Probing the stereoselectivity of OleD-catalyzed glycosylation of cardiotonic steroids

The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) are assessed. The results of this study demonstrated that the OleD-catalyze glycosylation of CTS is significantly influenced by the configuration at C-3 and the A/B fusion mode. 3β-OH and A/B ring cis fusion are favoured by OleD (ASP). An epoxide ring at C-14 and C-15 further increases the bioconversion rate; while an acetyl group at C-16 and lactone ring type at C-17 did not influence the biotransformation. A high conversion rate corresponded to a low Km value. A molecular docking simulation showed that filling of hydrophobic pocket II and interaction with residue Tyr115 may play an important role in the glycosylation reactions catalyzed by OleD glycosyltransferases. Furthermore, the glycosylation products showed a stronger inhibitory activity for Na+, K+-ATPase than the corresponding aglycones. This study provides the first stereoselective properties for OleD (ASP) catalyzed glycosylation.


Introduction
2][3][4] Recent studies revealed an enhanced triple mutant (A242V/S132F/P67T, OleD (ASP)) that displayed marked improvement in prociency and substrate promiscuity.The OleD (ASP) was found to show highly permissive properties and is capable of glycosylation on 100 diverse acceptors, e.g.erythromycin, 5 avones, 6 indole alkaloids 7 and steroids. 8Nowadays, 56% of the drugs in clinics are chiral compounds; however 88% of them are marketed as racemates consisting of an equimolar mixture of two enantiomers. 8Although OleD (ASP) was found to catalyze the glycosylation of cardenolide and bufadienolide aglycone with a bias toward the desired C-3 regiospecicity, 7 the stereospecicity of this enzyme remains unreported.
2][13][14][15] Natural CTS includes cardenolides and bufadienolides.Cardenolides, such as ouabain and digoxin, possess a ve-membered lactone ring at position C-17b of the steroidal skeleton.The natural cardenolide can be aglycone or glycosides with one or more sugar groups at C-3. 16,17 Bufadienolides, isolated from many plants and animals such as Bufo bufo gargarizans, bears a six-membered lactone ring at C-17b.9][20][21] For both bufadienolides and cardenolides, there is a hydroxy group at C-3, either bor a-conguration.Normally the CTS with a 3b-OH shows more remarkable activities than the 3a-diastereomer. 22Especially, the sugar groups of C-3 of CTS play a key role for the inhibitory activity of NKA. 23n this paper, we use OleD (ASP) to catalyze the glycosylation of four pairs of epimers of cardiotonic steroids.K m values of two pairs of epimers representing the bufadienolides and cardenolides were determined.Molecular docking was used to simulate the interactions between the substrate and enzyme.Finally, the inhibitory activities against NKA of CTS aglycone and the corresponding glycosides were compared.

General experimental procedures
All chemicals and reagents were purchased from Sigma unless otherwise stated.The NMR spectra were recorded on a Bruker AV-400 spectrophotometer (Bruker, Germany) with TMS as internal standard.Chemical shis (d) were expressed in ppm with reference to the solvent signals.HR-ESI-MS were determined on a Micromass Q-TOF mass spectrometer (Waters, USA).Analytical HPLC is run on Agilent 1200 system (Agilent, USA) with a Phenomenex Luna C-18 column (250 mm Â 4.6 mm, 5 mm, USA).Preparative HPLC was performed on a Wu Feng HPLC system (Shanghai, China) equipped with a preparative reverse phase column (20 Â 250 mm, 5 mm).

