Assessing the regioselectivity of oleD-catalyzed glycosylation with a diverse set of acceptors

Article abstract of DOI:10.1021/np300890h

To explore the acceptor regioselectivity of OleD-catalyzed glucosylation, the products of OleD-catalyzed reactions with six structurally diverse acceptors flavonesnY (daidzein), isoflavones (flavopiridol), stilbenes (resveratrol), indole alkaloids (10-hydroxycamptothecin), and steroids (2- methoxyestradiol)- were determined. This study highlights the first synthesis of flavopiridol and 2-methoxyestradiol glucosides and confirms the ability of OleD to glucosylate both aromatic and aliphatic nucleophiles. In all cases, molecular dynamics simulations were consistent with the determined product distribution and suggest the potential to develop a virtual screening model to identify additional OleD substrates.

Full text of DOI:10.1021/np300890h

Article
pubs.acs.org/jnp
Assessing the Regioselectivity of OleD-Catalyzed Glycosylation with
a Diverse Set of Acceptors
Maoquan Zhou,† Adel Hamza,‡ Chang-Guo Zhan,‡ and Jon S. Thorson*,‡
†Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, United States
‡Department of Pharmaceutical Sciences, Center for Pharmaceutical Research and Innovation, College of Pharmacy, University of
Kentucky, 789 S. Limestone Street, Lexington, Kentucky 40536, United States
S
* Supporting Information
ABSTRACT: To explore the acceptor regioselectivity of
OleD-catalyzed glucosylation, the products of OleD-catalyzed
reactions with six structurally diverse acceptors flavones
(daidzein), isoflavones (flavopiridol), stilbenes (resveratrol),
indole alkaloids (10-hydroxycamptothecin), and steroids (2-
methoxyestradiol)were determined. This study highlights
the first synthesis of flavopiridol and 2-methoxyestradiol
glucosides and confirms the ability of OleD to glucosylate
both aromatic and aliphatic nucleophiles. In all cases,
molecular dynamics simulations were consistent with the
determined product distribution and suggest the potential to develop a virtual screening model to identify additional OleD
substrates.
he glucosyltransferase OleD from Streptomyces antibioticus
acceptor architecture. A parallel molecular dynamics simulation
Tcatalyzes the glucosylation of oleandomycin using UDP-D- for each reaction studied was consistent with the corresponding
product distribution observed and sets the stage to use virtual
screening as a means to identify additional OleD substrates.
glucose (UDP-Glc) as the glycosyl donor (Figure 1). This
enzyme, first studied by Salas and co-workers, exists as part of a
prototype system for macrolide inactivation and secretion in
macrolide-producing microorganisms.1 Consistent with its role
in detoxification, wild-type OleD (wtOleD) displays relatively
broad substrate tolerance with a bias toward small aromatic
hydroxy groups, and recent OleD-directed evolution and
engineering efforts dramatically improved upon this catalyst’s
proficiency and range of accessible substrates.2,3 The availability
of enhanced OleD mutants and simple activated aromatic
glycoside donors also enabled efforts to modulate the
corresponding reaction equilibrium as a unique strategy for
sugar nucleotide synthesis, glycodiversified small molecules, and
even a high-throughput screen for glycosylation.3c Cumula-
tively, these studies revealed OleD variants to function as a
multifunctional and iterative O-/S-/N-GT capable of glucosy-
lating well over 100 diverse acceptors. However, with a few
exceptions2,3b,4 the product characterization for these studies
was limited to LC-MS, and thus, the regio-/stereospecificity of
OleD-catalyzed glycosyltransfer with “non-native” substrates
remains poorly understood.5 To address this limitation, herein
we describe the characterization of glycosides produced via the
OleD-catalyzed glucosylation of a set of six representative
structural classes: flavones (daidzein), isoflavones (flavopiridol),
stilbenes (resveratrol), indole alkaloids (10-hydroxycamptothe-
cin), and steroids (2-methoxyestradiol). This study revealed
OleD to glycosylate both aromatic and aliphatic nucleophiles,
the regioselectivity of which was dictated by a range factors,
including reaction thermodynamics, enzyme mutation, and the
RESULTS AND DISCUSSION
■
Glucosylation of Daidzein. Isoflavones occur naturally in
legumes and are consumed regularly in the human diet.6
Isoflavones often exist naturally as O-glycosides and have
attracted considerable pharmaceutical interest.7 Daidzein is one
of the most commonly occurring isoflavones, with the
corresponding 4′- and 7-O-glucosides as well as the 7,4′-di-O-
glucosides of daidzein isolated from numerous sources.8
Daidzein and its corresponding O-glucosides are believed to
be the major effective components of a traditional Chinese
medicine, Kudzu (Pueraria lobata), for the treatment of a wide
range of disorders since 600 A.D.9 While daidzein has limited
solubility in water, the aqueous solubility of the corresponding
7-O-glucoside is ∼30-fold improved.10 The unique activities of
these glycosides have inspired several targeted syntheses of
daidzein 4′- and 7-O-glucosides.11 However, a convergent
method to produce both mono- and diglucosides in parallel has
not been reported.
