An Efficient Strategy for the Glycosylation of Total Bufadienolides in

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Article Cite This: ACS Omega 2019, 4, 6819−6825

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An Efficient Strategy for the Glycosylation of Total Bufadienolides in Venenum Bufonis Zhi-Hao Fu,†,§ Chao Wen,†,§ Qing-Mei Ye,‡,§ Wei Huang,† Xuan-Ming Liu,† and Ren-Wang Jiang*,† †

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Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, and International Cooperation Laboratory of TCM Modernization and Innovation Drug Development, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China ‡ Department of Pharmacy, Hainan General Hospital, Haikou 570311, P. R. China S Supporting Information *

ABSTRACT: Drug discovery process and biological research critically depend on the access to libraries of molecules with interesting biomolecular properties. Thus, it is extremely important to develop robust and efficient strategies to access structurally diverse druglike compound collections. We introduce here a strategy for glycosylation of the total bufadienolides in Venenum Bufonis (VB) by using a permissive glycosyltransferase YjiC1 with conversion rates up to 90%, which was more efficient than other two enzymes. Compared to the crude extract, the glycosylated VB showed lower toxicity against zebrafish and more potent inhibitory activity on Na+,K+-ATPase. The results demonstrated the great advantages of using permissive enzymes as an alternative strategy for producing structurally diverse natural product-like libraries.

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INTRODUCTION Venenum Bufonis (VB), also called ChanSu or toad venom, is an important Traditional Chinese Medicine derived from the parotid gland secretion of giant toads (Bufo bufo gargarizans or Bufo melanostictus).1−3 It is applied in various preparations, such as Liu-Shen-Wan, She-Xiang-Bao-Xin pill, and ChanSu injection, for the treatment of heart failure, pains, and various cancers in clinics.4,5 The total bufadienolides are known to be the major active constituents of VB in these preparations and more than 50 bufadienolides have been isolated.6 However, the high toxicity, low bioavailability, and poor water solubility of total bufadienolides limit the clinic applications.7,8 Glycosylation is a universal approach to increase the water solubility, which would also improve their pharmacodynamic and pharmacokinetic properties.9−11 Therefore, it is of great significance to develop an efficient strategy for the glycosylation of total bufadienolides in VB, which would possibly alleviate its toxicity and facilitate the clinical application. Chemical synthesis of steroid glycosides is hindered by many drawbacks, such as poor regio- and stereoselectivities, requirement of protection and deprotection steps, use of toxic catalysts, and production of chemical waste.12−14 Instead, biocatalysis has been recognized as an alternative way to solve these problems, which is environment-friendly and costeffective.15,16 It was reported that the cell suspension cultures of several specific plant species are capable of glycosylation of cinobufagin, such as Catharanthus roseus, Platycodon grandiflorus, and Saussurea involucrata.17−19 However, low yields and © 2019 American Chemical Society

generation of byproducts make it hard to purify the target steroid glycosides. Recently, significant progress has been made in enzymatic glycosylation of steroid compounds. For example, the microbial glycosyltransferase (GT) OleD-ASP, YjiC1, and plant GT UGT74AN1 showed robust catalytic efficiency toward cardenolide or bufadienolide aglycones,20−22 which makes it possible to establish an efficient strategy for the glycosylation of total bufadienolides in VB. In this paper, the glycosylated VB was produced by the glycosylation of crude VB extracts through the enzymatic transformation of bufadienolide aglycones into the corresponding monoglycosylated or diglycosylated compounds by using permissive GTs (Figure 1). Three GTs were first compared and YjiC1 was found to be the most efficient one. The results showed that the main components were glycosylated with high conversion rates (90%) with YjiC1 catalysis. Compared to the starting extract, the resulting glycosylation products showed more potent activity on the molecular target Na+,K+-ATPase and much lower toxicity on the embryo of zebrafish.

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RESULTS AND DISCUSSION Structural Characterization of Bufadienolides in the VB Extract. The ethanol extract of VB was analyzed by liquid chromatography−mass spectrometry (LC−MS). Compounds gamabufotalin (2), arenobufagin (3), telocinobufagin (4), Received: February 11, 2019 Accepted: April 2, 2019 Published: April 15, 2019 6819