Cloning and expression of variant OleD (ASP) glycosyltransferase
The OleD (ASP) glycosyltransferase gene was generated by Genscript Biotechnology (Nanjing, China) and was cloned into DH5a (Takara, Japan) and pET28a (Novagen, USA) expression vector.Single colony of Escherichia coli BL21 (DE3) pLysS (Tiangen, China) transformed with pET28a/OleD vector was inoculated in Luria Bertani (LB) medium (3 mL) supplemented with 50 mg mL À1 kanamycin.The medium was cultured overnight at 37 C with shaking (200 rpm).The entire starter culture was then transferred to 1 L LB medium supplemented with 50 mg mL À1 kanamycin and grown at 37 C with shaking (200 rpm) until the OD 600 reached 0.6.Isopropyl b-D-thiogalactoside (IPTG) was subsequently added (nal concentration to 0.4 mM) and the culture was incubated at 18 C for 18 h.Then the cell pellets were collected by centrifugation at 10 000g at 4 C for 20 min and the supernatant was discarded. 24Pellets were resuspended in 10 mL PBS buffer (20 mM phosphate buffer, 0.2 M NaCl, 2 mM KCl, pH 7.4) and then lysed by sonication.Cell debris was removed by centrifugation at 10 000g at 4 C for 20 min and the clear supernatant was immediately submitted to His60 Ni super ow resin (Clontech, USA).The resin was balanced by the equilibration buffer (50 mM sodium phosphate buffer, 0.3 M NaCl, 20 mM imidazole, pH 7.4).The enzyme was allowed to bind for 1 h at 4 C with gentle agitation, and the resin was washed with 10 mL mild buffer (20 mM phosphate buffer, 0.5 M NaCl, 50 mM imidazole, pH 7.4).Finally, the protein was eluted by with 20 mL strong buffer (50 mM sodium phosphate buffer, 0.3 M NaCl, 300 mM imidazole, pH 7.4).The enzyme aliquots were immediately frozen in liquid nitrogen and stored at À80 C. Protein purity was conrmed by SDS-PAGE to be >95% and protein concentration for all studies was determined using the Bradford Protein Assay Kit from Bio-Rad (TransGen Biotech, China).

Preparation of epimers of bufadienolides
CTS (1b, 2b and 3b, 10.7 mM) was dissolved in CH 2 Cl 2 (35 mL) in a round bottom ask.Pyridinium chloride hydrochloride (PCC) (21.4 mM) was added and stirred constantly to dissolve.The solution was stirred for 2 h at room temperature.Then the solvent was removed under reduced pressure and the residue was dissolved in anhydrous tetrahydrofuran (5 mL) in a round bottom ask.Sodium borohydride (NaBH 4 ) was added and stirred constantly to dissolve.The solution was stirred for 1 h at room temperature.Then 5 mL of water was added slowly to the reaction solution.Ethyl acetate (10 mL) was used to extract the mixture and the solvent was removed under reduced pressure.The nal residue was puried by preparative HPLC eluting with acetonitrile to yield C-3a CTS (1a, 2a, 3a).

General pilot-scale reaction
In vitro glycosylation reactions were carried out in 500 mL reaction buffer (50 mM Tris-HCl, pH 8.0) containing 250 mg of enzyme, 2.5 mM of UDPG, 1 mM aglycon and 5 mM MgCl 2 .The mixture was incubated at 37 C for 16 h.The reaction mixture was subsequently frozen and lyophilized, and the residue was resuspended in 500 mL ice cold methanol and ltered.One portion of each claried reaction mixture was analyzed by analytical reverse-phase HPLC equipped with a Phenomenex Luna-C18 column (250 mm Â 4.6 mm, 5 mm).The ow rate was 1 mL min À1 Detections were set at 220 and 296 nm.Conversion rate was calculated by the corresponding HPLC peak area percentage using the Agilent Chromatography Workstation Soware.The LC-ESI-MS analysis was accomplished using standard C-18 reversed-phase chromatography with diode array detection wherein 5% of the ow was diverted to time-of-ight (TOF) mass spectrometer.

Preparative scale glycosylation reaction
Aglycons (10 mg) were dissolved in 5% of DMSO and transferred to pH 8 buffer solution (50 mM Tris-HCl, 5 mM MgCl 2 ).UDPG was added followed by OleD (ASP) catalyst.Aer 16 h incubation at 37 C, the reaction was stopped with equal volume of ice cold methanol.Then the reaction mixture was centrifuged at 10 000g for 30 min and the supernatant was concentrated under reduced pressure, and the debris was resuspended in 5 mL of ice-cold methanol and ltered with 0.22 mM membrane.The ltrate was subjected to preparative HPLC (20 Â 250 mm, 5 mm; ow rate: 5 mL min À1 ; A 296 and A 220 detection) using water/ acetonitrile as the eluent to afford the corresponding of glucosides.The compound was then characterized using high resolution MS, 1 D and 2 D NMR, including 1 H, 13 C and HSQC. 1b-glu.

