Enzymatic Synthesis of Novel and Highly Soluble Puerarin Glucoside by Deinococcus geothermalis Amylosucrase

Puerarin (daidzein-8-C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties; however, the poor solubility of puerarin limits its applications. To resolve this poor solubility, Deinococcus geothermalis amylosucrase (DgAS) was used to modify puerarin into more soluble derivatives. The results showed that DgAS could biotransform puerarin into a novel compound: puerarin-4′-O-α-glucoside. The biotransformation reaction was manipulated at different temperatures, pH values, sucrose concentrations, reaction times, and enzyme concentrations. The results showed that the optimal reaction condition was biotransformed by 200 μg/mL DgAS with 20% (w/v) sucrose at pH 6 and incubated at 40 °C for 48 h, and the optimal production yield was 35.1%. Puerarin-4′-O-α-glucoside showed 129-fold higher solubility than that of puerarin and, thus, could be further applied for pharmacological use in the future.


Introduction
Puerarin (daidzein-8-C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties and was approved for clinical trials in diabetes mellitus (DM) by the Chinese Ministry of Health in 1993 [1]. Recently, puerarin has proved to be a potential anticancer drug [2]. However, its water solubility remains low although puerarin contains a glucosyl residue attached to the parent isoflavone [3]. Thus, its low solubility makes puerarin difficult to apply in food processing and results in poor absorption after oral administration. Only two dosage forms could be found on the market until now: puerarin injections and eye drops [4]. Therefore, improving the solubility of puerarin could strengthen its applications.
Molecular modifications could be achieved by either chemical or enzymatic approaches [5]. However, chemical modifications sometimes involve multiple steps of protection/deprotection to control regioselectivity. Multiple-step reactions usually reduce the final product yield. In contrast, one-step enzymatic modification could promote regio-and enantioselective catalytic activity. For example, glycosylation can significantly improve the solubility of molecules [6]. Glycosylation modulates the solubility, bioavailability, and chemical properties of many natural products, such as flavonoids and steroids [7]. Many novel bioactive flavonoid glycosides have been obtained by enzymatic glycosylation for developing new and potential drugs [8].

Biotransformation of Puerarin by DgAS
Puerarin was biotransformed by DgAS to modify puerarin. The biotransformation products were analyzed by HPLC. The results showed that DgAS could biotransform puerarin into a major product, compound (1) ( Figure S1, see Supplementary Materials). Fifteen point six percent of puerarin was converted to compound (1) by DgAS under the original testing conditions.
To improve the efficiency of the biotransformation by DgAS, the reaction conditions were optimized with different sucrose concentrations, temperatures, pH values, and times. The results showed that the optimal reaction conditions were 20% (w/v) sucrose and pH 6 at 40 • C for 48 h (Figure 1). Under the optimal reaction conditions, the yield of compound (1) from the biotransformation of puerarin by DgAS was increased to 24.1%. To improve the production yield in advance, the DgAS enzyme concentration was increased from 25 µg/mL to 200 µg/mL in reactions. The results showed that the highest yield of compound (1) can reach 35.1% when DgAS was increased to 200 µg/mL ( Figure 2).

