*3.6. Isolation and Identification of Mangiferin Glycosides by PgMA*

The glycosylation of mangiferin by *Pg*MA was scaled up to 100 mL. The products M1 and M2 were purified using preparative HPLC. From the 100 mL reaction, 20.1 mg of compound (**1**) (M1) and 9.3 mg of compound (**2**) (M2) were isolated. The molecular weights of the purified products were then determined with mass spectrometry. The mass spectrometry of compound (**1**) revealed an [M–H]− ion peak at *m/z*: 583.4 in the electrospray ionization mass spectrum (ESI-MS) corresponding to the molecular formula C25H28O16 (Figure S1). The mass data imply that M1 contains one glucosyl moiety attached to the mangiferin structure. In the mass data of M2, an [M–H]− ion peak at *m/z*: 745.3 in the ESI-MS corresponded to the molecular formula C31H38O21 (Figure S2), which implies that compound (**2**) contains two glucosyl moieties attached to the mangiferin structure. To identify the structures in advance, the structures of both compounds were determined using NMR spectroscopy. 1H and 13C NMR, including the DEPT, HSQC, HMBC, COSY, and NOESY spectra, were obtained.

The characteristic 1H and 13C NMR sugar signals in compound (**1**) were assigned to *C*-glucosyl and *O*-glucosyl moieties by one-dimensional (1-D) and 2-D NMR experiments. The 1H spectrum of compound (**1**) in DMSO-*d6* showed three singlets at 6.36, 6.86, and 7.37 ppm and a complex 10-spin system between 3.0 and 5.0 ppm. Analysis of this secondorder system revealed coupling constants typical of two glucose moieties. The compound (**1**) glucosidic linkage of the *C*-glucosyl moiety on the xanthone C-2 was revealed by the presence of HMBC correlations between C-2/H-1 (107.5/4.58 ppm), and the anomeric proton H-1 at 4.58 (d, *J* = 9.1 Hz) indicated a *C*-*β*- configuration of mangiferin that was confirmed by the data reported in the literature [44]. The mangiferin *O*-glucosyl moiety was a doublet signal at H-1 (4.73 ppm, d, *J* = 4.2 Hz) with the corresponding carbon atom at C-1 (98.7 ppm) assigned to the anomeric proton and indicating an *O*-*α*-configuration by HSQC, which is in the *O*-*α*-configuration. The H-1 (*δ* = 4.73 ppm) of mangiferin and the HMBC cross signaled H-1/C-6 (4.73/66.9 ppm) and H-6 a, 6 b/C-1 (3.46, 3.52/98.7 ppm). The significant downfield shift of the 13C signal of C-6 indicated the connection of the second glucosyl moiety, which confirmed the *α*-(1→6) linkage of the second glucosyl moiety. The NMR signals were identified as shown in Table S3. The compound (1) was thus confirmed as glucosyl-*α*-(1→6)-mangiferin (Figures S3–S9).

The 1H spectrum of compound (**2**) in the same compound (**1**) solvent also showed three singlets at 6.36, 6.86, and 7.37 ppm and a complex 11-spin system between 3.0 and 5.0 ppm. Analysis of this second-order system revealed coupling constants typical of three glycose moieties, which included the chemical shifts listed in Table S3. The glucosyl moiety chemical shifts of C-2 at 107.4 ppm and H-1 at 4.58 ppm (d, *J* = 9.8 Hz) according to the corresponding HMBC indicated a C-C bond between the sugar and the aglycone of mangiferin (*C*-glucosyl-xanthone) and were confirmed by the data reported in the literature [44]. The *O*-maltosyl moiety connected to mangiferin was confirmed by HMBC from the anomeric carbon C-6 (66.9 ppm), and the corresponding anomeric proton H-1 at 4.75 (d, *J* = 3.5 Hz) indicated an *O*-*α*-configuration. The maltose doublet signal at *δ*<sup>H</sup> H-1 (d, *J* = 3.5 Hz) and H-1 4.95 (d, *J* = 3.5 Hz) with the corresponding carbon atom at C-1 (98.6 ppm) and C-1 (100.9 ppm) was assigned to the anomeric proton and indicated two *O*-*α*-configurations by HSQC. The HMBC cross peaks of C-1/H-6 (98.6/3.64, 3.71 ppm) and C-1/H-4 (100.9/3.34 ppm) confirmed the *α*-(1→4) between the two-glucosyl moiety. Our experimental 1H and 13C chemical shifts listed in Table S2 confirmed compound (**2**) as maltosyl-*α*-(1→6)-mangiferin (Figures S10–S16). Figure 8 summarizes the biotransformation process of mangiferin by *Pg*MA.