DOI: 10.1021/acsomega.9b00386 ACS Omega 2019, 4, 6819−6825

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OleD-ASP, and UGT74AN1 could not accept desacetylcinobufagin (5) when they were in mixture form (Figure 4). In contrast, the above three compounds can be effectively transformed by OleD-ASP and UGT74AN1 when using the corresponding single compound as the substrate.20,22 YjiC1 was found to accept all these bufadienolides when using a compound library as the substrate. According to the MS data of each enzymatic product (Tables S2−S4), both monoglycosylated and diglycosylated bufadienolides were detected when using YjiC1 or OleD-ASP as the catalyst. In comparison, only monoglycosylated bufadienolides were present when using UGT74AN1 as the catalyst, which further confirmed that UGT74AN1 was a regiospecific 3-O-GT.22 It is noteworthy that 5 was completely converted to desacetylcinobufagin 3,16di-O-β-D-glucoside (5b) under the catalysis of YjiC1, which was a new compound with two sugar units. The above results definitely demonstrated that YjiC1 was the most suitable GT for the glycosylation of the total bufadienolides in VB because of the higher conversion rate and the wider substrate tolerance. Optimization of the Conditions of YjiC1-Catalyzed Glycosylation. To determine the optimal reaction conditions, the conversion rate of YjiC1 at different pH, temperature, divalent metal ions, and reaction time was compared (Figure S3). The results indicated that the optimal pH of YjiC1 was 7.0 with 90% conversion rate, and YjiC1 exhibited over 62% conversion rate among the pH of 6−10, which is similar to its homologous enzyme.26 Moreover, the optimum temperature of YjiC1 was approximately 45 °C, which was higher than many other GTs.27−29 YjiC1 showed the most potent catalytic activity with the addition of Ca2+. The reaction time was tested from 5 min to 8 h, and the best reaction time was 1 h. The conversion rate decreased as the reaction time exceeded 1 h.30 In order to figure out the possible reason, two control experiments were done. Arenobufagin 3-O-β-D-glucoside was incubated with (A) or without (B) the enzyme YjiC1 for 1 h under the same conditions as the bioconversion reaction, and the hydrolyzed peak arenobufagin was observed in the chromatogram of A, whereas it was not observed in the chromatogram of B (Figure S16). These two control experiments indicated that the conversion decrease was due to enzymatic hydrolysis. Establishment of the One-Pot Reaction System. UDPG is the most expensive ingredient in the reaction.31

Figure 1. Schematic representation of the biotransformation of crude VB extract to glycosylated VB via enzymatic catalysis.

desacetylcinobufagin (5), bufalin (8), cinobufagin (9), and resibufogenin (10) were identified by direct comparison with the mixed standard, while compounds bufarenogin (1), bufotalin (6), and cinobufotalin (7) were tentatively characterized based on their UV spectra, full scan mass spectra, and MS/MS spectra (Figure 2 and Table S1) and compared with the previous reports.23,24 The characterization of these peaks enabled us to determine the changes of chemical profiles after the enzymatic glycosylation.25 Selection of GT for the Glycosylation of VB. One plant GT (UGT74AN1 from Asclepias curassavica) and two microbial GTs (OleD-ASP from Bacillus antibioticus and YjiC1 from Bacillus subtilis) were cloned, expressed, and purified (Figure S2). The results showed that the expression level of YjiC1 was apparently higher than the other two GTs, and UGT74AN1 presented the lowest level. The screening reaction was conducted using VB as the substrate, UDP-Glc as the sugar donor, and 1000 μg of purified enzyme as the catalyst. The enzymatic reaction mixture of the experiment was tested on high-performance liquid chromatography (HPLC)diode array detector (DAD) and LC−MS. The conversion rate of VB was represented by the peak area ratios of the bufadienolide aglycones versus corresponding glycosylated products. As shown in the HPLC-DAD chromatogram (Figure 3), a large number of glycosylated bufadienolides were generated according to the retention time, UV absorption, and MS data. It was noteworthy that in our experiment, bufotalin (6) and cinobufotalin (7) were not accepted by

Figure 2. HPLC trace of the EtOH extract of VB. Compounds 2−5 and 8−10 were identified by direct comparison with the mixed standard, while 1, 6, and 7 were tentatively assigned based on the online UV spectra and mass spectra data. 6820

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Figure 3. Selection of GT for the glycosylation of VB. (A) HPLC trace of VB and glycosylated VB by YjiC1, UGT74AN1, and OleD ASP. (a) EtOH extract of VB. (b) Glycosylated VB by YjiC1. (c) Glycosylated VB by UGT74AN1. (d) Glycosylated VB by ASP. Arabic numerals marked with asterisks represent EtOH extract of VB. (B) Structure of bufadienolides and corresponding glycosylated products.