Identification of the Biotransformation Product
To reveal the chemical structures of compound (1), the biotransformation was scaled up to 40 mL. The biotransformation product was purified by preparative HPLC. The chemical structure of the purified compound was then resolved using mass and nucleic magnetic resonance (NMR) spectral analyses. The molecular formula of compound (1) Figure S2). The functional groups of compound (1) were analyzed by infrared (IR) spectroscopy. The results revealed significant absorption at 3273 cm −1 , representing the typical hydroxyl groups, and another at 1623.9 cm −1 , representing the typical carbonyl groups in compound (1) ( Figure S3). The compound (1) characteristic 1 H and 13 C NMR sugar signals have been assigned to C-glucosyl and O-glucosyl moieties by 1D and 2D NMR experiments. The 1 H spectrum of compound (1) in DMSO-d 6 showed one singlet at 8.42 ppm; eight doublets at 4.81, 5.42, 6.99, 7.13, 7.13, 7.52, 7.52, and 7.94 ppm; and a complex 10-spin system between 3.0 and 5.0 ppm. An analysis of this second-order system revealed coupling constants typical of two glucose moieties. The compound (1) glycosidic linkage of the C-glucosyl moiety on puerarin C-8 has been revealed by the presence of heteronuclear multiple bond connectivity (HMBC) correlations between C-8/H-1 (112.7/4.81 ppm) and anomeric proton H-1 at 4.81 (d, J = 9.1 Hz), indicating a C-β-configuration of puerarin, supported by data from previous literature [30]. The puerarin O-glucosyl moiety was a doublet signal at H-1 (5.42 ppm, d, J = 3.5 Hz), with the corresponding carbon atom at C-1 (98.0 ppm) assigned to the anomeric proton, and indicated an O-α-configuration by heteronuclear single quantum coherence (HSQC), which is in the O-α-configuration, and the H-1 (δ = 5.42 ppm) of puerarin, as well as the HMBC cross signals H-1 /C-4 (5.42/156.9 ppm). The significant downfield shift in the 13 C signal of C-4 indicated the connection of the second glucosyl moiety. The NMR signals were fully identified, as shown in Table S1. Compound (1) was thus confirmed to be puerarin-4 -O-α-glucoside (Table S1 and Figures S4-S10). Figure 3 illustrates the biotransformation process of puerarin by DgAS. Previously studied GH enzymes catalyzed two major glycosylations-α-(1→6 )puerarin and α-(1→4 )-puerarin-on the C-glucoside residue of puerarin [9][10][11][12][13][14][15][16][17]. In contrast, DgAS preferred catalyzing glycosylation on the 4 -hydroxyl group of puerarin and produced a novel derivative. Our previous study also showed that DgAS catalyzed glycosylation on the 4 -hydroxyl group of 8-OHDe-7-O-glucoside and produced 8-OHDe-7,4 -O-diglucoside [29]. Both studies suggest that DgAS is a good enzyme for producing isoflavones-4 -glucoside.

Aqueous Solubility of Puerarin and Its Derivatives
The solubility of puerarin and its derivatives was determined by HPLC analysis. The results showed that puerarin-4 -O-α-glucoside had a solubility 129 folds higher than that of puerarin ( Table 1). The well soluble puerarin-4 -O-α-glucoside could present an alternative application to the industry. It is known that the more sugars are attached, the higher the solubility of the modified molecules. Table 2 summarizes various puerarin glycosides produced by different GH enzymes. All reported puerarin-monoglycosides linked to the C8-glucoside of puerarin possessed glycosyl attached to one site (C8). Most puerarin-monoglycosides possessed 14-fold to 23-fold higher aqueous solubility than that of puerarin. Furthermore, puerarindiglycosides or puerarin-triglycosides possessed over 100-fold higher aqueous solubility than that of puerarin. On the other hand, the solubility of puerarin-4 -O-α-glucoside (puerarin-monoglycoside) was 129 folds higher than that of puerarin. A possible reason for such higher aqueous solubility might be the mono-sugar linked to the two glucosyl sites (C8 and C4 ) of puerarin-4 -O-α-glucoside. Higher solubility of a similar linkage was also found in the 8-OHDe-7,4 -O-α-diglucoside, which was linked to the two glucosyl sites (C7 and C4 ); it was seven folds higher than that of daidzin-4 -O-α-glucoside [29]. This study revealed that glycosylation at the 4 -hydroxyl group on the isoflavone skeleton would yield better solubility than glycosylation at the hydroxyl groups on the C-glucoside of puerarin. This study highlighted a novel and unique action of DgAS on the 4 -glycosylation of isoflavones. Previous studies revealed that O-glycosylated flavonoids (vitexin, isovitexin, and isoorientin) could be deglycosylated to recover the bioactivities of the parental flavonoids via in vitro fecal fermentation [31] or in vivo intestinal microbes [32]. In contrast, it has been reported that C-glucosides (e.g., puerarin) are more resistant to acidic and enzymatic hydrolysis [31]. Thus, the novel puerarin glucoside (O-glucoside) might also be in vivo digested to the parental puerarin as previous studies has shown [31,32]. Nevertheless, we expect that the high solubility of puerarin glucosides is an advantage for clinical therapy, in which the puerarin glucosides could be absorbed more easily and deglycosylated into the human body with a wider range of pharmaceutical dosages. Further clinical experiments are needed to confirm the bioactivities in the future.

