An Efficient Strategy for the Chemo-Enzymatic Synthesis of Bufalin Glycosides with Improved Water Solubility and Inhibition against Na+, K+-ATPase

Article abstract of DOI:10.1002/cbdv.202000529

In this study, bufalin was glycosylated by an efficient chemo-enzymatic strategy. Firstly, 2-chloro-4-nitrophenyl-1-O-β-D-glucoside (sugar donors) was obtained by chemical synthesis. Then, the glycosylation of the bufalin was achieved with the synthesized sugar donor under the catalysis of two glycosyltransferases (Loki and ASP). Finally, two glycosides, i. e., bufalin-3-O-β-D-glucopyranoside and bufalin-3-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside)], were obtained by preparative HPLC. Compared to our previously reported sole chemical (total yield 10 % in four steps) or enzymatic methods (30 %), our combined chemo-enzymatic strategy in this article greatly improves the yields of monoglycoside (68 %) and diglycoside (21 %) and decreased the experimental cost (90 %). Furthermore, we tested the water solubility of these glycosides and found that the water solubilities of the two glycosides were 13.1 and 53.7 times of bufalin, respectively. In addition, the inhibitory activity of these glycosides against Na⁺, K⁺-ATPase were evaluated. The mono-glycosylated compound showed more potent activity than bufalin, while the diglycosylated compound was less potent.

Full text of DOI:10.1002/cbdv.202000529

Title: An efficient strategy for the chemo-enzymatic synthesis of bufalin
glycosides with improved water solubility and inhibition against
Na+,K+-ATPase
Authors: Ren-Wang Jiang, Yan Liu, Wei Xu, Zhao-He Huang, and Jun
Guo
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000529
Link to VoR: https://doi.org/10.1002/cbdv.202000529

10.1002/cbdv.202000529
Chemistry & Biodiversity
Chem. Biodiversity
An efficient strategy for the chemo-enzymatic synthesis of bufalin
glycosides with improved water solubility and inhibition against Na+,
K+-ATPase
Yan Liu a,b,c, Wei Xu c, Zhao-He Huangb, Jun Guoa, Ren-Wang Jiangc*
a Department of Cardiology, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong 510630.
b Department of Cardiology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi 533000, P.R. China.
c Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, and International Cooperative
Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development of Chinese Ministry of Education, Jinan
University, Guangzhou 510632, People’s Republic of China.
Correspondence: trwjiang@jnu.edu.cn (R.W.J.)
In this study, bufalin was glycosylated by an efficient chemo-enzymatic strategy. Firstly, 2-chloro-4-nitrophenyl-1-O--D-glucoside (sugar
donors) was obtained by chemical synthesis. Then the glycosylation of the bufalin was achieved with the synthesized sugar donor under the
catalysis of two glycosyltransferases (Loki and ASP). Finally, two glycosides, i.e. bufalin-3-O--D-glucopyranoside (3) and bufalin-3-O-[-
D-glucopyranosyl(1→2)--D-glucopyranoside)] (4), were obtained by preparative HPLC. Compared to our previously reported sole chemical
(total yield 10% in four steps) or enzymatic methods (30%), our combined chemo-enzymatic strategy in this paper greatly improves the yields
of monoglycoside (68%) and diglycoside (21%) and decreased the experimental cost (90%). Furthermore, we tested water solubility of these
glycosides and found that the water solubilities of 3 and 4 were 13.1 and 53.7 times of bufalin, respectively. In addition, the inhibitory activity
of these glycosides against Na+, K+-ATPase were evaluated. The mono-glycosylated compound 3 show more potent activity than bufalin, while
the diglycosylated compound 4 was less potent.
Keywords: Bufalin, Na+, K+-ATPase, Glycosylation, Water solubility
Introduction
Heart failure is a clinical syndrome with impaired ventricular filling and ejection ability caused by abnormal heart structure or function,
which ultimately leads to a decline in ventricular pumping function. It is the end stage of cardiovascular disease, with a high mortality rate and
poor prognosis.[1] Digitalis glycosides, e.g. digoxin and other cardiac glycoside drugs are used to treat heart failure by inhibiting the activity of
Na+, K+-ATPase. But such drugs have safety issues such as small safety windows and large toxic and side effects.[2-3] It is of great clinical
significance to develop high-efficient and low-toxic cardiotonic drugs.
