Id was analyzed additional. Samples had been taken at suitable occasions to measure the initial reaction prices (1 h) and to determine the course of conversion (up to 24 h). Reversed-Phase HPLC Analytics. Samples had been analyzed on an Agilent 1200 HPLC method (Santa Clara, CA, USA) equipped with a Kinetex EVO C18 column (five m, one hundred 150 four.6 mm; Phenomenex, Aschaffenburg, Germany) and also a UV detector. The column temperature was 45 . The injection volume was 5-10 L. The eluent flow price was 1 mL/min. The column was equilibrated in water containing 0.1 formic acid. Elution was accomplished with an growing gradient of acetonitrile (0.1 formic acid), beginning from ten . Assays. Reactions with 4-methylumbelliferone and 7-amino-4methylcoumarin have been analyzed with ten -60 acetonitrile more than 15 min. The column was washed with 90 acetonitrile for two min and equilibrated with 10 acetonitrile for three min. The acceptors as well as the corresponding -D-glucosides were detected at 220 and 320 nm. Reactions with phloretin were analyzed as described inside the literature.32,33 Detection from the acceptor and the corresponding -Dglucoside was at 288 nm. Reactions with COMT Inhibitor Gene ID 15-hydroxy Cinmethylin. A gradient of 20-75 acetonitrile more than 5.5 min was used. The column was washed with 75 acetonitrile for two min and equilibrated with 20 acetonitrile for four.5 min. 15-Hydroxy cinmethylin and its mono–D-glucoside and di–Dglucosides were detected at 203 nm. NMR. The -D-glucoside isolated from the reaction with 15hydroxy cinmethylin (ten mM) was analyzed. Briefly, the solution washttps://doi.org/10.1021/acs.jafc.1c01321 J. Agric. Meals Chem. 2021, 69, 5491-Journal of Agricultural and Meals Chemistrypubs.acs.org/JAFCArticleFigure two. HPLC evaluation of enzymatic glycosylation of 15-hydroxy cinmethylin. (A) 15-hydroxy cinmethylin (acceptor, orange) and 15-hydroxy cinmethylin -D-glucoside (product, black). (B,C) Samples of reactions of C. tinctorius UGT71E5 (B; pH 7.4; 0 h, orange; 0.five h, green; two h, blue; 6 h, purple) and BcGT1 (C; pH 7.4; 0 h, orange; 1 h, green; six h, purple) with 15-hydroxy cinmethylin (1 mM) and UDP-glucose (2 mM). The insets show close-ups in the HPLC traces to highlight further items (peaks at three.7 and four.1 min) formed by BcGT1 but not by UGT71E5. (D) Glycosylation products (HPLC traces at 0 h, orange; 0.5 h, green; and three h, purple) formed upon reaction of BcGT1 with 15-hydroxy cinmethylin D-glucoside (1 mM) and UDP-glucose (two mM). From their elution, the solutions formed will be the exact same because the additional glycosylation items in the reaction of 15-hydroxy cinmethylin (panel C). Disaccharide-bearing merchandise in the general form 15-hydroxy cinmethylin -D-glucosyl–Dglucoside are suggested. purified by reversed-phase HPLC utilizing the Kinetex EVO C18 column utilized also for analytical determinations. Pooled fractions (15 mL) were concentrated to about one-twentieth of your original volume using a SHP2 custom synthesis Heidolph Laborota 4000 rotary evaporator equipped having a Vacuubrand PC2001 pump and CVC2000II controller (Wertheim, Germany; 40 , 230 mbar) then lyophilized overnight with an Alpha 1-4 freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at -40 and 0.020 mbar. The solution was taken up in DMSO-d6 (99.8 D) for NMR measurements. A Varian Unity Inova 500 MHz spectrometer (Agilent Technologies) and VNMRJ 2.2D software program were utilised. 1H- and 13C NMR, COSY, HSQC, and HMBC spectra have been recorded. The spectra with the enzymatically synthesized item enabled unambiguous assignment.