Determination of kinetic parameters
Assays were performed in a nal volume of 200 mL 50 mM Tris-HCl (pH 8.0), and contained constant concentrations of OleD (ASP) (40 mg) and UDPG (2.5 mM) while varying the concentration (0.01-1.2 mM) of 2b, 2a, 4b and 4a.Aliquots (100 mL) were removed every 15 min, mixed with an equal volume of ice cold methanol, and centrifuged at 10 000g for 10 min.Supernatants were analyzed by analytical reverse-phase HPLC.Conversion rate is calculated by the corresponding HPLC peak area percentage using the Agilent Chromatography Workstation Soware. 25All experiments were performed in triplicate.Initial velocities were tted to the Michaelis-Menten equation using Origin Pro 7.0 soware.

Molecular docking
The program Autodock Vina was used for docking simulations.For docking purpose, the crystal structure of OleD (PDBID 4M60) was retrieved from Protein Data Bank.To create receptor and ligand structures for docking, the following procedure was conducted.Firstly, the 3D structures of the ligands were prepared using the Gaussian 09 program at the B3LYP/6-31G(d) level.Harmonic vibration frequencies were calculated to conrm the stability of these conformers.Then the receptor and optimized structure of the ligands were converted to required pdbqt format using Autodock Tools 1.5.4.The Autodock Vina parameters were set as follow, box size: 15 Â 15 Â 15 A, the center of box: x ¼ 38.96, y ¼ 61.05, z ¼ 13.83, the exhaustiveness: 100, and number of output conformations was set to 20.The calculated geometries were ranked in terms of free energy of binding and the best poses were selected for further analysis.All molecular visualizations were carried out in PyMOL soware.

Assessment inhibition NKA activity of CTS
The inhibitory effects of CTS on NKA were determined essentially as previously reported method. 11,26,27

Preparation of epimers of bufadienolides
Three epimers, i.e. a-bufalin (1a), a-resibufogenin (2a) and acinobufagin (3a) were synthesized from b-bufalin (1b), b-resibufogenin (3b) and b-cinobufagin (3b), respectively, by an oxidation with pyridinium chloride hydrochloride followed by a reduction with sodium borohydride.Uzarigenin (4a) and digitoxigenin (4b), two natural cardenolides with a trans and cis A/B fusion mode (4a, 4b Fig. 1), respectively, were identied from the whole herb of Asclepias curassavica 28 and the roots of Streptocaulon juventas, 29 respectively.These probes are suitable for us to investigate whether the congurations at C-3 and the fusion mode of A/B ring inuence the OleD-catalyze glycosylation.

Glycosylation of 1b and 1a
Both 3a-and 3b-hydroxylated bufalin are endogenous in Bufo bufo gargarizans.Though only 3b-hydroxylated bufalin is present as a toxic chemical defence in toad venom, both epimers were found to occur at a 2 : 3 ratio in the heart and a 1 : 2 ratio in the blood. 223b-Hydroxylated bufalin is the major active component of the toad venom and exhibits potent cardiotonic activity. 30,31Its structural core includes a cis-transcis fused steroid core with two hydroxyl groups at C-3 and C-14.
However, it has serious toxicity because there is no distinct selectivity for a1 and a2 subunits of NKA.Fortunately, a lot of bufalin analogues including glycosides have been prepared by chemical and biological transformation.Recently, our group reported synthesis of 3b-N-methoxy-N-b-D-glucoside of bufalin which could enhance its inhibition on NKA. 22The latest crystal structure demonstrated that the level of glycosylation affect the depth of CTS binding and that the steroid core substituents ne tune the conguration of transmembrane helices aM1-2. 27urthermore, the sugar unit could enhance the selectivity on the alpha2 isoform of NKA; however, chemical synthesis of bufalin glycoside was laborious and consumed a lot of toxic reagents.It is necessary to develop a green method for glycosylation of bufadienolides.
The pilot biotransformation of 1b and 1a were carried out using UDPG as the sugar donor and OleD (ASP) as the catalyst under the standard conditions (0.5 mM UDPG, 0.1 mM aglycon, 16 h). 5The result of LC-MS showed that OleD (ASP) could catalyze the generation of monoglycoside of 1b with a conversion rate 30%; while glycosylation product of 1a was not detected (Fig. 2).Thus the bioconversion of 1b and 1a catalyzed by OleD (ASP) was inuenced by the C-3 conguration.The conversion rates of different isomers were compared in Table 1.
To maximize the production of bufalin-3-O-b-D-glucoside (1b-glu), a 20 h reaction was carried out at 37 C. Compound 1b (10 mg, 40 mM) was dissolved in DMSO (0.625 mL) and diluted with buffer solution (50 mM Tris-HCl, 5 mM MgCl 2 , pH 8.0, 25 mL total volume).UDPG (38 mg, 50 mM) was added along with OleD (ASP) (13 mg).The reaction was stopped with 25 mL of ice cold methanol.Then the reaction mixture was centrifuged at 10 000g for 30 min and supernatant was concentrated under reduced pressure.The residue was dissolved in 3 mL methanol, and ltered with 0.22 mM membrane.The ltrate was subjected to preparative HPLC using water/acetonitrile as the eluent.