conversion rate of 80%. However, the corresponding monoglycosylated products of bufarenogin (1) and gamabufotalin (2) were not detected. This is the first report using mixed steroids as reactants in the one-pot reaction system. Inhibitory Activities on Na+,K+-ATPase. Cardiotonic steroids, for example, digoxin, are important drugs for the treatment of heart failure owing to their potent inhibition of cardiac Na+,K+-ATPase (NKA), the integral membrane protein that maintains ionic gradients in all superior eukaryotic cells.32 At a dose of 5 μg mL−1, Na+,K+-ATPase activities were inhibited 42 and 86% by the original and the glycosylated extracts of VB, respectively. Then we compared the inhibitory activities of two pairs of compounds, for example, arenobufagin (3) and arenobufagin 3-O-β-D-glucoside (3a) and cinobufagin (9) and cinobufagin 3-O-β-D-glucoside (9a) (Figure 6). The bufadienolides 3 and 9 were isolated from the crude VB extract, while the glycosides 3a and 9a were isolated from the glycosylated VB catalyzed by the enzyme YjiC1. The bioassay results showed that the glycosylated products demonstrated much stronger (about 3-fold) activities than the corresponding aglycones (Table 1). Toxicity Assay of Bufadienolides and the Corresponding Glycosylated Products on Zebrafish Embryo. Zebrafish has become one of the most popular animal models in the world. It is especially a useful tool for the toxicity evaluation of drug molecules.33−35 In the study, first, we compared the toxicity of the crude VB extract and the glycosylated VB at 20 μM and found that the former one demonstrated strong toxicity (all zebrafish showed pericardial edema and bent spine), whereas the latter one was nontoxic. Then we compared the toxicities of the same two pairs of compounds as those in the NKA inhibitory assay and found that the glycosides showed much weaker (>5-fold) toxicity as compared to the corresponding bufadienolide aglycones (Table 1). Thus, toxicity assay on the mixture and single compounds illustrated that glycosylation could notably reduce the toxicity of bufadienolides. In summary, we compared the glycosylation rate of bufadienolides in VB catalyzed by three GTs, and YjiC1 was found to display the highest catalytic efficiency and can accept all bufadienolides when using a compound library as the

Figure 4. Conversion rate of bufadienolides in VB catalyzed by enzymes OleD-ASP, UGT74AN1, and YjiC1. Bufotalin (6) and cinobufotalin (7) were not accepted by OleD ASP, and UGT74AN1 could not accept desacetylcinobufagin (5) when they were in mixture form. In contrast, YjiC1 was found to accept all bufadienolides even using a compound library as the substrate.

The consumption of UDPG is much more in the catalysis of mixture glycosylation than the single substrate. To establish a green, cost-effective enzymatic platform for the glycosylation of total bufadienolides, we exploited the catalytic reversibility of YjiC1 for one-pot transglycosylation reactions. As shown in Figure 5, the reaction was carried out in the presence of only a catalytic amount of UDP and led to the formation of expected products 3a, 4a, 6a, 8a, 10a, and 5b. In order to achieve the highest conversion rate, we explored UDP concentration in the range of 1−1000 μM. The best reaction system was found to be as follows: 50 mM Tris-HCl buffer, pH 8.0; β-D-glucose, 2chloro-4-nitrophenyl (11) 2 mM; UDP 500 μM; VB (1 mM), enzyme YjiC1 500 μg; incubation at 37 °C for 8 h. The reaction products were identified by HPLC−MS/MS. Under this condition, arenobufagin (3) could be glycosylated at a 6821

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Figure 5. Exploiting the catalytic reversibility of YjiC1 for the one-pot glycosylation of the total bufadienolides in VB. (A) One-pot reaction with 2chloro-4-nitrophenyl-β-D-glucopyranoside (11), VB extract and UDP. (B) HPLC trace of the glycosylated products indicating the presence of glycosylated derivatives 3a, 4a, 6a, 8a, 10a, and 5b.

