Glycosylation of the polyphenols from Resina draconis by glycosyltransferase YjiC1

Abstract Resina Draconis (RD), also known as ‘dragon's blood’, contains a broad range of natural compounds, such as flavonoids, stilbenes and dihydrochalcones. It is clinically used to enhance blood circulation. However, the major components of RD suffer from relatively poor water solubility. Glycosylation is a critical determinant for modulating solubility and improving bioavailability and bioactivity of natural products. Herein, we report a novel method to efficiently synthesize glycosidic derivatives of the major polyphenols in RD using a microbial glycosyltransferase, i.e., YjiC1. Solubility test showed that the synthetic glycosidic derivatives displayed higher water solubility than the raw materials. This research sheds light on the structural modification of natural products for higher water solubility, which is important for innovative drug discovery. Graphical Abstract


Introduction
Resina Draconis (RD) is a kind of resin extracted from Dracaena cochinchinensis (Lour.) S.C.Chen, a plant in Agavaceae family (Liu et al. 2006;Chen et al. 2018). As a traditional Chinese medicine (TCM), the chemical compositions of RD include a variety of polyphenolic compounds Sun et al. 2019Sun et al. , 2021. Many important ingredients in RD, such as pterostilbene and loureirin B, perform strong pharmacological activities in clinical studies, while their applications are limited due to the poor water solubility and low bioavailability. Glycosylation is a prominent strategy to increase solubility and pharmacodynamic properties of chemicals (Thibodeaux et al. 2007;H€ arle and Bechthold 2009;Williams et al. 2011). Chemical glycosylation is achieved through multi-steps of protection and deprotection of functional groups, and often suffers from low yield and limited regiospecificity (He et al. 2019;Ruprecht et al. 2019). Moreover, when synthesizing complex scaffolds, the high-cost problem must be overcome. In contrast, structurally diverse substrates can be readily modified by enzymatic catalysis in vitro (Ye et al. 2002;Chen et al. 2016). Compared with chemical synthesis, biocatalysis shows higher catalytic efficiency and is more environmentally friendly.
The glycosyltransferase YjiC1, derived from Bacillus subtilis, can efficiently catalyse the glycosylation of various substrates, including flavonoids, steroids and terpenoids . Moreover, YjiC1 can also catalyse the formation of O-, N-and S-bonded glycosides (Ramesh et al. 2014). Our previous work showed that YjiC1 could be used to transform various bufadienolide aglycones into the corresponding glycosides , indicating that YjiC1 not only had a high catalytic ability, but also had a broad substrate promiscuity. Therefore, we hypothesized that this promiscuous enzyme could be used to modify the active polyphenols in RD to obtain the glycosylated derivatives with higher water solubility.
Herein, the glycosides of main polyphenolic compounds in RD were obtained using the glycosyltransferase YjiC1. Structures of the glycosylated products were identified by mass spectrometry and extensive NMR analyses. Furthermore, compared with their aglycones, the hydrophilicity of the corresponding glycosides was found to be significantly enhanced, as evidenced by the increased polarities in reversed-phase chromatography. In addition, the water solubility was also tested by subsequent quantitative analysis.

Heterologous expression of YjiC1
To characterize the catalytic function of YjiC1 in vitro, we constructed an expression plasmid of YjiC1, which contains an open reading frame (ORF) of 1281 bp encoding 392 amino acids. The plasmid was introduced into E. coli BL21 (DE3) cells for heterologous expression. Notably, as a water-soluble protein with His 6-tagged label (N-terminal), YjiC1 could be readily purified by Ni-NTA metal affinity chromatography. Convincingly, the result of SDS-PAGE indicated that the molecular weight of YjiC1 was approximately 47 kDa, which was consistent with the predicted molecular weight ( Figure S1). This purified recombinant enzyme was used for the glycosylation of polyphenols from RD.

