 10% ACN. The compounds were monitored by UV 241 nm, and the fractions collected at the retention time 13.5 min. The resulting fractions were lyophilized and analyzed by NMR.