Glycosylation of 2b and 2a
Resibufogenin (2b) is another type of bufadienolides from the venom of Bufo bufo gargarizans with an 14,15-epoxide ring in contrast to the 14-OH in bufalin.Compound 2b has been reported to exhibit a wide range of activities such as cardiotonic, renal sodium excretion, blood pressure stimulating, antitumor activity and immunoregulatory activity. 32However, the application of resibufogenin is restricted because of its strong toxicity. 33In the past several years, several structure modications on 2b was performed which generate more than 30 derivatives. 23In order to obtain the glycosylation derivative of 2b and compare the NKA inhibitory activities of these derivatives with different congurations, OleD (ASP) catalysed biotransformation on 2b and 2a were carried out.
The pilot reaction conditions are the same as those described for 1b and 1a.According to the result of LC-MS analysis, OleD (ASP) catalyzed the formation of monoglucoside (major) and diglucoside (minor) of 2b with a total conversion rate 80% which was much higher than that of 1b; while the conversion rate of monoglucoside 2a-glu is about 1% in contrast to the absence of 1a glucoside (Fig. 3).Thus similar to 1b and 1a, the conguration at C-3 was also important for conversion rate of 2b and 2a.The much high conversion rate of 2b and 2a might be due to the 14,15-epoxide moiety as compared to the 14-OH for 1b and 1a.
To maximize the production of resibufogenin-3-O-b-Dglucoside (2b-glu), a preparative scale experiment was undertaken.Using the same condition as 1b, while 2b (10 mg, 40 mM) was served as substrate, we obtain 8.4 mg 2b-glu with a conversion rate 65%.The identication of compounds 2b-glu was determined by 1 D NMR and HR-ESI-MS analysis.The pseudo-molecular ion m/z 547.2902 [M + H] + in the HR-ESI-MS corresponded to a formula C 30 H 42 O 9 (calcd for 547.2938) which was 162 unit larger than the parent compound 2b.Compared with 2b, the chemical shi C-3 of 2b-glu was shied to downeld by 3.9 ppm and the chemical shi H-3 was shied to downeld by 0.02 ppm, conrming the glycosylation site was also at 3-OH.The b-conguration was determined by the large coupling constant of the anomeric proton (d H 4.32, d, J ¼ 7.8 Hz).Accordingly, it was concluded that OleD (ASP) could catalyze the transformation of 2b to its glucoside 2b-glu with a much higher rate than 1b.It is noteworthy that bioconversion of 2b also lead to the generation of a diglucoside (2b-diglu) which was conrm by the HR-MS m/z 709.3421 [M + H] + .Due to the small amount, it was not determined by NMR.