analyses were carried out on Bruker AV-400 spectrometers using tetramethylsilane as an internal standard (Bruker, Fällanden, Switzerland). Chemical shifts (δ) were referenced to internal solvent resonances and given in parts per million (ppm). Coupling constants (J) were given in hertz (Hz). Gene Cloning, Expression, and Purification. UGT74AN1 (GenBank accession number MF942416) was subcloned into pET28a as previously reported. The genes of YjiC1 (GenBank accession number JX982974)36 and Oled (ASP) (GenBank accession number DQ195536) were synthesized and subcloned into pET28a by GenScript (Nanjing, China). The recombinant UGT74AN1-pET28a, YjiC1-pET28a, and Oled (ASP)-pET28a plasmid were transferred into Transetta (DE3) Escherichia coli (TransGen Biotech, China), respectively. The transformation of pET28a control vector was conducted in parallel. A single colony of each transformant was grown in 5 mL of Luria-Bertani (LB) medium supplemented with 50 μg mL−1 kanamycin and 34 μg mL−1 chloromycetin and cultured overnight at 37 °C with shaking (220 rpm). The overnight culture was transferred to 1 L LB medium containing 50 μg mL−1 kanamycin and 34 μg mL−1 chloromycetin and then grown at 37 °C and 220 rpm until the OD600 reached 0.4−0.6.37 For UGT74AN1, isopropyl β-D-thiogalactoside (IPTG) was subsequently added to a final concentration of 0.25 mM to induce the cells. For YjiC1 and Oled (ASP), 0.5 mM IPTG was added. The cells were grown for additional 16 h at 18 °C and 180 rpm. Then the cells were collected by centrifugation at 3000 rpm, 4 °C for 15 min and stored at −80 °C. Frozen cells were resuspended in 10 mL of chilled lysis buffer (20 mM phosphate buffer, 50 mM NaCl, 10 mM imidazole, pH 7.5) and disrupted by sonication in an icewater bath. The cell lysate was centrifuged at 12 000 rpm, 4 °C

substrate. The conditions for YjiC1-catalyzed glycosylation were optimized, and the highest conversion rate was achieved at 45 °C, pH 7.0 for 1 h. Furthermore, a one-pot reaction system was established by using UDP instead of UDPG to catalyze the glycosylation of total bufadienolides in VB. Finally, our zebrafish embryo toxicity assays showed that both monoglycosylated and diglycosylated bufadienolides were much less toxic than the corresponding aglycones. This is the first report on the glycosylation of total bufadienolides in VB by enzymatic catalysis.

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EXPERIMENTAL SECTION

Materials and Reagents. UDP-Glc was purchased from Sigma-Aldrich (Shanghai, China). The standard compounds, that is, bufalin, arenobufagin, cinobufagin, resibufogenin, gamabufotalin, telocinobufagin, and desacetylcinobufagin, were purchased from Chenguang Bio-Tech Ltd. (Baoji, China). HPLC analysis was carried out using an Agilent 1200 series system (Agilent Technologies, USA). HPLC−MS/ MS analysis was performed on a mass spectrometer X500 QTOF (AB Sciex, USA) coupled with Nexera Prominence LC (Shimadzu, Japan). Samples were analyzed on a phenomenex Luna 5 μm C18 analytical column (250 mm × 4.6 mm, 5 μm). Mass spectrometer acquisition parameters: ion source temperature, 550 °C; ion spray voltage, 5500 V; time-of-flight mass range, 50−1000 Da; collision energy for MS, 10 V; collision energy for MS/MS, 30 V. Semi-preparative HPLC was performed on a WUFENG LC-100 system (Wufeng Scientific Instruments Co., Shanghai, China) equipped with a UV detector using a COSMOSIL Packed C18-MS-II column (250 mm × 10.0 mm i.d., 5 μm, Nacalai Tesque, Inc., Japan). NMR 6822

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Figure 6. Comparison of the toxicity of bufadienolides and the corresponding glycosides on zebrafish embryo. (A) Dose-dependent curves for the mortality rate; (B) malformation of zebrafish embryos exposed to the solution of different compounds arenobufagin (3) and arenobufagin 3-O-β-Dglucoside (3a); cinobufagin (9) and cinobufagin 3-O-β-D-glucoside (9a). Malformations are indicated by red arrows. PE, pericardial edema; BS, bent spine.

phoresis (SDS-PAGE), and protein concentration for all studies was determined using Easy Protein Quantitative Kit (Bradford method) (TransGen Biotech, China).38 Identification of Natural Bufadienolides in VB by HPLC−MS/MS. Standard solution preparation: the seven standard compounds gamabufotalin (2), arenobufagin (3), telocinobufagin (4), desacetylcinobufagin (5), bufalin (8), cinobufagin (9), and resibufogenin (10) were dissolved in methanol to prepare a standard solution (40 mM). Then, aliquots of each stock solution were mixed and diluted with methanol up to volume 500 μL. An amount of 50 g of VB was smashed and extracted with 95% EtOH for five times (1 h per extraction).39 The organic extracts were combined and concentrated under reduced pressure to give a brown solid (14.38 g). It was dissolved in dimethyl sulfoxide to prepare a VB mixture (40 mM, considering the average molecular weight 400 Da for the bufadienolides in VB). The VB mixture was filtered through a 0.22 μm micropore membrane filter (Jinteng Corp., Tianjin, China), and a volume of 30 μL was injected for HPLC−MS/MS analysis. Flow rate: 0.7 mL/min; gradient of solvents A (0.1% formic acid aqueous solution) and B (100% acetonitrile): (a) 0−15 min, 28−54% B; (b) 15−30 min, 54% B; (c) 30−35 min, 54−100% B; the UV detection wavelength is 296 nm.23 The mixed reference solution was analyzed using the same conditions as the VB mixture.

