T with the product formation kinetics in the BcGT1 CXCR1 custom synthesis reaction (Figure 3C): the doubly glycosylated items only appeared in the mixture following the mono-glycoside had been released in substantial amounts. Additionally, we showed that purified 15-hydroxy cinmethylin -D-glucoside (Figure 1) was the substrate for additional glycosylation from UDP-glucose catalyzed by the BcGT1 (Figures 2D and 4). Reaction with 15-hydroxypubs.acs.org/JAFCArticlePreparative Synthesis of 15-Hydroxy Cinmethylin The UGT71A15 showed low activity for glycosylation of 15-hydroxy cinmethylin (Table 1), along with the yield of 15-hydroxy cinmethylin -D-glucoside didn’t exceed 60 (0.6 mM; Figure 3B). To examine limitations on UGT71A15 synthetic utility triggered by the reaction conditions, we carried out the synthesis inside the presence of an enzyme stabilizer [tris(2-carboxyethyl)phosphine; up to 5.0 mM] and used varied concentrations (1.0-5.0 mM) of UDP-glucose. We also applied in situ formation of UDP-glucose via the sucrose synthase reaction (Figure 1B). The results are shown in the Supporting Facts Figures S6-S9. The formation of 15-hydroxy cinmethylin -D-glucoside was marginally improved by these adjustments in reaction situations. We hence concluded that UGT71A15 was not a probably candidate enzyme for thriving application in the synthesis of 15-hydroxy cinmethylin -D-glucoside. Possessing chosen UGT71E5, we analyzed the impact of the DMSO co-solvent around the enzyme activity. The co-solvent was essential to boost the 15-hydroxy cinmethylin solubility to a minimum target concentration of 10 mM. UGT71E5 activity was strongly inhibited by DMSO (Figure five), with half of theD-Glucoside.Figure four. Glycosylation of 15-hydroxy cinmethylin -D-glucoside by BcGT1. The reaction made use of two mM UDP-glucose and 0.5 mg/mL BcGT1. The symbols show 15-hydroxy cinmethylin -D-glucoside (open circles, 1 mM) as well as the putative disaccharide glycosides of 15hydroxy cinmethylin (closed circles). The concentration in the disaccharide-modified 15-hydroxy cinmethylin was obtained as the sum in the two product peaks at 3.7 and 4.1 min, as shown in Figure 2C. The handle lacking BcGT1 is shown in open triangles.cinmethylin -D-glucoside gave the same disaccharide glycoside products as identified from reaction with 15-hydroxy cinmethylin (Figure 2D). The rate of glycosylation of 15hydroxy cinmethylin -D-glucoside determined from Figure 4 (6.5 mU/mg) was 9.2-fold decrease than the glycosylation rate of 15-hydroxy cinmethylin. Interestingly, BcGT1 reaction with 15-hydroxy cinmethylin stopped just after 1 h (Figure 3C), regardless of the fact that a substantial portion from the acceptor substrate (35 ) was nevertheless remaining. We noted that the UDPglucose was largely depleted at this point, implying that the substrate had been utilized in methods (e.g., hydrolysis of UDPglucose) not fully accounted for by our analytical procedures. Thinking of the focus of this study on the synthesis of 15-hydroxy cinmethylin -D-glucoside, we did not pursue these qualities in the BcGT1 reaction, leaving them for future study. Reactions of your OleD enzymes (Figure 3D,E) involved ROR Purity & Documentation Iterative glycosylation of your 15-hydroxy cinmethylin similarly as with BcGT1. The conversion of 15hydroxy cinmethylin was 86 , greater than in the BcGT1 reaction. Iterative glycosylation of small-molecule acceptors was previously reported for both BcGT1 and OleD. The flavonoid kaempferol was converted into the di- or tri-O–Dglucoside by BcGT1.49 Glycosylation of thiophenol by OleD gave.