The pilot reaction for this study utilized UDP-Glc as the
donor and OleD ASP3 as the catalyst under standard conditions
(0.5 mM UDP-Glc, 0.1 mM aglycon, 16 h). On the basis of LC-
MS, three products were observed (two monoglucosides and
one diglucoside), with the diglucoside emerging as the major
Received: December 21, 2012
Published: January 29, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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Figure 1. The native OleD-catalyzed reaction as a macrolide-producing host resistance mechanism.
Figure 2. (a) Products deriving from OleD ASP-catalyzed glucosylation of daidzein. (b) Product distribution over time (the standard deviation of
three trials was 3%).
Figure 3. (a) Products deriving from OleD ASP-catalyzed glucosylation of resveratrol. (b) Product distribution over time (the standard deviation of
three trials was 2.5%).
1
product over time (Figure 2b). To maximize the production of
all three products for subsequent characterization, a 25 h
reaction was selected for the preparative-scale reaction. For this,
daidzein (6.4 mg, 28.0 μmol) was dissolved in 1.25 mL of
DMSO and transferred to 25 mL of assay buffer solution (50
mM Tris HCl, 5 mM MgCl2, pH 8.0). The reaction was
initiated via the addition of UDP-Glc (38 mg, 62.3 μmol) and
25 mg of OleD ASP. After 25 h of gentle agitation at room
temperature (rt), the reaction was frozen and lyophilized to
dryness. HPLC purification of the crude reaction mixture
provided daidzein 7,4′-di-O-β-D-diglucoside 6 (5 mg, 8.6 μmol,
31%), daidzein 4′-O-β-D-glucoside 5 (1 mg, 2.4 μmol, 9%), and
daidzein 7-O-β-D-glucoside 4 (2 mg, 4.8 μmol, 17%). The H
and 13C NMR and HR-MS data of the isolated glucosides were
consistent with previously reported characterization data.11
Glucosylation of Resveratrol. Resveratrol, a naturally
occurring phytoalexin found in various plants, grape skin,
peanuts, cranberries, and red wine,12 reportedly exhibits
multiple effects including life extension,13 neuroprotection,14
anti-inflammatory activity,15 cardioprotection,16 antidiabetic
activity,17 viral inhibition,18 and cancer chemoprevention.19
Recent work also revealed resveratrol to inhibit Wnt/β-catenin
signaling, which inspired the synthesis of stilbene analogues
that inhibit a unique target (methionine adenosyltransferase)
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Figure 4. (a) OleD-catalyzed glucosylation of flavopiridol. (b) Flavopiridol glucosylation mediated by different OleD variants.