Table 1. Inhibitory Activity on NKA and Toxicity on Zebrafish of Two Pairs of Bufadienolides compound 3 3a 9 9a

inhibitory activity on NKA IC50 (μM) 1.65 0.52 3.40 1.22

± ± ± ±

0.10 0.04 0.32 0.10

toxicity on zebrafish LD50 (μM) 7.57 83.37 11.92 65.06

± ± ± ±

0.05 0.04 0.07 0.04

for 40 min to obtain the supernatant (the crude enzyme solution). After filtration through a 0.45 μm syringe filter, the supernatant was immediately loaded onto 5 mL nickel− nitrilotriacetic acid resin (GE Healthcare, USA) pre-equilibrated with the binding buffer (20 mM phosphate buffer, 50 mM NaCl, 10 mM imidazole, pH 7.5) of five column volumes (CVs). The recombinant proteins were allowed to bind for 1 h at 4 °C by slowly inverting the column on a vertical mixer. After washing with five CVs’ wash buffer (20 mM phosphate buffer, 50 mM NaCl, 50 mM imidazole, pH 7.5), the proteins were eluted from the resin using three CVs’ elution buffer (20 mM phosphate buffer, 50 mM NaCl, 250 mM imidazole, pH 7.5). Finally, the proteins were concentrated, and the buffer was exchanged to desalting buffer (50 mM Tris-HCl, pH 7.5) using Amicon Ultra-30K (Millipore, USA). Aliquots of the enzymes were stored at −80 °C. Protein purity was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electro6823

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Screening of the Most Efficient Enzyme. To explore the most efficient enzyme for the VB glycosylation reaction, three equal amounts of pure enzymes (OleD-ASP, YjiC1, and plant UGT74AN1) were separately performed in the range of 125− 2500 μg (Figure S1). For each enzyme, the reactions were performed at a final volume of 500 μL containing 50 mM TrisHCl (pH 8.0), 1 mM VB mixture, 2.5 mM UDP-Glc, 1000 μg of purified enzyme, and 5 mM CaCl2. All the reactions were incubated at 37 °C for 12 h and terminated by adding an equal volume of MeOH. After centrifugation at 12 000 rpm for 30 min, the supernatant of each reaction was analyzed by HPLC− MS/MS. Flow rate: 0.7 mL/min; gradient of solvents A (0.1% formic acid aqueous solution) and B (100% acetonitrile): (a) 0−10 min, 10−20% B; (b) 10−30 min, 20−28% B; (c) 30−45 min, 28−54% B; (d) 45−60 min, 54% B. The control reaction was carried out in the absence of either enzyme or UDP-Glc. For comparison, three parallel assays with different enzymes were carried out at the same conditions. The conversion rates (%) were measured by the peak area ratios of substrates (e.g., arenobufagin) versus corresponding products in HPLC chromatograms. Optimization of the Enzymatic Reaction Conditions. The enzyme amount was optimized, and 1000 μg of the purified enzyme was chosen for the following study. To acquire the optimal pH value of YjiC1 activity, the reaction was carried out in two types of reaction buffers with pH value in the scopes of 3.0−6.0 (citric acid−sodium citrate buffer) and 7.0−11.0 (Tris-HCl buffer), respectively. To measure the best reaction temperature for YjiC1 activity, the reaction was incubated at different temperatures (30−60 °C). To assay the impact of divalent metal ions, 5 mM CaCl2, MgCl2, MnCl2, PbCl2, ZnCl2, CuCl2, NiCl2 or ethylenediaminetetraacetate was individually added to the reaction mixture. To determine the best reaction time, the reaction was terminated at different times (5 min to 8 h). UDPG was used as the sugar donor and VB was used as the aglycone acceptor. The conversion rate was calculated by the peak area ratios of arenobufagin versus arenobufagin-3-O-β-D-glucoside. Preparative-Scale Reaction. The preparative-scale reactions were conducted in 100 mL of assay buffer solution containing 360 μmol of VB mixture, 180 μmol of UDP-Glc, and 150 mg of purified YjiC1. The reactions were gently agitated at 37 °C for 12 h, followed by termination with an equal volume of MeOH and centrifugation at 12 000 rpm for 30 min. The organic solvent was removed under reduced pressure, and samples were redissolved in 10 mL MeOH. To obtain the representative glycosylated compounds, the glycosylated products were separated by reverse-phase semipreparative HPLC which was equipped with an UV detector using a COSMOSIL C18-MS-II column (250 mm × 10.0 mm i.d., 5 μm, Nacalai Tesque, Inc., Japan) at a flow rate of 3 mL/ min. The obtained products were confirmed by MS and 1H and 13C NMR spectroscopic analyses (Figures S4−S15). Na+,K+-ATPase Inhibition Assay. The NKA inhibitory activities were determined essentially as previously reported.40 Toxicity Evaluation on Zebrafish Embryo. Zebrafish embryos were collected after 1 h postfertilization. The embryos were incubated for 24 h at 28.5 °C before exposure to drugs. Then, the embryos were selected under stereomicroscope and transferred to a 96-well plate (100 μL per well). Each drug concentration was tested on 60 embryos divided into three replicated groups. The 96-well plate was incubated at 28.5 °C.