Bufalin is one of the major constituents from Bufonis Venenum, a Traditional Chinese Medicine used for the treatment of heart failure. It
has a strong cardiotonic effect with potent Na+, K+-ATPase inhibitory activity.[4] But clinical application of bufalin is limited due to poor water
solubility and large toxic side effects.[5,6] Glycosylation modification can enhance the water solubility,[7] targeting[8] and reduce the toxic and
side effects of drugs.[9] Both chemical method[10] and enzymatic method[11] can achieve glycosylation, but enzymatic glycosylation has the
advantages of green and high efficiency,[12] and thus its application is increasingly widespread. This paper intends to modify bufalin via the
chemo-enzymatic strategy. Furthermore, the water solubility of glycosylation products and the inhibitory activity against Na+, K+-ATPase were
evaluated and compared with the parent compounds.
1
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Results and Discussion
Design of the chemo-enzymatic glycosylation route
Chemoenzymatic glycosylation in this study utilizes a combination of Loki- and ASP- glycosyltransferases. The formal enzyme
contained the mutations P67T/I112P/T113M/S132F/A242I and was found to be capable of recognizing 2-chloro-4-nitrophenyl glycoside as
the sugar donor;[13] while the latter enzyme contained triple mutant (A242V/S132F/P67T) and was found to displayed marked improvement
in proficiency and substrate promiscuity as compared with the wild-type OleD glycosyltransferase.[14] The synthesis route for chemical-
enzymatic glycosylation of bufalin is shown in Scheme 1. First, we synthesized the 2-chloro-4-nitrophenol sugar donor (1). The sugar donor
reacted with UDP (uridine diphosphate) under the catalysis of glycosyltransferase 1 (Loki) to form a highly active UDP-α-D-sugar donor
intermediates, which then reacted with the aglycon substrate (bufalin, 2) under the catalytic action of glycosyltransferase 2 (ASP) [15]
.
Identification of the glycosylation products was achieved by LC-MS. Then we use preparative HPLC to separate and prepare the
glycosylation products (3 and 4). Finally, the structures of glycosylation products were confirmed by 1H NMR, 13C NMR and HR-MS.
Scheme 1 The synthesis route for chemical-enzymatic glycosylation of bufalin.
Synthesis of sugar donor
In addition to UDPG (uridine diphosphate glucose), glycosyltransferase can accept 2-chloro-4-nitrophenol glycoside (1) as a sugar
donor.[16] Through a simple four-step reaction: acetylation, bromination, glycosylation, and deacetylation, the sugar donor 1 is obtained
(Scheme 2). For the detailed syntheis of intermedates and spectral data, please refer to the experimental section.
Scheme 2 Synthesis route of sugar donor (2-chloro-4-nitrophenol glucoside). Reaction conditions and reagents：(i) Ac2O-AcONa, Reflux,
140℃; (ii) 33% HBr-AcOH, CH2Cl2, rt; (iii)Ag2CO3, 4Å MS, DCM, Ar, rt; (iv) MeONa-MeOH, rt.
2
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Synthesis and characterization of bufalin glycosides
The glycosylation of bufalin (2) with the synthesized sugar donor 2-chloro-4-nitrophenol glucoside (1) was shown in Figure 1. The LC-
MS chromatogram showed two new peaks with retention times at 14.8 min (3) and 12.9 min (4), respectively. The characteristic UV absorption
of these two peaks at 296 nm was consistent with the substrate bufalin (2). The preparative HPLC was used to separate and purify these two
new peaks.
Figure 1 HPLC chromatogram and ESI-MS of the glycosylation products.