and display dramatic anticancer activity.20 Despite these
beneficial effects, the low bioavailability of resveratrol limits
therapeutic application.21 In humans and rats, less than 5% of
an oral dose was observed as free resveratrol, with the most
abundant metabolites comprising resveratrol 3-O-glucuronide
and resveratrol 3-O-sulfate.22 The β-D-glucosides of resveratrol,
8,23 9,24 and 1025 (Figure 3), are also naturally occurring
products that possess antiplatelet,26 antioxidant,27 and prolyl
endopeptidase inhibitory activities,28 and these activities
stimulated the pursuit of various resveratrol glycosylation
strategies.29 Among these, the four glycosides 8, 9, 10, and 11
were synthesized in parallel using multistep trifluoroacetimidate
methodology.29d
range of cancer cell lines and initiates cell cycle arrest and p53-
independent apoptosis through down-regulation of Mcl-1 and
X-linked inactivator of apoptosis (XIAP).31 Preclinical studies
demonstrated the capacity of flavopiridol to induce pro-
grammed cell death, promote differentiation, inhibit angiogenic
processes, and modulate transcriptional events.32 These unique
characteristics inspired extensive clinical investigation of
flavopiridol.33 Flavopiridol is eliminated via excretion in the
form of both the parent drug and the C-5- or C-7-
glucuronide.34
Using UDP-Glc as the donor and OleD ASP as catalyst (1.25
mM UDP-Glc, 0.25 mM aglycon, 16 h), the formation of a
single monoglucoside (10% conversion) was observed by
HPLC and LC-MS analysis (Figure 4). In an effort to boost
production of this desired product, reactions catalyzed by a
panel of OleD mutants were examined (Figure 4b), which
surprisingly revealed wtOleD to enable the best conversion
(35%). Thus, flavopiridol (11.1 mg, 25.0 μmol) was dissolved
in 1.25 mL of DMSO and transferred to 50 mL of assay buffer
solution (50 mM Tris HCl, 5 mM MgCl2, pH 8.0). The
reaction was initiated via addition of UDP-Glc (76 mg, 0.125
mmol) and 15 mg of wtOleD. After 24 h of gentle agitation at
rt, the reaction was frozen and lyophilized to dryness. The
residue was dissolved in MeOH and subjected to HPLC
purification to give flavopiridol monoglucoside product (4.9
mg, 8.7 μmol) in 35% yield.
The pilot reaction utilized UDP-Glc as the donor and OleD
ASP as catalyst under standard conditions (2.5 mM UDP-Glc, 1
mM aglycon). The reaction was nearly complete within 3 h,
leading to two diglucosides and two monoglucosides based
upon LC-MS. A continuation of the reaction for longer periods
of time (48 h) led to the production of two diglucosides, 10
and 11, as the only products (data not shown). On the basis of
this pilot reaction, 3 h was selected as the optimal reaction time
for a preparative-scale reaction. For the preparative-scale
reaction, resveratrol (5.7 mg, 25.0 μmol) was dissolved in
1.25 mL of DMSO and transferred to 25 mL of assay buffer
solution (50 mM Tris HCl, 5 mM MgCl2, pH 8.0). The
reaction was initiated via addition of UDP-Glc (38 mg, 62.3
μmol) and 30 mg of OleD ASP. After 3 h of gentle agitation at
rt, the reaction was frozen and lyophilized to dryness. HPLC
purification of the crude reaction gave four products:
resveratrol 4′-O-β-D-glucoside (8, 1.8 mg, 4.6 μmol, 18%),
resveratrol 3-O-β-D-glucoside (9, 2.0 mg, 5.1 μmol, 20%),
resveratrol 3,4′-di-O-β-D-glucoside (10, 2.2 mg, 4.0 μmol, 16%),
and resveratrol 3,5-di-O-β-D-glucoside (11, 1.2 mg, 2.2 μmol,
HRESIMS analysis of purified glucoside yielded an [M + H]+
ion at m/z 564.1644, confirming a monoglucoside of
flavopiridol with a formula of C27H30ClNO10. 1D and 2D
NMR data support the 3′-O-β-D-glucosidic structure presented
in Figure 4a. The key evidence for C-3′ glucosylation derives
from the HMBC correlation between the anomeric proton and
the C-3′ carbon, with the large coupling constant (8.0 Hz) of