Microorganism and Chemicals
Puerarin was purchased from Baoji Herbest Bio-Tech (Xi-An, Shaanxi, China). Recombinant DgAS was obtained from our previous studies [28,29]. One unit of DgAS activity was defined as the amount of the enzyme that hydrolyzed sucrose into 1 µmol of fructose per minute. The specific sucrose hydrolysis activity of the purified recombinant DgAS was determined to be 6.6 U/mg. The other reagents and solvents used were commercially available.

Biotransformation Using DgAS
The reaction mixture (0.1 mL) comprised 25 µg/mL of DgAS, 1 mg/mL of the tested substrate compound (diluted from a stock of 20 mg/mL in DMSO), 50% (w/v) sucrose, and 50 mM of phosphate buffer (PB) at pH 7 and was incubated at 40 • C for 24 h. The reaction was stopped by adding an equal volume of methanol and was analyzed using high-performance liquid chromatography (HPLC). To optimize the reaction conditions, different temperatures, pH values, sucrose concentrations, and reaction times were used. The buffers used were 50 mM of acetate buffer (pH 5), PB (pH 6 and 7), and Tris buffer (pH 8).

HPLC Analysis
HPLC was performed with the Agilent 1100 series HPLC system (Santa Clara, CA, USA) equipped with a gradient pump (Waters 600, Waters, Milford, MA, USA). The stationary phase was a C18 column (5 µm, 4.6 i.d. × 250 mm; Sharpsil H-C18, Sharpsil, Beijing, China), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The elution condition was a linear gradient from 0 min with 40% B to 20 min with 70% B, an isocratic elution from 20 min to 25 min with 70% B, a linear gradient from 25 min with 70% B to 28 min with 40% B, and an isocratic elution from 28 min to 35 min with 40% B. All eluants were eluted at a flow rate of 1 mL/min. The sample volume was 10 µL. The detection condition was set at 254 nm.

Purification and Identification of the Biotransformation Metabolite
The purification process followed a previously described method [33]. To purify compound (1), the biotransformation reaction was scaled up to 40 mL (1 mL per tube), and the 40-vial reactions were incubated with 180 rpm of shaking at 40 • C for 24 h. After the reaction, compound (1) was purified by a preparative YoungLin HPLC system. The fraction with the compound (1) was collected, condensed under a vacuum, and then dehydrated by freeze drying. In total, five batches of the 40 mL reactions were purified for 60.1 mg of compound (1). The production yield was 60.1 mg/277.8 mg = 21.6%, which is the purified product (60.1 mg) divided by that theoretical value of 100% conversion rate [(40 mL × 1 mg/mL puerarin)/(416 molecular weight of puerarin) × [578 molecular weight of compound (1)] × 5 batches] = 277.8 mg]. The structures of the compound were confirmed with NMR and mass spectral analyses. Mass analyses were performed using the Finnigan LCQ Duo mass spectrometer (ThermoQuest Corp., San Jose, CA, USA) with electrospray ionization (ESI). 1 H-and 13 C-NMR, distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded on a Bruker AV-700 NMR spectrometer at ambient temperature. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million (ppm, δ). Puerarin

Determination of Solubility
The aqueous solubility of puerarin and its glucoside were determined according to a previous method with slight modification [34]. Twenty milligrams of the tested compound were resuspended in 50 µL of double-deionized H 2 O with 180 rpm of shaking at 25 • C for 1 h. The mixture was then centrifuged at 10,000× g at 25 • C for 30 min. The supernatant was filtrated with 0.2 µm of nylon membrane for HPLC analysis. For HPLC analysis, 10 µL of the puerarin filtrate or 2 µL of the puerarin-4 -O-α-glucoside filtrate were 100-fold or 500-fold diluted with 50% methanol, respectively. The concentrations of the tested compounds were determined with calibration curves of authentic samples. The final concentrations of the standard solutions were prepared with 4, 8, 12, 16, or 20 mg/L of puerarin (from 20 mg/mL of stock in DMSO) or with 100, 200, 300, or 400 mg/L of puerarin-4 -O-α-glucoside (from 20 mg/mL stock in DMSO) in 50% methanol.

Conclusions
Puerarin possesses important pharmacology activities for clinical usage. However, the poor solubility property of puerarin limits its dosage. In the present study, puerarin could be glycosylated by DgAS to produce a new compound, puerarin-4 -O-α-glucoside. Puerarin-4 -O-α-glucoside showed 129-fold higher solubility than that of puerarin. Such higher solubility could be further applied for pharmacological therapy in the future.