The new peak at Rt = 14.8min (3), showed a pseudomolecular ion at [M+H]+ 549.3052. The IR spectrum of 3 indicated the presence of
1
hydroxyl (3422 cm-1) and carbonyl (1715 cm-1) groups. The H-NMR showed an anomeric proton signal δH 4.31 (d, 1H, J=7.8 Hz, H-1’),
indicating that 3 is a monoglycoside. The low field region showed proton resonance for a characteristic six-membered lactone ring：δH: 7.99
(dd, 1H, J=9.7, 2.5 Hz, H-22), 7.42 (d, 1H, J=1.6 Hz, H-21) and 6.27 (d, 1H, J=9.7 Hz, H-23). The 13C-NMR spectrum showed signals for an
anomeric carbons 102.7 (C-1’) and a group of glucose carbon signals 102.7 (C-1’), 78.2 (C-5’), 77.8 (C-2’), 75.5 (C-4’), 75.2 (C-3’) and 62.8
(C-6’), confirming the presence of a glucose sugar unit, and carbon resonances for a characteristic six-membered lactone ring: δC 164.9, 150.5,
3
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149.4, 125.1 and 115.4. Comparison with the parent aglycone, the carbon signal of C-3 was shifted to downfield 0.4 ppm (from 71.3 to 71.7),
indicating that the sugar chain was linked to the aglycone at C-3. All other signals of 3 from was consistent with the parent aglycon. From the
above analysis, we confirmed that 3 was a monoglucoside of bufalin at C-3.
The new peak at Rt= 12.9 min (4) showed a pseudomolecular ion at [M+H]+ 711.3583. The IR spectrum of 4 indicated the presence of
hydroxyl (3380 cm-1) and carbonyl (1700 cm-1) groups. The 1H-NMR spectrum was similar to that of 3 except that there were two anomeric
proton signals δH 4.69 (d, 1H, J=7.8 Hz, H-1’) and 4.59 (d, 1H, J=7.5 Hz, H-1’’), indicating that 4 is a diglucoside, which was confirmed by
the 13C-NMR spectrum showing signals for two anomeric carbons δC 104.1 (C-1’) and 100.7 (C-1’). Similarly, comparison with the parent
aglycone, the proton signal of H-3 was shifted to downfield 0.06 ppm (from 4.07 to 4.13), and the carbon signal of C-3 was shifted to downfield
0.5 ppm (from 71.3 to 71.8), indicating that the sugar chain was linked to the aglycone at C-3. It is noteworthy that the carbon signal of C-2’
was shifted to downfield 0.3 ppm (from 77.6 to 77.9), indicating that the second glucose unit was linked to the first glucose unit at C-2’.
Accordingly, the two new peaks 3 and 4 were identified as the mono-glucoside and di-glucoside of bufalin, respectively (Figure 2).
Figure 2 Structures of 3 and 4.
Solubility test
When the concentration of bufalin is in the range of 09.65 mg/ml, its content has a good linear correlation with the peak area. The
linear regression equation is: y = 40.878x + 30.375 (R2 = 0.999). So the solubility of bufalin and its glycosylation products can be determined
by high performance liquid chromatography.
Through HPLC analysis, the chromatographic peak areas of 2, 3 and 4 were: 33.2, 66.8, 179.6, and the solubilities of 2, 3 and 4 in water
were calculated to be 0.068, 0.892 and 3.652 mg/mL, respectively. The water solubilities of 3 and 4 increased by 13.1 and 53.7 times as
compared to bufalin (2), respectively (Table 1). These results indicated that the addition of glucosyl group could remarkably increase the
water solubility of bufalin, which was important for achiving desired concentration in systemic circulation.[17] These bufalin glucosides with
higher water solubility might have broad applications in industry.
Table 1 Solubility of bufalin and its glycosides
Compound
Solubility in water (mg/mL)
Ratio
1.0
2
3
4
0.068
0.892
3.652
13.1
53.7
4
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Effects of bufalin glycosides on Na+, K+-ATPase
The glycosylation products of bufalin were tested for Na+, K+-ATPase inhibitory activity, and the results are shown in the Figure 3.
It can be seen that bufalin monoglucoside (3) showed more potent inhibition on Na+, K+-ATPase than that of bufalin, but the diglucosylation
product (4) was less potent.
Figure 3 Na+, K+-ATPase inhibitory activities of bufalin glucosides.
