Probing the regiospecificity of enzyme-catalyzed steroid glycosylation

Article abstract of DOI:10.1021/ol3024924

The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-O-β-d-glucoside.

Full text of DOI:10.1021/ol3024924

ORGANIC
LETTERS
2012
Vol. 14, No. 21
5424–5427
Probing the Regiospecificity of
Enzyme-Catalyzed Steroid Glycosylation
Maoquan Zhou,† Yanpeng Hou,† Adel Hamza,‡ Chang-Guo Zhan,‡ Tim S. Bugni,*,† and
Jon S. Thorson*,‡
Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin,
Madison, Wisconsin 53705, United States, and Center for Pharmaceutical Research and
Innovation, University of Kentucky, College of Pharmacy, 789 South Limestone Street,
Lexington, Kentucky 40536-0596, United States
tbugni@pharmacy.wisc.edu; jsthorson@uky.edu
Received September 10, 2012
ABSTRACT
The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability
of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While
a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked
diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin
12-O-β-D-glucoside.
Steroidal glycosides such as digitoxin, digoxin, and pro-
scillaridin have been used for centuries to treat congestive
heart failure and more recently noted to display highly
potent anticancer activity.1 These ligands are known to
bind the Naþ,Kþ-ATPase alpha subunit with high affinity
where they function to control a range of intracellular
signaling cascades critical to cell proliferation and regulate
intracellular Naþ and Kþ concentrations.2 C3 glycosylation
of the steroidal core among this natural product class has a
dramatic effect upon the delicate balance between signaling
(antiproliferative) versus inotropic (cardiotoxicity) activi-
ties.3 Thus, there remains notable interest in the develop-
ment of simple glycosylation platforms as a means to
improve the putative therapeutic index of steroidal glyco-
side anticancer preclinical leads. Using a novel high
throughput screen and LC-MS,4 representative steroidal
aglycons were recently identified as substrates for a set
of highly permissive glycosyltransferase variants derived
from the macrolide-inactivating glucosyltransferase OleD.5
† University of Wisconsin.
‡ University of Kentucky.
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r
10.1021/ol3024924
Published on Web 10/18/2012
2012 American Chemical Society

Scheme 1. OleD Catalyzed Glucosylation of Digitoxigenin
Herein we extend this preliminary study through the appli-
cation of an LC-UV/MSSPE-NMR platform6 to rapidly
probe the regio-/stereospecificity of OleD-catalyzed glyco-
sylation of cardenolide and bufadienolide aglycons. While
this study reveals a bias toward the desired C3 regiospeci-
ficity in the context of a range of non-native substrates
for this enzyme, it also highlights how subtle modifica-
tions of the steroidal aglycon can dictate and/or prohibit
glycosyltransfer.
To explore the feasibility of the LC-UV/MS-SPE-NMR
platform for microscale structural elucidation of steroidal
glycosides, an initial pilot study was conducted using
digitoxigenin as the model (Scheme 1). For this study,
the reaction contained 600 μg of OleD ASP, 2.5 mM UDP-
Glc, 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, and 1 mM of
digitoxigenin in a total volume of 1 mL. The reaction was
allowed to proceed for 16 h at 25 °C and subsequently
frozen and lyophilized, and the debris was resuspended in
2 mL of ice cold MeOH, filtered, and concentrated to
150 μL for LC-UV/MS-SPE-NMR analysis. The HPLC
component of the subsequent analysis platform was ac-
complished using standard C18 reversed-phase chromato-
graphy with diode array detection wherein ∼5% of the
flow was diverted to quadrupole time-of-flight mass
(QTOF) detection. Fractions containing detected peaks
were automatically diverted to preconditioned solid-phase
extraction (SPE, C18) cartridges, which were subsequently
dried via N2 gas and then eluted with 30 μL of CD3CN into
1.7 mmNMR tubesfor direct analysisvia a BrukerAvance
Figure 1. Potential substrates and determined products of OleD-
catalyzed test reactions.
an observed HMBC correlation between the sugar anome-
ric C10 proton and the C3 of digitoxigenin. An observed
large anomeric proton coupling constant (δH 4.32, dou-
blet, 8.0 Hz) supported the formation of the β-anomer,
consistent with an established inverting mechanism for
OleD variants studied to date.8 C3 glucosylation also led
to a notably consistent large downfield 13C shift (∼7 ppm)
of the C3 carbon (‘glycosylation shift’),9 which served as a
convenient indicator in probing OleD-catalyzed glycosy-
lation of alternative steroidal aglycons (Table 1).
1
III 600 MHz spectrometer with a 1.7 mm H{13C/15N}
cryogenic probe.
LC-MS analysis of the pilot reaction described above
revealed the formation of a single glycoside in 20% yield.
Structure elucidation of this glycoside, based upon 1HNMR,
1
13
13
1Hꢀ H COSY, 1Hꢀ C HSQC, and 1Hꢀ C HMBC, was
consistent with the 3-O-β-D-glucoside 2 (Scheme 1).7 Key
support for the regiospecificity assignment derived from
ꢀ
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Org. Lett., Vol. 14, No. 21, 2012
5425