Glycosylation catalysed by YjiC1
In order to explore the catalytic activity of YjiC1, we tested the conversion rates using the polyphenols from RD as the substrates and uridine-5 0 -diphosphate glucose (UDP-Glc) as the sugar donor. Subsequently, the corresponding glycosylation products were analysed by high performance liquid chromatography-diode array detector (HPLC-DAD, Figure S2) and LC-MS (Table S1). As shown in Table S1, all components were effectively converted (>70%), indicating the high glycosylation ability of YjiC1. In addition, we prepared the glycosylation products of some polyphenols in RD (1a, 2a, 3a, 4a) (Scheme 1) for detailed structural characterization using HR-ESI-MS and NMR (for original spectra, see supporting materials showed a fragment ion unit 162 Da, indicating that 1a was a monoglycoside. 1 H and 13 C NMR spectra of 1a were similar to those of its aglycone pterostilbene (1), except for the presence of one sugar unit (Agnes et al. 1994). The 13 C-NMR spectrum showed a resonance at d C ¼ 101.57 ppm, ascribed to the anomeric carbon of the sugar unit. The 1 H NMR signals d H 4.98-4.88 were correspondent to the protons on the glucose moiety. The large coupling constant J ¼ 7.6 Hz of the anomeric proton indicated b-orientation of the sugar moiety. The HMBC correlation between H-1 0 ' and C-4 0 confirmed the glycosylation site at C-4 0 . Furthermore, H-3 0 (d H 7.09) and H-5 0 (d H 7.07) moved to the low field by 0.42 ppm and 0.41 ppm, respectively, further confirming the glycosylation site at 4 0 -OH. Combination of the spectra data and the retention time in HPLC, product 1a was finally confirmed to be pterostilbene- carbon of the sugar unit. The large coupling constant J ¼ 7.6 Hz of the anomeric proton (d H 5.03, 1H, d, J ¼ 7.6 Hz, H-1 0 ') indicated the b-orientation of sugar moiety. The HMBC correlation between H-1 0 ' and C-4 0 confirmed the glycosylation site at C-4 0 . Combination of the spectra data, product 2a was finally confirmed to be loureirin B- The loss of 162 Da in MS/MS spectra of 3a and 4a indicated that they were monoglycosides. 1 H and 13 C NMR spectra of 3a and 4a were similar to those of loureirin A (3) (Meksuriyen and Cordell 1988) and trans-resveratrol (4) , respectively, except for the presence of one sugar unit. The large coupling constants (7.2 Hz for 3a and 4a) of the anomeric protons suggested that the glucose moieties in 3a and 4a were b-oriented. The low field shift for H-3 0 and H-5 0 after a detailed comparison of the corresponding protons between the aglycones and glycosides (3: d H-3 0 ¼6.84, d H-5 0 ¼6.81; 3a: d H-3 0 ¼7.16, d H-5 0 ¼7.16; 4: d H-3 0 ¼ 6.76, d H-5 0 ¼6.73; 4a: d H-3 0 ¼7.00, d H-5 0 ¼6.97) revealed that the glucose moieties were attached to 4 0 -OH for both compounds. Taking these data together, product 3a was confirmed to be loureirin A-4 0 -O-b-D-glycoside, which is a new compound. Product 4a was finally confirmed to be trans-resveratrol-4 0 -O-b-D-glucoside, consistent with the reported data (Wang et al. 2013).
Accordingly, all four glycosides (1a, 2a, 3a, 4a) were determined. Our results showed that YjiC1 exhibited excellent glycosylation ability for the stilbenes and dihydrochalcones (>70%) structural types. As the main polyphenols of RD, these compounds were mainly catalysed to produce O-glycosides at 4 0 -OH. To the best of our knowledge, it is the first time to report the glycosylation of dihydrochalcone compounds by enzymatic catalysis in vitro, though glycosylated dihydrochalcones had ever been obtained by cultures of entomopathogenic filamentous fungi (Agnieszka et al. 2021).

Optimization of glycosylation conditions
To explore the effects of temperature, pH, metal ions and reaction time on catalytic efficiency of YjiC1, we conducted a series of experiments using compound 2 as the substrate ( Figure S3). YjiC1 had strong conversion efficiency at 25 $ 45 C (>80%), and the maximum conversion efficiency was achieved at 40 C (90.1%). YjiC1 displayed remarkable activity when the pH of Tris-HCl buffer was in the range of 7 to 9, and the optimal pH was 8.5. It is noteworthy that YjiC1 still showed certain catalytic activity without adding any divalent cations. However, Mg 2þ , Mn 2þ and Co 2þ could increase the activity of YjiC1, while Cu 2þ , Zn 2þ and Pd 2þ showed inhibition of the enzyme. Particularly, when Pd 2þ was involved in the reaction, there was almost no transformation. This indicates that metal ions, as cofactors, may affect or occupy the active sites of YjiC1, and thereby affect the protein conformation of YjiC1. In addition, YjiC1 could complete the reaction in a very short time (about 10 min). Taking these results together, the optimized reaction was carried out in Tris-HCl buffer (pH 8.5) at 40 C for10 min with some divalent metal ions (Mg 2þ , Mn 2þ and Co 2þ ).