After 72 h postfertilization exposure, the mortality rate was calculated and compared to the positive control.41,42

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00386. 1 H and 13C spectra, HPLC−MS/MS data, and SDSPAGE analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 8620-85221016. Fax: 8620-85221559. Author Contributions §

Z.-H.F., C.W., and Q.-M.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the National Natural Science Foundation of China (81573315 and 81872760) and the Hainan Provincial Natural Science Foundation of China (no. 817307).

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REFERENCES

(1) China Pharmacopoeia Committee. Chinese Pharmacopoeia; China Pharmaceutical Science and Technology Press: Beijing, 2010; p 360. (2) Tang, H. J.; Ruan, L. J.; Tian, H. Y.; Liang, G. P.; Ye, W. C.; Hughes, E.; Esmann, M.; Fedosova, N. U.; Chung, T. Y.; Tzen, J. T. Novel stereoselective bufadienolides reveal new insights into the requirements for Na+, K+-ATPase inhibition by cardiotonic steroids. Sci. Rep. 2016, 6, 29155. (3) Tian, H. Y.; Ruan, L. J.; Yu, T.; Zheng, Q. F.; Chen, N. H.; Wu, R. B.; Zhang, X. Q.; Wang, L.; Jiang, R. W.; Ye, W. C.; Bufospirostenin, A.; Bufogargarizin, C. Bufospirostenin A and Bufogargarizin C, Steroids with Rearranged Skeletons from the Toad Bufo bufo gargarizans. J. Nat. Prod. 2017, 80, 1182−1186. (4) Yu, Z.; Guo, W.; Ma, X.; Zhang, B.; Dong, P.; Huang, L.; Wang, X.; Wang, C.; Huo, X.; Yu, W.; Yi, C.; Xiao, Y.; Yang, W.; Qin, Y.; Yuan, Y.; Meng, S.; Liu, Q.; Deng, W. Gamabufotalin, a bufadienolide compound from toad venom, suppresses COX-2 expression through targeting IKKβ/NF-κB signaling pathway in lung cancer cells. Mol. Canc. 2014, 13, 203. (5) Prassas, I.; Diamandis, E. P. Novel therapeutic applications of cardiac glycosides. Nat. Rev. Drug Discovery 2008, 7, 926. (6) Gao, H.; Popescu, R.; Kopp, B.; Wang, Z. Bufadienolides and their antitumor activity. Nat. Prod. Rep. 2011, 28, 953−969. (7) Bick, R. J.; Poindexter, B. J.; Sweney, R. R.; Dasgupta, A. Effects of Chan Su, a traditional Chinese medicine, on the calcium transients of isolated cardiomyocytes: Cardiotoxicity due to more than Na+, K+ATPase blocking. Life Sci. 2002, 72, 699−709. (8) Barrueto, F.; Kirrane, B. M.; Cotter, B. W.; Hoffman, R. S.; Nelson, L. S. Cardioactive steroid poisoning: A comparison of plantand animal-derived compounds. J. Med. Toxicol. 2006, 2, 152−155. (9) De Bruyn, F.; Maertens, J.; Beauprez, J.; Soetaert, W.; De Mey, M. Biotechnological advances in UDP-sugar based glycosylation of small molecules. Biotechnol. Adv. 2015, 33, 288−302. (10) Feng, J.; Zhang, P.; Cui, Y.; Li, K.; Qiao, X.; Zhang, Y. T.; Li, S. M.; Cox, R. J.; Wu, B.; Ye, M.; Yin, W.-B. Regio- and Stereospecific OGlycosylation of Phenolic Compounds Catalyzed by a Fungal Glycosyltransferase from Mucor hiemalis. Adv. Synth. Catal. 2017, 359, 995−1006. 6824

