Of its structure as HDAC1 site 15-hydroxy cinmethylin -D-glucoside. The spectra are shown inside the Supporting Details Figures S2-S4, as well as the spectral information are summarized inside the Supporting Info Table S1. LC-MS. A Shimadzu LCMS-2020 program (Kyoto, Japan) equipped with a Nucleodur C18 gravity column (3 m, 110 150 three mm, Macherey-Nagel, Duren, Germany) was made use of. A linear gradient (ten to 85 ) of acetonitrile in ammonium acetate buffer (five mM, pH 6.67) over 5 min was utilized. The column was washed with ten acetonitrile for 2.five min. The flow rate was 0.7 mL/min. The column temperature was 30 . A UV detector tuned to 210 and 262 nm was applied. Masses have been scanned over the selection of 150-800 in the constructive mode. The masses of mono-glucoside (452.five; [M + H]+, 453.5; [M + Na]+, 475.5; and [M + K]+, 491.five) and bis-glucoside (614; [M + H]+, 615; [M + Na]+, 637.6; and [M + K]+, 653.six) have been also analyzed within the SIM mode. The obtained data are shown inside the Supporting Information and facts Figure S5.Final results AND DISCUSSION Panel of Leloir GTs for 15-Hydroxy Cinmethylin Glycosylation. To recognize enzyme(s) for -glycosylation of 15-hydroxy cinmethylin, we selected a representative panel of eight GTs (Table 1) active with UDP-glucose and showing broad specificity for bulky acceptor substrates. We chose a balanced distribution between GTs of plant (4 enzymes) and microbial origin (3 enzymes). Amongst the bacterial GTs, the OleD from S. antibioticus is often a well-characterized enzyme which has been widely employed for small-molecule glycosylation.38,39 Its triple variant was laboratory-evolved for even broadened donor and acceptor scope.38,39 OleD wildtype and its triple mutant ASP are active with major alcohols and benzyl alcohols as in 15-hydroxy cinmethylin in particular.38,39 We furthermore employed the human GT UGT1A9 to examine glucuronidation of 15-hydroxy cinmethylin from UDPglucuronic acid. Working with Cytochrome P450 medchemexpress recombinant production in E. coli, we obtained the GTs within a hugely purified kind (Supporting Info Figure S1). Reaction using the regular acceptor substrate from the literature revealed that each and every enzyme was functional, displaying the required activity for glycosylation from UDP-glucose (Table 1) and appropriate for test of reactivity with 15-hydroxy cinmethylin. HPLC trace with the sample from the UGT71E5-catalyzed conversion of 15-hydroxy cinmethylin inside the presence of UDPglucose revealed the look of a brand new compound peak (Figure 2) that elevated in abundance because the 15-hydroxy cinmethylin consumption progressed. The mass data (452.5;https://doi.org/10.1021/acs.jafc.1c01321 J. Agric. Meals Chem. 2021, 69, 5491-Journal of Agricultural and Food Chemistrypubs.acs.org/JAFCArticleFigure three. Time courses of enzymatic glycosylation of 15-hydroxy cinmethylin. Reactions utilized two mM UDP-glucose. The 15-hydroxy cinmethylin D-glucoside (open circles), the 15-hydroxy cinmethylin (closed circles), and the putative disaccharide glycoside of 15-hydroxy cinmethylin (open triangles) are shown. (A) UGT71E5; (B) UGT71A15; (C) BcGT1; (D) OleD wildtype; and (E) OleD triple variant ASP. The concentration on the putative disaccharide glycosides of 15-hydroxy cinmethylin was obtained because the sum with the two product peaks at three.7 and 4.1 min, as shown in Figure 2C. Initial prices of 15-hydroxy cinmethylin -D-glucoside formation were calculated from the data and are shown in Table 1.[M + H]+, 453.5; [M + Na]+, 475.five; and [M + K]+, 491.five) for the product are fully consistent with these of singly glycosylated 15-.