the anomeric proton (δH 4.28, doublet) as a key signature for
the β-anomer (Figure S1). That OleD catalysis led to the
glucosylation of the C-3′ aliphatic hydroxy was surprising given
the typical bias of OleD for aromatic nucleophiles and the
previously reported accessibility of flavopiridol C-5- and C-7-
OH for glucuronidation.34
1
9%). The H and 13C NMR and HR-MS data were consistent
with previously reported data.29d
Glucosylation of Flavopiridol. Flavopiridol (also known
as Alvocidib, HMR-1275, NSC 649890) is a semisynthetic
analogue of the alkaloid rohitukine, a compound derived from
the indigenous Indian plant Dysoxylum binectarife.30 Flavopir-
idol is a cyclin-dependent kinase inhibitor that targets the
positive transcription elongation factor P-TEFb, preventing
activation of RNA polymerase II. Flavopiridol is cytotoxic to a
Glucosylation of 10-Hydroxycamptothecin. The indole
alkaloid 10-hydroxycamptothecin from the Chinese tree
Camptotheca acuminata inhibits the activity of DNA topo-
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Figure 5. (a) OleD ASP-catalyzed glucosylation of 10-hydroxycamptothecin. (b) Glucosylation of 10-hydroxycamptothecin mediated by different
OleD variants.
Figure 6. (a) OleD-catalyzed glucosylation of 2-methoxyestradiol. (b) Glucosylation of 2-methoxyestradiol catalyzed by a panel of OleD variants
(the standard deviation of 3 trials was 1.5%).
isomerase I and has a broad spectrum of anticancer activity in
vitro and in vivo.35 The unique mode of action of 10-
hydroxycamptothecin has inspired many structure activity
relationship studies, which ultimately led to the discovery of
two water-soluble drugs used for the treatment of ovarian and
lung cancer (topotecan)36 and colon cancer (irinotecan).37
Although these drugs are notably effective, their use suffers
from dose-limiting toxicities, prompting continuing efforts to
improve upon the properties of this drug class via structural
modification, including glyconjugation.38
Using both wtOleD and OleD ASP, the pilot glucosylation of
10-hydroxycamptothecin under standard conditions (2.5 mM
UDP-Glc, 1 mM aglycon) revealed the OleD ASP-catalyzed
production of a single monoglucoside product (Figure 5, 50%
conversion). In contrast, OleD ASP glucosylation of topotecan
(Figure 5a) was low (<3%, data not shown), suggesting steric
infringement imposed by substitution at C-9. Thus, OleD ASP
was selected to catalyze the preparative-scale reaction. For
subsequent product characterization, the preparative-scale
reaction was conducted with 10-hydroxycamptothecin (6 mg,
16.25 μmol), UDP-Glc (50 mg, 81.9 μmol), and OleD ASP (16
mg) in the assay buffer solution (50 mM Tris HCl, 5 mM
MgCl2, pH 8.0, 50 mL total volume). After 20 h of gentle
agitation at rt, the reaction was frozen and lyophilized to
dryness. The residue was dissolved in MeOH and subjected to
HPLC purification to give monoglucoside 15 (1.1 mg, 2.1
μmol) in 13% yield.
HRESIMS analysis of purified glucoside yielded an [M +
Na]+ ion at m/z 549.1484, confirming a monoglucoside of 10-
hydroxycamptothecin with a formula of C26H26N2O10. 1D and
2D NMR data support the 10-O-β-D-glucoside structure
presented in Figure 5a. The key evidence for C-10
glucosylation derives from the HMBC correlation between
the anomeric proton and the C-10 carbon (Figure S1). The
anomeric β-configuration is supported by the large coupling
constant of the anomeric proton (δH 5.12, d, J = 6.0 Hz)
(Figure S1). The 1H NMR spectrum was consistent with
previously reported data.38e,f
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Figure 7. Binding modes based upon molecular dynamics simulations. A common extended binding mode (a) was observed for all compounds
highlighted within this study with the exception of flavopiridol, which adapted a truncated binding mode (b).