Discussion
This study adopts a chemical-enzymatic method to carry out the glycosylation of bufalin. The advantages of this method included high
efficiency, environmentally friendly and mild reaction conditions. Firstly, we obtained 2-chloro-4-nitrophenol glucoside by chemical synthesis.
Then the synthesized 2-chloro-4-nitrophenol glucoside was used as the sugar donor in the glycosylation reaction catalyzed by the two
glycosyltransferases (Loki and ASP). Finally, the glycosylation products were purified by preparative HPLC. It is noteworthy that synthesized
2-chloro-4-nitrophenol glucoside instead of UDPG was used as the sugar donor.[16] Compared to our previously reported sole chemical (total
yield 10% in four steps)[18] or enzymatic methods (30%)[19], the combined chemo-enzymatic strategy in this paper greatly improved the yields
of monoglycoside (68%) and diglycoside (21%) and decreased the experimental cost (90%) because of the much higher price of UDPG. During
the biosynthesis of bufalin glycosides, similar to the one-pot chemical reaction,[20] an one-pot enzymatical reaction incoporating the Loki- and
ASP-glycosyltransferases was performed, which led to the direct synthesis of 3 and 4 without the need to isolate the intermediates.
We tested the water solubility of the two glycosylated products. The results show that the water solubilities of monoglycosylation and
diglycosylation products were 13.1 and 53.7 times higher than the parent compound, respectively. The Na+, K+-ATPase inhibitory activity of
the two glycosylation products were tested. The results showed that the monoglycosylation product had stronger inhibitory activity than the
parent compound 2, but the diglycosylation product was less potent.
Conclusions
In this study, bufalin, one of the major constituents from Bufonis Venenum, was glycosylated by an efficient chemo-enzymatic strategy. The
sugar donor 2-chloro-4-nitrophenyl-1-O--D-glucoside was obtained by chemical synthesis. Then the glycosylation of the bufalin was achieved
under the catalysis of two glycosyltransferases (Loki and ASP) using the synethsized sugar donor, which led to the biosynthesis of glycosides
3 and 4. Compared to the starting material 2, the water solubilities of 3 and 4 were increased 13.1 and 53.7 times, respectively. Futhermore.
5
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the mono-glycosylated compound 3 show more potent inhibitory activity against Na+, K+-ATPase than the starting material 2, while the
diglycosylated compound 4 was less potent. In summary, this study provides an efficient strategy for the chemo-enzymatic synthesis of bufalin
glycosides with improved water solubility and inhibition against Na+, K+-ATPase and extends the application of bufalin in industry.
Experimental Section
General
Bufalin (2) was purchased from Baoji Chenguang Biotech Co, Ltd. (Baoji, China). D-glucose was purchased from J & K Scientific Ltd.
(Beijing, China). High-performance liquid chromatography (HPLC)-grade acetonitrile was purchased from Merck KGaA (Darmstadt,
Germany). Water was prepared with a Milli-Q plus Ultrapure Water System (Millipore, Billerica, MA). Other reagents and chemicals were
purchased from Sigma-Aldrich or J & K reagents.
Expression of glycosyltransferase (Loki and ASP)
The two recombinant plasmids of ASP-pET28a and Loki-pET28a (synthesized by Nanjing Genscript Co., Ltd.) were transferred into
Transetta (DE3) expression cells (TransGen Biotech, China) for recombinant protein expression. The next day, single colonies were selected
from the plates and inserted into 5 mL Luria-Bertani (LB) medium containing 50 μg·mL-1 kanamycin and 34 μg·mL-1 chloramphenicol, and
cultured in incubator overnight at 37°C with shaking (220 rpm). Then the overnight culture was inoculated (1/100) into 1L LB medium
containing 50 μg·mL-1 kanamycin and 34 mL-1 chloramphenicol, and cultured at 37°C and 220 rpm until the OD600 value reached 0.4-0.6.
Subsequently, isopropyl β-D-thiogalactoside (IPTG) was added (final concentration of 0.4 mM) to induce the expression of recombinant
proteins (ASP and Loki), and the cells were grown at 18°C and 180 rpm for another 16 hours. Finally, the cells were collected by
centrifugation at 3,000 rpm, 4°C for 15 minutes and stored at -80°C.