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Promiscuous Glycosyltransferase from Carthamus tinctorius. Adv. Synth. Catal. 2017, 359, 603−608. (30) Zhang, C.; Griffith, B. R.; Fu, Q.; Albermann, C.; Fu, X.; Lee, I. K.; Li, L.; Thorson, J. S. Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions. Science 2006, 313, 1291−1294. (31) Xie, K.; Chen, R.; Li, J.; Wang, R.; Chen, D.; Dou, X.; Dai, J. Exploring the catalytic promiscuity of a new glycosyltransferase from Carthamus tinctorius. Org. Lett. 2014, 16, 4874−4877. (32) Skou, J. C. The Identification of the Sodium-Potassium Pump (Nobel Lecture). Angew. Chem., Int. Ed. 1998, 37, 2320−2328. (33) Vong, L. B.; Kobayashi, M.; Nagasaki, Y. Evaluation of the toxicity and antioxidant activity of redox nanoparticles in zebrafish (Danio rerio) embryos. Mol. Pharm. 2016, 13, 3091−3097. (34) Oliveira, R.; Domingues, I.; Koppe Grisolia, C.; Soares, A. M. V. M. Effects of triclosan on zebrafish early-life stages and adults. Environ. Sci. Pollut. Res. 2009, 16, 679−688. (35) van Aerle, R.; Lange, A.; Moorhouse, A.; Paszkiewicz, K.; Ball, K.; Johnston, B. D.; de-Bastos, E.; Booth, T.; Tyler, C. R.; Santos, E. M. Molecular mechanisms of toxicity of silver nanoparticles in zebrafish embryos. Environ. Sci. Technol. 2013, 47, 8005−8014. (36) Luo, S. L.; Dang, L. Z.; Zhang, K. Q.; Liang, L. M.; Li, G. H. Cloning and heterologous expression of UDP-glycosyltransferase genes fromBacillus subtilisand its application in the glycosylation of ginsenoside Rh1. Lett. Appl. Microbiol. 2015, 60, 72−78. (37) Chen, D.; Chen, R.; Wang, R.; Li, J.; Xie, K.; Bian, C.; Sun, L.; Zhang, X.; Liu, J.; Yang, L.; Ye, F.; Yu, X.; Dai, J. Probing the Catalytic Promiscuity of a Regio- and Stereospecific C-Glycosyltransferase fromMangifera indica. Angew. Chem. Int. Ed. 2015, 54, 12678−12682. (38) Lee, S. C.; Knowles, T. J.; Postis, V. L. G.; Jamshad, M.; Parslow, R. A.; Lin, Y. P.; Goldman, A.; Sridhar, P.; Overduin, M.; Muench, S. P.; Dafforn, T. R. A method for detergent-free isolation of membrane proteins in their local lipid environment. Nat. Protoc. 2016, 11, 1149. (39) Chen, Y. L.; Bian, X. L.; Guo, F. J.; Wu, Y. C.; Li, Y. M. Two new 19-norbufadienolides with cardiotonic activity isolated from the venom of Bufo bufo gargarizans. Fitoterapia 2018, 131, 215−220. (40) Middleton, D. A.; Rankin, S.; Esmann, M.; Watts, A. Structural insights into the binding of cardiac glycosides to the digitalis receptor revealed by solid-state NMR. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13602−13607. (41) Dogra, Y.; Scarlett, A. G.; Rowe, D.; Galloway, T. S.; Rowland, S. J. Predicted and measured acute toxicity and developmental abnormalities in zebrafish embryos produced by exposure to individual aromatic acids. Chemosphere 2018, 205, 98−107. (42) Wang, Z. G.; Zhou, R.; Jiang, D.; Song, J. E.; Xu, Q.; Si, J.; Chen, Y. P.; Zhou, X.; Gan, L.; Li, J. Z.; Zhang, H.; Liu, B. Toxicity of Graphene Quantum Dots in Zebrafish Embryo. Biomed. Environ. Sci. 2015, 28, 341−351.

