OcUGT1-Catalyzed Glucosylation of Sulfuretin Yields Ten Glucosides

Sulfuretin glucosides are important sources of innovative drugs. However, few glucosides of sulfuretin have been observed in nature. Therefore, it is urgent to diversify sulfuretin glycosides. Herein, glycosyltransferase (GT)-catalyzed glycodiversification of sulfuretin was achieved. Specifically, a flavonoid GT designated as OcUGT1 was used as a biocatalyst for the glucosylation of sulfuretin with UDP-Glc. The OcUGT1-assisted glucosylation of sulfuretin yielded ten glycosylated products, including three monoglucosides, five diglucosides and two triglucosides. The three monoglucosides were thus identified to be sulfuretin 3′-, 4′and 6-glucoside according to HR-ESI-TOFMS data and their coelution with respective standards. A major diglucoside was assigned as sulfuretin 4′,6-diglucoside by HR-ESI-TOFMS in conjunction with NMR analysis. The exact structure of the other four diglucosides was not well characterized due to their trace amount. However, they were reasonably inferred as sulfuretin 3′,6-diglucoside, sulfuretin 3′,4′-diglucoside and two disaccharide glucosides. In addition, the structural identification of the remaining two triglucosides was not performed because of their small amount. However, one of the triglucosides was deduced to be sulfuretin 3′,4′,6-triglucoside based on the catalytic behavior of OcUGT1. Of the ten sulfuretin glucosides, at least six were new compounds. This is the first time to obtain monoglucosides, diglucosides and triglucosides of sulfuretin simultaneously by a single glycosyltransferase.


Introduction
Glycodiversification is a collective strategy of natural product glycosylation, in which varied activated sugars are attached to natural-product acceptors by enzymatic or chemical means, thereby providing diverse carbohydrate structures and functions [1,2]. The resultant glycosylated bioactive compounds have been shown to exert various biological and pharmacological activities with improved physicochemical characters, such as solubility and stability [3,4]. Many glycosides are thus developed to clinical drugs, e.g., rutin [5][6][7], puerarin [8] and scutellarin [9]. Hence, glycodiversification of natural products is deemed an effective strategy to broaden the scope of new compounds [2].
Owing to the structural complexity of many glycosylated compounds, glycodiversification of natural products by chemical synthesis may be a formidable task [2]. Conversely, enzymatic glycodiversification is becoming a main strategy for diversifying glycosylated natural products due to the great strides made in the generation of glycosyltransferase with catalytic promiscuity [10][11][12][13].

Protein Expression and Purification
After induction by IPTG, total proteins of Escherichia coli strain BL21(DE3) [pET28a-OcUGT1 + pKJE7] were subject to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis [13]. As shown in Figure 2A, an intense band with 53 kDa was detected in the sample. No corresponding band was present in the control strain, suggesting a soluble OcUGT1 was expressed in E. coli (Figure 2). The expressed OcUGT1 was thus purified to near homogeneity and its concentration was determined for glucosylation reaction. OcUGT1 (Ornithogalum caudatum UDP-glycosyltransferase), isolated from O. caudatum previously [13], is a flavonoid glycosyltransferase (GT) with catalyzing promiscuity. OcUGT1 can glucosylate diverse sugar acceptors including flavonoids. Moreover, OcUGT1 has been observed to function on multiple sites of flavonoids, yielding a number of flavonoid glycosides [13]. Both indicate OcUGT1 is an ideal tool for glycodiversification of small molecules. OcUGT1 was used as a biocatalyst for the glucosylation of sulfuretin with UDP-D-glucose (UDP-Glc). OcUGT1-assisted glucosylation of sulfuretin resulted in the formation of ten glucosides including three monoglucosides, five diglucosides and two triglucosides. Of these ten newly formed glycosides, at least six glucosides were new compounds ( Figure 1). Thus, the use of single glycosyltransferases capable of forming multiple glycosides is an effective way to achieve glycosidic diversification, and can significantly increase the probability of drug discovery.

Protein Expression and Purification
After induction by IPTG, total proteins of Escherichia coli strain BL21(DE3) [pET28a-OcUGT1 + pKJE7] were subject to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis [13]. As shown in Figure 2A, an intense band with 53 kDa was detected in the sample. No corresponding band was present in the control strain, suggesting a soluble OcUGT1 was expressed in E. coli ( Figure 2). The expressed OcUGT1 was thus purified to near homogeneity and its concentration was determined for glucosylation reaction.

OcUGT1-Catalyzed Glycosylation towards Sulfuretin
After incubated at 50 °C for 2 h, the reaction mixture containing purified OcUGT1, sulfuretin and UDP-Glc was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). As shown in Figure 3, ten new peaks 1a-j were present in the reaction mixture ( Figure 3), while there were no new peaks in the control reaction harboring no purified OcUGT1 ( Figure 3) suggesting the ten peaks might be glucosylated metabolites of sulfuretin.