The frozen cells were resuspended in 10 mL of lysis buffer (20 mM phosphate buffer, 50 mM NaCl, 10 mM imidazole, pH 7.5) and
sonicated in an ice-water bath for 20 min. The cell lysate was centrifuged (12,000 rpm and 4°C) for 40 minutes to obtain a supernatant (crude
enzyme solution). After filtration through a 0.45 μm filter membrane, the supernatant was immediately added to a Ni column (GE Healthcare,
USA) pre-equilibrated with 5  equilibration buffer (20 mM). The Ni column was turned over to allow the recombinant protein to bind at 4°C
for 1 hour. After washing the Ni column with 5  washing buffer, the target protein is eluted using 3 elution buffer. Finally, Amicon Ultra-
30 K (Millipore, USA) was used to concentrate the target protein (centrifuged at 3500 rpm, 10 min and 4°C). The protein purity was analyzed
by 12% SDS-PAGE, and the concentration of the target protein was determined using the Easy Protein Quantitative Kit (Bradford method).
Finally, the concentrated protein was stored at -80°C.
Synthesis of sugar donor
The synthesis route of the sugar donor was shown in Scheme 2.
Synthesis of 6. D-glucose (5, 4.0 g, 22.20 mmol) was accurately weighted and added in a 100 mL reaction flask. Acetic anhydride (222
mmol) and sodium acetate (3.64 g, 44.4 mmol) were added to the flask. The mixture was stirred to dissolve at room temperature, then the
reaction was heated to reflux at 140 ℃, and monitored by TLC (Developing agent: petroleum ether: ethyl acetate=2:1). The reaction was
completed after 1.5 h, and the reaction mixture was cooled to room temperature. Ethyl acetate and water were added to the mixture for extraction.
The aqueous layer was further extracted with ethyl acetate, and the organic layers were combined. The organic layer was washed three times
with saturated aqueous sodium bicarbonate solution and then three times with saturated aqueous sodium chloride solution. The organic layer
6
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was dried over anhydrous Na2SO4, filtered, concentrated and dried under reduced pressure to obtain 7.62 g of light yellow oil, which was
directly used in the next reaction without purification.
Synthesis of 7. The crude product 6 (6.0 g, 15.37 mmol) from the previous step was put in a 100 mL reaction flask, and dichloromethane
(40 mL) was added to dissolve 6. Then hydrobromic acid-acetic acid solution (33%, 23.45 mL) was added dropwise. The reaction was carried
out at 0 ℃ for 30 min under the protection of argon gas, and monitored by TLC (Developing agent: petroleum ether: ethyl acetate = 2:1). The
reaction was completed in 2 hours. The reaction solution was poured into ice water (50 mL), and extracted with dichloromethane. The organic
layer was washed three times with saturated aqueous sodium bicarbonate solution, followed by three times with aqueous sodium chloride
solution. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated, and dried under reduced pressure to obtain 4.8 g light
yellow oil 7, which was used directly in the next reaction without purification.
Synthesis of 8. The crude product 7 (4.8 g, 11.67 mmol) from the previous step was put in a 100 mL reaction flask, and 2-chloro-4-
nitrophenol (3.04 g, 17.51 mmol) was added. The mixture was dried under reduced pressure to remove water by adding activated molecular
sieve. Then dry dichloromethane was added to dissolve the mixture. Silver carbonate (0.64 g, 2.33 mmol) was added to initiate the reaction
under the protection of argon gas. The reaction was carried out overnight at room temperature. After completion, the reaction solution was
filtered through celite, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography
(petroleum ether: ethyl acetate = 5:1) to obtain a white solid product 8 (2.41 g, 40.9%). The NMR data of 8 were shown in the supporting
materials.
Synthesis of 1. The solid product 8 (2.0 g, 3.97 mmol) was dissolved in a 100 mL reaction flask with methanol, then sodium methoxide
(428.9 mg, 7.94 mmol) was added. The reaction was carried out at room temperature and monitored by TLC (developing agent:
dichloromethane: methanol = 10:1). The reaction was completed in two hours, and the solution was concentrated under reduced pressure. The
residue was separated by silica gel column chromatography (dichloromethane: methanol = 30:1) to obtain a white solid product 1 (1.19 g,
89.5%). The NMR data of 1 were shown in the supporting materials.