Figure 2. Simulated ligand-bound models for OleD-1 (a) and OleD-3 (b). Ligands are represented in cyan, OleD in green, key active-site
residues in purple, and H-bonds as dashed lines. The calculated binding energy of the compounds 1 and 3 with OleD are ꢀ12.1 and
ꢀ11.9 kcal/mol.
Table 1. C3 13C Chemical Shifts of Substrates and Products
Table 2. In Vitro Cytotoxicity (μM)
compound
C3 shift (ppm)
compound
C3 shift (ppm)
compound
H460a
A549b
1
3
4
7
8
66.7
2
73.5
66.0b
73.6
75.6
73.7
74.8
73.2
74.1
3
0.38 ( 0.05
3.60 ( 0.24
0.30 ( 0.02
1.49 ( 0.07
66.3a
66.810
67.73c
66.711
10
11
12
13
14
15
16
10
a H460, large cell lung carcinoma. b A549, small cell lung carcinoma.
toward C3 glycosylation was observed with one surprising
exception: digoxigenin, which led to C12 glucosylation
exclusively in high efficiency.
9
66.612
a C12 13C chemical shift 73.7 ppm. b C12 13C chemical shift 82.1 ppm.
The docking and molecular dynamics (MD) simulation of
OleD complexed with 1 or 3 are consistent with our experi-
mental data (Figure 2). In comparison to the binding mode
of 1, the binding model of 3 invokes an ‘inverse binding
mode’ due to electrostatic and steric repulsions between the
steroid C12-OH and the Tyr140 side chain -OH (Figure S3).
Similar docking and MD simulations with ligands 4ꢀ9, 13,
and 15 revealed the following key observations. The pre-
dicted binding of ouabaingenin (5) and strophanthidin (6)
Given the success of the digitoxigenin pilot study, ste-
roidal aglycons 3ꢀ9 (Figure 1) were subsequently treated
in an identical manner. Five (aglycon 3, 4, 7, 8, and 9) out
ofthe seven putative aglycons led toglucosylated products,
three (3, 4, and 7) of which led to monoglucosides (10,13
11,14 and 1215) and two (8 and 9) of which provided both
mono- (13 and 1516) and diglucosides (14 and 16).17 A bias
˚
are misaligned and distant (∼4.7 A, Figure S4, panels b and
c) with the active-site base, the His19 side chain imidazole,
due in part to the electrostatic attraction between the Asn80
side-chain carboxamide and functional groups at C19 in
5 or 6. In contrast, the predicted binding modes of 8 and
9 highlight favorable dipoleꢀquadrupole stabilization
between the substrate C16 methyl ester and the Tyr140
(Figure S4), also prevalent in the binding models for the
corresponding monoglucosides forms 13 and 15, which may
be important to the observed iterative (steroid C3 and
subsequent glucoside C20) glucosylation of 8 and 9.
(13) For the synthesis of digoxigenin 3-Gc, see: Reference 7a and
Brown, L.; Boutagy, J.; Thomas, R. Arzneim.-Forsch. 1981, 31, 1059–
10604. For the chemical synthesis of the 3,12-diglucoside of digoxigenin:
Makarevich, I. F Pharm. Chem. J. 1969, 5, 266–269.
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Med. 1994, 60, 63–69.
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2002, 43, 8535–8538. (b) Ye, M.; Han, J.; Guo, H.; Guo, D. Magn.
Reson. Chem. 2002, 40, 786–788.
(17) The diglucosylation was evidenced by the HMBC coupling of
the anomeric proton of the second sugar with the C2 carbon of the first
sugar.
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Org. Lett., Vol. 14, No. 21, 2012

