*2.2. Transglycosylation of IFAs with DGAS*

Three IFAs (daidzein, glycitein, and genistein) are found in soybeans, and their glucosides are linked by β-glycosidic bonds at C-7 (Figure 2) [4]. Glycosylation carried out by glycosyltransferases, such as AS, generates two types of *O*-glucosides, which are commonly defined as α- and β-linked glucosides [35]. The detailed glycosylation mechanism has not been clearly elucidated, but the glycosylation reaction usually occurs on the anomeric carbon with the attack of weak nucleophiles, such as the −OH group of sugars [35]. Glycosylation reactions often involve a unimolecular (SN1) or bimolecular (SN2) nucleophilic substitution at the anomeric center [35].

**Figure 2.** Structures of isoflavone glucosides (daidzin, glycitin, and genistin) present in soybeans.

DGAS used in this study produced various transglycosylated isoflavones with α-1,4-glycosidic linkages from IFAs (Figure 3). The transglycosylation of IFAs (such as in genistein) using DGAS are likely to occur at the C-7 or C-4 positions rather than the C-5 position. Previous studies reported that recombinant AS created various stereostructures of acceptor compounds due to position-specific transglycosylation [27,33] (Figure 3). Moreover, the transglycosylation of flavonoids using DGAS has been reported to occur primarily at the C-7 position of the A ring and at the C-4 position of the B ring [27,32,36]. DGAS has been reported to attach four or fewer glucose moieties to flavonoids in transglycosylation [21,32]. Thus, we expected the total number of glucose moieties attached to IFAs in the DGAS transglycosylation process to be four. For example, if four glucoses bind to the C-7 position, the number of glucoses that can attach to the C-4 position is zero, and if three glucoses bind to the C-4 position, one additional glucose may bind to the C-7 position (Figure 3).

**Figure 3.** Scheme of transglycosylation reactions of isoflavone aglycones (IFAs) and sucrose using DGAS. G, glucose; F, fructose.

To identify reactants and products after transglycosylation, 12 isoflavone standards found in soybeans were separated by high-performance liquid chromatography (HPLC; peaks labeled: GD, daidzin; GL, glycitin; GG, genistin; MD, malonyldaidzin; ML, malonylglycitin; AD, acetyldaidzin; AL, acetylglycitin; MG, malonylgenistin; DN, daidzein; LN, glycitein; AG, acetylgenistin; GN, genistein) (Figure 4a). The results of transglycosylation using DGAS of the three IFA standards are shown chromatographically in Figure 4b–d. Compared to the 12 isoflavone standards, six to eight new peaks were detected, indicating that various transglycosylated products were produced from each aglycone during the enzymatic process (Figure 4b–d). Transglycosylated products from daidzein tended to be more polar (faster retention time) than transglycosylated glycitein and genistein glucosides for the same HPLC analysis conditions. This tendency was the same as the elution order of the three aglycones: daidzein > glycitein > genistein (Figure 4a). We attributed the different elution order of transglycosylated IFAs to their polarity differences, which are mainly influenced by the polarity of the parent compounds (acceptors in this study). If patterns of bound positions and the number of glucose moieties were the same in each aglycone, the elution order of transglycosylated IFAs according to increasing retention time would have been as follows: transglycosylated daidzein > transglycosylated glycitein > transglycosylated genistein. It was previously reported that α-glycosylisoquercitrin with more glucose moieties produced by the enzymatic transglycosylation of isoquercitrin eluted earlier than α-glycosylisoquercitrin with fewer glucose moieties [37]. Furthermore, after the transglycosylation of daidzin using DGAS, daidzein triglucoside eluted faster than daidzein diglucoside [36]. Therefore, in this study, transglycosylated IFAs with more glucose moieties had a faster elution time in reversed-phase HPLC analysis than transglycosylated IFAs with fewer glucose moieties (Figure 4b–d).