Glycosylation of 3b and 3a
Cinobufagin is also a major bufadienolides (4-6% dry weight) from the venom of Bufo bufo gargarizans with an 14,15-epoxide ring and an acetyl group at C-16. 34 Cinobufagin was found to show potent cardiotonic, blood pressure-stimulating, antiviral, local anesthetic and antineoplastic activities. 34,35However, the poor water solubility restricted its clinical use.Though a series of analogues of cinobufagin has been generated by chemical synthesis and cell suspension cultures, 33,36 the glycosylation method is rarely reported.Furthermore, difference between the glycosidation prole on the 3b and 3a isomers of cinobufagin is unclear.Using the same reaction conditions as described for 2b and 2a, OleD (ASP) was found to catalyze the glycosylation of 3b leading to both monoglucoside (3b-glu) and diglucoside (3bdiglu) of 3b with a total conversion rate of 75%; while at the same condition as 3b, only the monoglucoside of (3a-glu) was detected with a low conversion rate 2% (Fig. 4).Similarly, this phenomenon further conrmed the importance of conguration at C-3 for the conversion rate.It is noteworthy that with an additional acetyl group at C-16 compound 3b showed similar conversion rate as 2b.Similarly, 3b also generate a diglucoside (3b-diglu) which was conrm by the HR-ESI-MS m/z 767.3479 [M + H] + .Due to the small amount, it was not determined by NMR.
Using the same conditions as 2b, we prepared 6.9 mg 3b-glu with a conversion rate 50% (10 mg 3b was served as substrate).The identication of the product 3b-glu was also conrmed by 1

Glycosylation of 4b and 4a
As compared to the three pairs of bufadienolides 1b and 1a, 2b and 2a, 3b and 3a, digitoxigenin (4b) and uzarigenin (4a) were two cardenolides with the same substitution pattern.The only difference between 4b and 4a is the A/B ring fusion modes, which are cis and trans, respectively.Compound 4b was the aglycone of digoxin, a commonly used cardiotonic drug. 37ecently, it is reported that introduction of a sugar unit at C-3 of cardenolides could improves NKA isoform selectivity (a2/a3 over a1). 38,39In order to compare the glycosylation proles of 4b and 4a with different A/B ring fusion mode, the OleD (ASP) catalyzed bioconversion was carried out.
Using the same reaction conditions as described for the bufadienolides, OleD (ASP) catalyzed the generation of monoglucoside of 4b-glu with a conversion rate of 26%; while the conversion rate for 4a is only 2% (Fig. 5).Thus the A/B ring fusion mode was also important for the conversion rate, and the lactone rings (either six-membered or ve membered) at position C-17 did not affect the biotransformation.
We prepared the monoglucoside of 4b (4b-glu) for structural analysis.Compound 4b-glu (3.6 mg) was obtained by preparative HPLC with conversion rate 25% (10 mg 4b of was used as the substrate).Similar to the bufadienolide glycosides 1b-glu, 2b-glu and 3b-glu, the monoglucoside structure of 4b-glu was conrmed by 1   glycosylation rate.In order to compare the dynamic process, K m value was measured.
Epimers 2b and 2a representing the high conversion bufadienolides and 4b and 4a representing the cardenolides were chosen in this study.A series of concentrations of the substrates (0.01-1.2 mM) were incubated with the OleD (ASP) enzyme and UDPG, and the conversion rate was calculated by the corresponding HPLC peak area using the Agilent Chromatography Workstation Soware.K m value was determined based on the Michaelis-Menten equation. 25The result indicated that a high conversion rate corresponded to a low K m value.Particularly, when the conversion rate is less than 2%, K m value is toward a large value more than 100 mM (Table 2).

Molecular modeling
Molecular docking studies shed new light on the mechanism of stereoselective glycosylation of cardiotonic steroids derivatives.Similar for the K m study, we selected the high conversion 2a and 2b to explore the binding mode in the enzyme substrate complex.As can be seen from Fig. 6A, the 2Hpyran-2-one ring of compound 2a penetrated deeply into the hydrophobic region I dened by His20, Phe85 and Trp74 residues.The epoxide contacted via van der Waals interactions with residues lle112 and Val82.In comparison to 2a binding to OleD (ASP), compound 2b showed an 'inverse' binding pose that C-3 aliphatic hydroxyl was placed in the hydrophobic pocket I and the 2H-pyran-2-one moiety bound in a hydrophobic cavity II (lle112, Ser184, Fig. 6B).Moreover, the oxygen atom of epoxide group formed a hydrogen bond (2.5 A) with critical residue Tyr115.It can be inferred from docking results that the lling of hydrophobic pocket II and interacted with residue Tyr115 may play an important role in the O-linked glycosylation reactions catalyzed by OleD (ASP) glycosyltransferase, since the conversion efficiency is 1% for 2a and 80% for 2b, respectively.