OcUGT1-Catalyzed Glycosylation towards Sulfuretin
After incubated at 50 • C for 2 h, the reaction mixture containing purified OcUGT1, sulfuretin and UDP-Glc was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). As shown in Figure 3, ten new peaks 1a-j were present in the reaction mixture (Figure 3), while there were no new peaks in the control reaction harboring no purified OcUGT1 (Figure 3) suggesting the ten peaks might be glucosylated metabolites of sulfuretin.

OcUGT1-Catalyzed Glycosylation towards Sulfuretin
After incubated at 50 °C for 2 h, the reaction mixture containing purified OcUGT1, sulfuretin and UDP-Glc was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). As shown in Figure 3, ten new peaks 1a-j were present in the reaction mixture (Figure 3), while there were no new peaks in the control reaction harboring no purified OcUGT1 (Figure 3) suggesting the ten peaks might be glucosylated metabolites of sulfuretin.

Structural Identification of Sulfuretin Monoglucosides
The ten metabolites were then subjected to high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. The positive ion HR-ESI-MS spectrum of 1a displayed a molecular ion peak at m/z 455.0928 [M + Na] + corresponding to C 21 H 20 O 10 Na ( Figure S2).The major metabolite 1b exhibited a pseudomolecular ion peak [M + Na] + at m/z 455.0927, and the molecular formula C 21 H 20 O 10 Na was established by HR-ESI-MS ( Figure S2). The molecular formula of a minor product 1c was determined to be C 21 H 20 O 10 Na, by HR-ESI-MS at m/z 455.0943 [M + Na] + ( Figure S2). The evidence suggests that all three metabolites were monoglucosylated sulfuretins. Coelutions of these metabolites with their standards assigned 1a, 1b and 1c to be sulfuretin 3 -, 4 -and 6-glucoside, respectively [29].

Structural Identification of Sulfuretin Triglucosides
The HR-ESI-MS of 1i and 1j displayed molecular ion [M + Na] + peaks at m/z 779.2011 and 779.2031, respectively, both corresponding to the molecular formula of C 33 H 40 O 20 Na, which indicated that both compounds were triglucosides of sulfuretin ( Figure S6). The structures of the two triglucosides were not well characterized due to their trace amount. According to the catalytic behavior of OcUGT1 [13], one of the triglucosides was sulfuretin 3 ,4 ,6-triglucoside. The other triglucoside could not been deduced from the HR-ESI-MS data. To the best of our knowledge, the two triglucosides were also new compounds.

Structural Identification of Sulfuretin Triglucosides
The HR-ESI-MS of 1i and 1j displayed molecular ion [M + Na] + peaks at m/z 779.2011 and 779.2031, respectively, both corresponding to the molecular formula of C33H40O20Na, which indicated that both compounds were triglucosides of sulfuretin ( Figure S6). The structures of the two triglucosides were not well characterized due to their trace amount. According to the catalytic behavior of OcUGT1 [13], one of the triglucosides was sulfuretin 3′,4′,6-triglucoside. The other Overall, OcUGT1-catalyzed glucosylation of sulfuretin led to the generation of ten glucosides including six new compounds. The data revealed that enzyme-mediated glucosylation is an effective way to diversify glucosides. Previously, glycosyltransferases capable of accepting glycosides for further glycosylation have been reported [3]. However, there are few glycosyltransferases that catalyze the formation of monoglycosides, diglucosides and triglycosides of a single substrate simultaneously. In this study, OcUGT1 has been demonstrated to catalyze sulfuretin to form corresponding monoglycosides, disaccharides and triglycosides simultaneously, indicating that OcUGT1 has a very wide substrate specificity. These results, together with previous reports [13,31,32], indicate that OcUGT1 has potential applications as a biocatalyst in glycodiversification of natural products.

Protein Expression and Purification
Heterologous expression and purification of OcUGT1 was performed as described previously [13]. As introduced by Yuan et al., an expression plasmid pET28a-OcUGT1 and a chaperone plasmid pKJE7 (Takara, Dalian, China) were co-transformed into E. coli strain BL21 (DE3) for soluble expression. Total protein extracts from isopropyl-β-D-thiogalactoside (IPTG)-induced bacterial cells were separated by SDS-PAGE. The expressed recombinant protein with His-Tag were purified by affinity chromatography. The concentration of the purified protein was determined based on the procedure introduced by Yin et al. [33]. The resultant purified OcUGT1 was applied as the biocatalyst for the glycosylation towards sulfuretin (1) ( Figure S1).

Glycosylation Assay
The reaction mixture and reaction conditions of OcUGT1-catalyzed glycosylation assay was the same as that of our previous reports [13]. In brief, a total of 100 µL phosphate buffer (10 mM, pH 8.0) harboring 10 mg purified OcUGT1, 1 mM sulfuretin and 1 mM UDP-Glc were incubated at 50 • C for 2 h. The glycosylation reaction was monitored by RP-HPLC. The HPLC conditions were the same as previously described by Yuan et al. [13].

Structural Identification
HR-ESI-MS spectra were recorded on A Triple TOF™ 5600 system (AB SCIEX, CA, USA) with a DuoSpray ionization source operating in the positive ESI mode.

Conflicts of Interest:
The authors declare no conflicts of interest.