**Figure 4.** High-performance liquid chromatography (HPLC) traces (254 nm) of IFA standards after transglycosylation using DGAS. (**a**) Isoflavone standard mixture, (**b**) transglycosylated glycitein, (**c**) transglycosylated daidzein, and (**d**) transglycosylated genistein. Peaks: GD, daidzin; GL, glycitin; GG, genistin; MD, malonyldaidzin; ML, malonylglycitin; AD, acetyldaidzin; AL, acetylglycitin; MG, malonylgenistin; DN, daidzein; LN, glycitein; AG, acetylgenistin; GN, genistein. Peaks labeled LN1 to LN6, DN1 to DN8, and GN1 to GN6 are transglycosylated products from each IFA. Enzymatic transglycosylation reactions were performed using 1.0 mM of glycitein, 5.0 mM of daidzein, or 5.0 mM of genistein with 5.0 U of DGAS and 2.0 M of sucrose.

Peaks observed at the very beginning of elution (up to 12 min of retention time in this study) of the chromatograms were considered to be transglycosylated IFAs with two, three, or four glucose moieties (Figure 4b–d). However, more polar products (transglycosylated IFAs with 2–4 glucose moieties) among the transglycosylated IFAs resulted in lower resolution in the chromatogram. It was previously reported that transglycosylated products generated from the DGAS-catalyzed transglycosylation of isoquercitrin had similar retention times to each other, which resulted in very low resolution and greater difficultly separating them [32]. In addition, some isoquercitrin glucosides with high polarity have been reported to exist as isomers with the same number of sugars, the molecular weights of which were the same in mass spectra [32]. Based on previously reported results [32], we hypothesized that the transglycosylated IFA glucosides that eluted at similar retention times in this study were likely to be isomers with the same number of glucose moieties bound at different positions.

Peaks (except for minor peaks < 5% of the total product area) of transglycosylated reaction products from glycitein, daidzein, and genistein after enzymatic modification were labeled (Figure 4b–d). Peak LN5 in Figure 4b had a retention time similar to glycitin (glycitein 7-β-*O*-glucoside; peak GL of Figure 4a). The retention times of peak DN7 in Figure 4c and peak GN6 in Figure 4d were similar to those of daidzin (daidzein 7-β-*O*-glucoside; peak GD of Figure 4a) and genistin (genistein 7-β-*O*-glucoside; peak GG of Figure 4a), respectively. Thus, we assumed that peaks LN5, DN7, and GN6 were α-glucosides of IFA with one glucose moiety attached. Standard compounds (daidzin, glycitin, and genistin) found in soybeans have a β-glycosidic bond between IFA and glucose. When IFAs are transglycosylated using DGAS, glucose moieties bound to the −OH group at the C-7 or C-4 position in IFAs are present in an α-glycosidic bond with IFAs [27,33]. The 7-α-monoglucosides (peaks LN5, DN7, and GN6) from the enzymatic modification of IFAs had different but similar retention times compared to standard isoflavone 7-β-monoglucosides (Figure 4b–d). Previously, daidzein triglucoside was reported to be more polar than daidzein diglucoside [36]. Due to their greater number of transglycosylated glucose moieties, LN1–LN4 from glycitein transglycosylation, DN1–DN6 from daidzein transglycosylation, and GN1–GN5 from genistein transglycosylation had higher polarities than LN5, DN7, and GN6, respectively (Figure 4b–d). DGAS produces higher amounts of flavonoid glucosides attached with a greater number of glucose molecules to parent flavonoids such as isoquercitrin and daidzin than glycosyltransferases such as CGTase and amyloglucosidase [27,32,36], which produce higher levels of flavonoid glucosides transglycosylated with fewer sugar moieties [22,24]. When UDP-glucose is used as a donor for glycosidic binding, only one or two new transglycosylated products are produced by glycosyltransferases [16,22]. In contrast, DGAS generated a variety of new transglycosylated products from the IFAs (Figure 4b–d). As shown in Figure 3, the number of transglycosylated glucoses has been reported to range from one to four [27,32], because the free hydroxyl (−OH) groups of flavonoid aglycones and their transglycosylated glucosides can be used as potential transglycosylation sites in the DGAS enzyme process.