Inhibition of NKA activity
To explore the inhibitory activity of glycosylated products on NKA, the inhibition activity of CTS (1b, 2b, 3b, 4b) and their glycosylation products (1b-glu, 2b-glu, 3b-glu, 4b-glu) were determined using previous reported method. 11,27As seen in Table 3, glycosylation products showed a stronger inhibitory activity for NKA than the corresponding aglycones.

Conclusions
In summary, this study demonstrated that the OleD-catalyze glycosylation of cardiotonic steroids are signicantly inuenced by the conguration at C-3 and the A/B fusion mode.3b-OH and A/B ring cis fusion are favoured by OleD (ASP), while an acetyl group at C-16 and lactone ring type at C-17 did not inuence the biotransformation.A high conversion rate corresponded to a low K m value.Molecular docking simulation showed that lling the hydrophobic pocket II and interaction with residue Tyr115 may play an important role in the glycosylation reactions catalyzed by OleD (ASP) glycosyltransferase.Furthermore, the glycosylation products showed a stronger inhibitory activity for NKA than the corresponding aglycones.This study provided the rst stereoselective properties for OleD a Conversion: calculated by the corresponding HPLC peak area percentage.(ASP) catalyzed glycosylation.It is noteworthy that glycosyltransferase has been used for the glycosylation of a wide variety of natural products; 40,41 however, glycosyltransferase for the cardiotonic steroids is rare.Results of this study rstly revealed the stereo-selectivity of OleD-catalyzed glycosylation of cardiotonic steroids.

Fig. 1
Fig. 1 Structures of cardiotonic steroid substrates and the corresponding glycosylation products.

Finally, 3 .
1 mg of 1b-glu was obtained with a conversion rate of 21%.The structure of 1b-glu was determined by 1 D NMR and HR-ESI-MS analysis.The pseudo molecular ion m/z 549.3035 [M + H] + in the HR-ESI-MS was corresponding to a formula C 30 H 44 O 9 (calcd for 549.3021), which was 162 unit larger than the parent compound bufalin, suggesting the formation of monoglucoside.Compared with 1b, the chemical shi d C-3 of 1b-glu was shied to downeld by 3.9 ppm and the d H-3 was shied to downeld by 0.02 ppm; while C-14 was unchanged, conrming the glycosylation site was at 3-OH.The b-conguration was determined by the large coupling constant of the anomeric proton (d H 4.37, d, J ¼ 7.8 Hz).Based on the above analysis, it was concluded that OleD (ASP) could catalyze the transformation of 1b to its monoglucoside 1b-glu at 3-OH.
D and 2 D NMR and HR-ESI-MS analysis.HR-ESI-MS showed a pseudo molecular ion m/z 605.2996 [M + H] + corresponding to a formula C 32 H 44 O 11 (calcd for 605.2938) which was 162 unit larger than the parent compound 3b.Similar to 2b-glu, the chemical shis of C-3 and H-3 of 3b-glu were shied to down-eld by 3.9 ppm and 0.02 ppm, respectively, conrming the glycosylation site at 3-OH.The b-conguration was also conrmed by the large coupling constant of the anomeric proton (d H 4.32, d, J ¼ 7.8 Hz).Accordingly, it was concluded that OleD (ASP) could catalyze the transformation of 3b into 3bglu.

Fig. 6 (
Fig. 6 (A) Selected docking poses compound 2a (depicted in yellow) and 2b (depicted in magenta) into the substrate cavity of OleD (ASP).The two ligands are shown in stick representation.The receptors are shown in cartoon representation with cyan alpha helices, green beta sheets and magenta loops.(B) OleD (ASP) (surface)-ligand (stick) complex.

Table 1
Conversion rates of individual compounds a N. T: not detected.

Table 2
Conversion rate and K m