While the impact of C3 glycosylation upon the anti-
cancer activity of steroidal glycosides has been previously
reported,3a,18 the influence of C12 glycosylation upon
the bioactivity of this natural product class had not. Thus,
the enzymatic synthesis of 10 was subsequently scaled to
enable bioactivity assessment. Specifically, digoxigenin 3
(10 mg, 25.6 μmol) was dissolved in 0.65 mL of DMSO and
transferred to 25 mL of assay buffer solution (50 mM Tris
HCl, 5 mM MgCl2, pH 8.0), and the reaction was initiated
via addition of UDP-Glc (62 mg, 0.124 mmol) and 16 mg
of OleD ASP. After 24 h of agitation at room temperature,
the reaction was quenched and flushed though a 12 mL
HLB column, which was subsequently dried with N2 gas
and then eluted with methanol. The methanolic eluent was
concentrated, and the collected residue was subjected to
flash chromatography using CH2Cl2/MeOH to afford
digoxigenin 12-O-β-D-Glc 10 (13 mg, 23.5 μmol, 92%),
highlighting the first synthesis of digoxigenin 12-O-β-D-
Glc. Consistent with the belief that steroidal glycosides
bind the Naþ, Kþ-ATPase alpha subunit with the lactone
(and presumably C12) buried deep within the ligand
binding pocket,19 the C12 glucoside was found to be
∼5ꢀ10-fold less active than the corresponding aglycon
based upon in vitro cytotoxicity against two lung cancer
cell lines (Table 2). The rather surprising fact that the
activity of the C12 glycoside was not further diminished
suggests notable flexibility in the Naþ, Kþ-ATPase alpha
subunit ligand-binding pocket.
In summary, this study highlights the capabilities of
OleD ASP in the context of steroidal glycoside construc-
tion and reveals how subtle alterations of the steroidal
architecture can dramatically influence the regioselectivity
of the reaction. As such, this study serves to provide
additional information for understanding the substrate
specificity of the uniquely permissive OleD ASP variant
and also sets the stage for rapid enzymatic glycorandomi-
zation of this class of natural product through the use of
alternative sugar donors.20
Acknowledgment. This work was supported by NIH
AI52218 (J.S.T.) and also made use of the National
Magnetic Resonance Facility at Madison, which is sup-
ported by NIH Grants P41RR02301 (BRTP/NCRR) and
P41GM66326 (NIGMS). Additional equipment was pur-
chased with funds from the University of Wisconsin, the
NIH (RR02781, RR08438), the NSF (DMB-8415048,
OIA-9977486, BIR-9214394), and the USDA.
Supporting Information Available. Experimental and
computational procedures, characterization, and biolog-
ical testing. This material is available free of charge via the
Internet at http://pubs.acs.org.
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L. J.; Franke, K.; Khine, M. M.; Kelter, G.; Fiebig, H. H.; Neumann, J.;
Wessjohann, L. A. J. Ethnopharmacol. 2011, 134, 781–8.
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Mol. Biol. 2009, 76, 55–119. (b) Gantt, R. W.; Peltier-Pain, P.; Thorson,
J. S. Nat. Prod. Rep. 2011, 28, 1811–1853.
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Natl. Acad. Sci. U.S.A. 2009, 106, 13742–13747. (b) Katz, A.; Lifshitz,
Y.; Bab-Dinitz, E.; Kapri-Pardes, E.; Goldshleger, R.; Tal, D. M.;
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The authors declare the following competing financialinterest(s): The
authors report competing interests. J.S.T. is a co-founder of Centrose
(Madison, WI).
Org. Lett., Vol. 14, No. 21, 2012
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