One-pot glycosylation of bufalin (2)
(1) Preparation of solutions. 100 mM MgCl2 and 25 mM UDP solutions were prepared with purified water; 40 mM substrate (bufalin 2) and
25 mM 2-chloro-4-nitrophenol glucoside (1) solutions were prepared with DMSO; 50 mM Tris-HCl solution was prepared with pure water,
and the pH was adjusted to 8.5.
(2) Glycosylation. Take three 2 mL EP tubes. The first EP tube was used as the experimental group, MgCl2 (25 μL, 100 mM), bufalin (12.5
μL, 40 mM), 2-chloro-4-nitrophenol sugar donor (40 μL, 25 mM), UDP (40 μL, 25 mM), Loki enzyme (40 μL, 10 μg), ASP enzyme (40 μL,
10 μg), and 50 mM Tris-HCl solution were added (the final volume is 500 μL); the second EP tube was used as the sugar donor-free control
group, MgCl2 (25 μL, 100 mM), substrate (12.5 μL, 40 mM), UDP (40 μL, 25 mM), Loki enzyme (40 μL, 10 μg), ASP enzyme (40 μL, 10 μg),
and 50 mM Tris-HCl solution was added (the final volume 500 μL); the third EP tube was used as an enzyme-free control group, MgCl2 (25
μL, 100 mM), substrate (12.5 μL, 40 mM), 2-chloro-4-nitrophenol sugar donor (40 μL, 25 mM), UDP (40 μL, 25 mM), and 50 mM Tris-HCl
solution were added (the final volume 500 μL). All three EP tubes were put in the same incubator (37°C, 16 h).
(3) Termination. After the incubation, equal volume of methanol (500 μL) was added to terminate the reaction.
(4) Purification of products. The reaction solution was centrifuged at 12,000 rpm for 30 min, and the supernatant was used for HPLC analysis
(Phenomenex Luna-C18 analytical column 250 mm×4.6 mm, 5 μm; mobile phase: water containing 0.1% formic acid (A)-acetonitrile (B);
elution conditions: 0-20 min 10→90% B, 21-22 min 90→100% B, 23-30 min 100% B, 31-40 min 100→10% B; flow rate: 1.0 mL/min;
detection at 296 nm.). The chromatogram of the experimental group was compared with the two control groups to confirm whether there were
7
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glycosylation products. Finally, the glycosylation products 3 and 4 were obtained preparative HPLC and determined according to LC-MS, 1H
NMR and 13C NMR analysis.
1
Spectral data for 3. H NMR (CD3OD-d4, 500 MHz) δ: 7.99 (dd, 1H, J=9.7, 2.5 Hz, H-22) , 7.42 (d, 1H, J=1.6 Hz, H-21), 6.27 (d, 1H,
J=9.7 Hz, H-23), 4.31 (d, 1H, J=7.8 Hz, H-1’), 4.07 (brs, 1H, H-3), 3.84 (dd, 1H, J=11.8, 1.9 Hz, H-6’), 3.65 (dd, 1H, J=11.8, 5.2 Hz, H-
6’) . 13C NMR (CD3OD-d4, 125 MHz) δ: 164.9 (C-24), 150.5 (C-21), 149.4 (C-22), 125.1 (C-20), 115.4 (C-23), 102.7 (C-1’), 86.2 (C-14),
78.2 (C-5’), 77.8 (C-2’), 75.5 (C-4’), 75.2 (C-3’), 71.7 (C-3), 62.8 (C-6’), 52.3 (C-17), 49.8 (C-13), 43.0 (C-9), 42.0 (C-12), 37.5 (C-8), 37.1
(C-4), 36.3 (C-1), 33.2 (C-15), 31.3 (C-2), 30.8 (C-16), 29.9 (C-16), 27.6 (C-6), 24.1 (C-19), 22.6 (C-7), 22.6 (C-11), 17.3 (C-18). IR: 3422
cm-1 (hydroxyl) and 1715 cm-1 (carbonyl). HRMS (ESI) calcd for C30H44O9 [M+H]+: 549.3058, found 549.3052.
