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Of its structure as 15-hydroxy cinmethylin -D-glucoside. The spectra are shown within the Supporting Data Figures S2-S4, plus the spectral data are summarized in the Supporting Information and facts Table S1. LC-MS. A Shimadzu LCMS-2020 method (Kyoto, Japan) equipped using a Nucleodur C18 gravity column (three m, 110 150 3 mm, Macherey-Nagel, Duren, Germany) was made use of. A linear gradient (ten to 85 ) of acetonitrile in ammonium acetate buffer (5 mM, pH six.67) more than 5 min was employed. The column was washed with ten acetonitrile for 2.five min. The flow price was 0.7 mL/min. The column temperature was 30 . A UV detector tuned to 210 and 262 nm was used. Masses had been scanned more than the range of 150-800 inside the positive mode. The masses of mono-glucoside (452.five; [M + H]+, 453.five; [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) were also analyzed in the SIM mode. The obtained information are shown in the Supporting Data Figure S5.Outcomes AND DISCUSSION Panel of Leloir GTs for 15-Hydroxy Cinmethylin Glycosylation. To determine enzyme(s) for -glycosylation of 15-hydroxy cinmethylin, we chosen a representative panel of eight GTs (Table 1) active with UDP-glucose and displaying broad specificity for bulky acceptor substrates. We chose a balanced distribution between GTs of plant (4 enzymes) and microbial origin (three enzymes). Amongst the bacterial GTs, the OleD from S. antibioticus is usually a well-characterized enzyme which has been extensively 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 primary alcohols and benzyl alcohols as in 15-hydroxy cinmethylin in certain.38,39 We moreover applied the human GT UGT1A9 to examine glucuronidation of 15-hydroxy cinmethylin from UDPglucuronic acid. Employing recombinant production in E. coli, we obtained the GTs in a extremely purified type (Supporting Info Figure S1). Reaction using the typical acceptor substrate in the literature revealed that each enzyme was functional, displaying the expected activity for glycosylation from UDP-glucose (Table 1) and ERRĪ± Accession appropriate for test of reactivity with 15-hydroxy cinmethylin. HPLC trace from the sample from the UGT71E5-catalyzed conversion of 15-hydroxy cinmethylin within the presence of UDPglucose revealed the appearance of a brand new compound peak (Figure two) that enhanced in abundance because the 15-hydroxy cinmethylin consumption progressed. The mass data (452.5; J. Agric. Meals Chem. 2021, 69, 5491-Journal of Agricultural and Food 3. Time courses of enzymatic glycosylation of 15-hydroxy cinmethylin. GLUT4 Molecular Weight Reactions utilised 2 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 in the putative disaccharide glycosides of 15-hydroxy cinmethylin was obtained because the sum of your two item peaks at three.7 and four.1 min, as shown in Figure 2C. Initial rates of 15-hydroxy cinmethylin -D-glucoside formation have been calculated from the information and are shown in Table 1.[M + H]+, 453.5; [M + Na]+, 475.five; and [M + K]+, 491.5) for the item are fully constant with those of singly glycosylated 15-.