Molecular docking
To explore the binding modes of YjiC1 with its substrates, molecular docking of compounds 1 or 2 with YjiC1 was performed ( Figure S4). The docking model showed that compound 1 was buried in a conserved active pocket of YjiC1. It formed intermolecular hydrogen bonds with Glu301 (2.037, 2.388 Å), Met296 (2.051 Å) and Thr229 (2.490 Å) of YjiC1, which might be the critical residues involved in the binding interactions of YjiC1 with 1. Similarly, three residues Thr229 (2.022 Å), Asn297 (1.977 Å) and Met296 (2.639 Å) of YjiC1 were found to show hydrogen bonding interactions with compound 2.

Water solubility
Generally, compared with the corresponding aglycone, the hydrophilicity of the glycoside is significantly enhanced as evidenced by the substantial increase of polarity in reversed-phase chromatography. Water solubilities of aglycone and the corresponding glycoside were determined by high performance liquid chromatography based on the linear regression equation between the content and peak area.
The linear ranges were found to be 10 $ 1250 lM for 1-3. Through HPLC analysis, the peak areas of 1 and 1a were 2427.2 and 4527.7, respectively. The calculated water solubilities of 1 and 1a were 0.285 and 0.865 mg/ml, respectively. Thus, the water solubility was relatively increased by 3.031 times after glycosylation. The peak areas of 2 and 2a were 874.1 and 1062.6, respectively, and the calculated water solubilities of 2 and 2a were 0.112 and 0.196 mg/ml, respectively (1.744 times increase). Similarly, the peak areas of 3 and 3a were 245.7, 738.4, respectively, and the calculated water solubilities of 3 and 3a were 0.050 and 0.141 mg/ml (2.848 times increase) (Table S2). These results indicated that the solubility of compounds could be effectively improved by glycosylation.

General
The substrate resveratrol and UDP-Glc were purchased from Aladdin (Shanghai city, China). The other substrates were purchased from Chenguang Bio-Tech Ltd. (Baoji city, China). The Agilent 1200 series system (Agilent Technologies, USA) equipped with a Kinetex EVO-C18 column (250 mm Â 4.6 mm, 5 lm) was used for HPLC analysis. Semipreparative HPLC was performed on the Wufeng LC-100 system (Shanghai Wufeng Scientific Instruments Co., Ltd., Shanghai city, China) equipped with a UV detector. The sample preparation was performed on a COSMOSIL C18-MS-II column (250 mm Â 10.0 mm inner diameter, 5 lm) (Nacalai Tesque, Inc., Japan). NMR analysis was performed with a Bruker AV-400 spectrometer, using tetramethylsilane as an internal standard (Bruker, Fallanden, Switzerland).

Enzyme preparation
Enzyme YjiC1 (GenBank Accession Number JX982974) was expressed in the E. coli cell (DE3) and purified by His-tag affinity chromatography according to the reported procedures ).

Molecular docking
Molecular docking was conducted according to the reported procedures (Gao et al. 2016) using the software Sybyl-X 2.0. The initial structures of ligands 1 or 2 were constructed with software Chemdraw 19.0. All ligands were subjected to energy minimization. The crystal structure of protein YjiC1 was obtained from the RCSB PDB Protein Data Bank (PDB code: 7bov).

Water solubility test
The water solubility test was conducted according to the reported procedures . In brief, the aglycones and their corresponding glycosylation products were dissolved in methanol, and then analysed by HPLC to calculate the regression equations. Then the excess samples were added to deionized water to reach saturation. After centrifugation, the supernatants were taken for HPLC analysis to get the peak areas. Then the concentration levels were calculated based on the regression equations and peak areas.

Conclusions
In summary, we have reported a method for the efficient biosynthesis of polyphenolic glycosides catalyzed by glycosyltransferase YjiC1. The current work shows that the drug-like molecules of polyphenols in RD can be decorated with sugars. We completed the glycosylation biosynthesis and afforded the corresponding O-glucosides which were not found in nature. Catalytic reaction could be achieved in high yield under the following conditions: temperature 40 C, Tris-HCl buffer at pH 8.5 with some divalent ions as the cofactor, and a reaction time of 10 min. Compared with aglycones, the corresponding glycosides exhibited significantly higher water solubility. This study highlighted that the diversity-oriented molecular modification could be generated through biosynthetic pathway catalyzed by enzymatic reactions (Xie et al. 2018).

Disclosure statement
No potential conflict of interest was reported by the authors.