1
Spectral data for 4. H NMR (CD3OD-d4, 500 MHz) δ: 8.02 (dd, 1H, J=9.5, 2.5 Hz, H-22) , 7.44 (d, 1H, J=1.7 Hz, H-21), 6.29 (d, 1H,
J=9.5 Hz, H-23), 4.69 (d, 1H, J=7.8 Hz, H-1’), 4.59 (d, 1H, J=7.5 Hz, H-1’’), 4.47 (m, 1H, H-2’), 4.13 (brs, 1H, H-3), 3.87 (dd, 2H, J=12.2,
1.5 Hz, H-6’, H-6’’ ), 3.67 (dd, 2H, J=12.2, 6.2 Hz, H-6’, H-6’’), 3.59-3.56 (m, 2H, H-5’, H-5’’). 13C NMR (CD3OD-d4, 125 MHz) δ:
164.2 (C-24), 150.2 (C-21), 149.1 (C-22), 122.4 (C-20), 115.1 (C-23), 104.1 (C-1’), 100.7 (C-1’’), 80.4 (C-14), 78.3 (C-5’), 78.2 (C-5’’), 77.9
(C-2’), 77.6 (C-2’’), 75.9 (C-4’), 71.8 (C-3), 71.4 (C-3’), 52.0 (C-13), 42.8 (C-9), 36.9 (C-4), 36.8 (C-1), 24.1 (C-15), 17.1 (C-2). IR: 3380 cm-
1 (hydroxyl) and 1700 cm-1 (carbonyl). HRMS (ESI) calcd for C36H54O14 [M+H]+: 711.3586, found 711.3583.
Determination of water solubilities of bufalin glycosides
Appropriate amount of bufalin (2) was accurately weighted and dissolved in DMSO. The it was diluted with methanol to a series of
concentrations: 0.01 mg/mL, 0.1 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL. After centrifugation and filtration through a microporous filter
membrane, 10 μL of supernatant was injected for HPLC analysis. The chromatographic peak area was used as the ordinate and the
concentration was used as the abscissa to obtain the regression equation.
Excess 2 and its glycosylation products 3 and 4 were placed in a 1.5 mL centrifuge tube. 1000 μL of deionized water was added and
sonicated for 1 h. The solution was let to stand overnight. After centrifugation and filtration through a microporous filter membrane, 10 μL of
supernatant was subjected to HPLC analysis under the standard chromatographic conditions. The corresponding solubility was calculated by
the chromatographic peak area.
Inhibitory activity on Na+, K+-ATPase
The Na+/K+ ATPase inhibitory activities of 2, 3 and 4 were determined by a reported method.[21] In brief, Na+, K+-ATPase was incubated
for 2 hours in the presence of 3 mM MgCl2, 3 mM Na-phosphate and 40 mM Tris (pH 7.0) together with increasing concentrations of
inhibitors, and the activity remaining after 2 hours was shown in the Fig. 3.
Supplementary Material
Supporting information including the IR, ESI-MS, 1H-NMR and 13C-NMR spectra of 2, 3 and 4 (Figure S1-S12) and the NMR and MS
data of compounds 1 and 8 is available under http://dx.doi.org/10.1002/MS-number.
Acknowledgements
This work was financially supported by grants from the National Natural Science Foundation of China (81872760 and 81573315) and
the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y036). The first author would like
to thank Jinan University for the PhD studentship. Thank is also due to Prof. Paul Pui-Hay But for the language improvement.
Author Contribution Statement
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Chem. Biodiversity
Yan Liu performed the enzyme expression and biotransformation.
Wei Xu finished the structure determination, bioassay and solubility test.
Zhao-He Huang and Jun Guo were involved in the discussion of project progress.
Ren-Wang Jiang designed the project and are responsible for the whole project.
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10.1002/cbdv.202000529
Chemistry & Biodiversity
Chem. Biodiversity
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Graphic illustration
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