(11) Zhou, M.; Thorson, J. S. Asymmetric enzymatic glycosylation of mitoxantrone. Org. Lett. 2011, 13, 2786−2788. (12) Mestre, J.; Matheu, M. I.; Díaz, Y.; Castillón, S.; Boutureira, O. Chemical Access to D-Sarmentose Units Enables the Total Synthesis of Cardenolide Monoglycoside N-1 from Nerium oleander. J. Org. Chem. 2017, 82, 3327−3333. (13) Ye, H.; Xiao, C.; Zhou, Q. Q.; Wang, P. G.; Xiao, W. J. Synthesis of Phenolic Glycosides: Glycosylation of Sugar Lactols with Aryl Bromides via Dual Photoredox/Ni Catalysis. J. Org. Chem. 2018, 83, 13325−13334. (14) Guo, J.; Tan, B.; Ye, Q.; Liang, G.; Yi, M.; Jiang, R. Synthesis and cytotoxic activities of spin-labeled derivatives of Cinobufagin. Chem. Res. Chin. Univ. 2017, 33, 581−586. (15) Chen, D.; Sun, L.; Chen, R.; Xie, K.; Yang, L.; Dai, J. Enzymatic Synthesis of Acylphloroglucinol 3-C -Glucosides from 2-O -Glucosides using a C -Glycosyltransferase from Mangifera indica. Chem. Eur. J. 2016, 22, 5873−5877. (16) Siitonen, V.; Nji Wandi, B.; Törmänen, A.-P.; Metsä-Ketelä, M. Enzymatic Synthesis of the C-glycosidic Moiety of Nogalamycin R. ACS Chem. Biol. 2018, 13, 2433−2437. (17) Ye, M.; Dai, J.; Guo, H.; Cui, Y.; Guo, D. Glucosylation of cinobufagin by cultured suspension cells of Catharanthus roseus. Tetrahedron Lett. 2002, 43, 8535−8538. (18) Zhao, J.; Guan, S. H.; Chen, X. B.; Wang, W.; Ye, M.; Guo, D. A. Two new compounds derived from bufalin. Chin. Chem. Lett. 2007, 18, 1316−1318. (19) Zhang, X.; Ye, M.; Dong, Y. H.; Hu, H. B.; Tao, S. J.; Yin, J.; Guo, D. A. Biotransformation of bufadienolides by cell suspension cultures of Saussurea involucrata. Phytochemistry 2011, 72, 1779− 1785. (20) Zhou, M.; Hou, Y.; Hamza, A.; Zhan, C. G.; Bugni, T. S.; Thorson, J. S. Probing the regiospecificity of enzyme-catalyzed steroid glycosylation. Org. Lett. 2012, 14, 5424−5427. (21) Li, K.; Feng, J.; Kuang, Y.; Song, W.; Zhang, M.; Ji, S.; Qiao, X.; Ye, M. Enzymatic Synthesis of Bufadienolide O -Glycosides as Potent Antitumor Agents Using a Microbial Glycosyltransferase. Adv. Synth. Catal. 2017, 359, 3765−3772. (22) Wen, C.; Huang, W.; Zhu, X. L.; Li, X. S.; Zhang, F.; Jiang, R. W. UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation. Org. Lett. 2018, 20, 534−537. (23) Ye, M.; Guo, H.; Guo, H.; Han, J.; Guo, D. Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine ChanSu by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections. J. Chromatogr. B: Biomed. Sci. Appl. 2006, 838, 86−95. (24) Ye, M.; Guo, D. A. Analysis of bufadienolides in the Chinese drug ChanSu by high-performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2005, 19, 1881−1892. (25) Hu, Y.; Yu, Z.; Yang, Z. J.; Zhu, G.; Fong, W. Comprehensive chemical analysis of Venenum Bufonis by using liquid chromatography/electrospray ionization tandem mass spectrometry. J. Pharm. Biomed. Anal. 2011, 56, 210−220. (26) Dai, L.; Li, J.; Yao, P.; Zhu, Y.; Men, Y.; Zeng, Y.; Yang, J.; Sun, Y. Exploiting the aglycon promiscuity of glycosyltransferase Bs-YjiC from Bacillus subtilis and its application in synthesis of glycosides. J. Biotechnol. 2017, 248, 69−76. (27) Sun, L.; Chen, D.; Chen, R.; Xie, K.; Liu, J.; Yang, L.; Dai, J. Exploring the aglycon promiscuity of a new glycosyltransferase from Pueraria lobata. Tetrahedron Lett. 2016, 57, 1518−1521. (28) Wang, R.; Chen, R.; Li, J.; Liu, X.; Xie, K.; Chen, D.; Yin, Y.; Tao, X.; Xie, D.; Zou, J.; Yang, L.; Dai, J. Molecular Characterization and Phylogenetic Analysis of Two Novel Regio-specific Flavonoid Prenyltransferases from Morus alba and Cudrania tricuspidata. J. Biol. Chem. 2014, 289, 35815−35825. (29) Xie, K.; Chen, R.; Chen, D.; Li, J.; Wang, R.; Yang, L.; Dai, J. Enzymatic N-Glycosylation of Diverse Arylamine Aglycones by a 6825

DOI: 10.1021/acsomega.9b00386 ACS Omega 2019, 4, 6819−6825