Glucosylation of 2-Methoxyestradiol. The naturally
occurring estrogen metabolite 2-methoxyestradiol exists at
low levels in human blood serum.39 This metabolite is notable,
as it displays antiproliferative, apoptotic, and antiangiogenesis
activities mediated via pathways independent of estrogen
receptors.40 Mechanistically, 2-methoxyestradiol was found to
invoke microtubule stabilization via the colchicine binding
site.41 The in vitro GI50 of 2-methoxyestradiol against the NCI
60 cancer cell ranges from 0.08 to 5.0 μM, and this molecule
has led to promising outcomes in phase I and II clinical trials as
a new cancer chemotherapy.40a,42 In addition, 2-methoxyestra-
diol inhibits vascular smooth muscle cell growth in arteries and
induces the expression of endothelial nitric oxide synthase and
production of nitric oxide.43 However, the low aqueous
solubility (5 nM in H2O) and rapid clearance of 2-
methoxyestradiol as the C-3- or C-17-glucuronide44 has
compelled continuing efforts to develop analogues with
improved properties.41,45
Using UDP-Glc as the donor, 2-methoxyestradiol was
assessed as a substrate for a panel of OleD variants under
standard pilot conditions (0.5 mM UDP-Glc, 0.1 mM aglycon,
4 h). Four products, two monoglucosides and two diglucosides,
were observed by HPLC and LC-MS analysis (Figure 6a),
wherein product distribution was dependent on the catalyst
employed. To maximize the yield of each product for
subsequent characterization, OleD ASP was selected for the
preparative-scale reaction.
The preparative-scale reaction utilized 2-methoxyestradiol
(7.6 mg, 25.0 μmol) and UDP-Glc (76 mg, 0.125 mmol) in 50
mM Tris HCl and 5 mM MgCl2, pH 8.0 (50 mL of total
volume), and was initiated by the addition of 20 mg of OleD
ASP. After gentle agitation at rt for 16 h, the reaction was
frozen and lyophilized to dryness. The residue was dissolved in
MeOH and subjected to HPLC purification to give 17 (4 mg,
8.6 μmol, 34.4%), 18 (0.1 mg, 0.2 μmol, 0.8%), 19 (1 mg, 1.6
μmol, 6.4%), and 20 (1 mg, 1.6 μmol, 6.4%). The identification
of compounds 17−20 was confirmed by 1D and 2D NMR and
HRESIMS analysis. The key evidence for C-3, C-17, or C-2′
glucosylation derives from the HMBC correlation between the
anomeric proton and the carbon that was glycosylated (Figure
S1). The anomeric β-configuration is supported by the large
coupling constant (7.0−8.0 Hz) of the anomeric proton
(Figure S1). Importantly, this study highlights the first synthesis
of 2-methoxyestradiol glucosides. Also particularly intriguing
from this study is the apparent C-3 glucosylation (17) en route
to disaccharide 19 versus a switch to C-17 glucosylation (18)
en route to the same product with the AIP variant.
Molecular Dynamics Simulations. The binding modes of
all 18 structures by OleD were studied by molecular docking
and included all five native aglycons (3, 7, 12, 14, and 16), all
monoglucoside products/intermediates (4, 5, 8, 9, 13, 15, 17,
and 18), and all diglucosides (6, 10, 11, 19, and 20) (Figure 7).
This cumulative analysis revealed all compounds studied, with
the exception of flavopiridol (12), to bind in a manner recently
described for steroidal glycosides.4b Specifically, ligand binding
within this model is mediated via distal hydrogen-bonding
interactions with key residues (Y114, Y162, and D179) deep
within a polar pocket located ∼16 Å from the proximal catalytic
H19. This analysis indicates an average length of substrates
examined to be ∼12.5 Å, and in cases where diglucosides were
formed, modeling revealed the distal pocket to readily
accommodate the larger glucosides with only minor shifts in
the H19-aglycon nucleophile distance/alignment. Also con-
sistent with the product distribution observed, docking
simulations with 3, 7, and 16 each revealed two modes of
binding (head first versus tail first) with similar binding affinity.
While flavopiridol was found to occupy the same substrate
cavity, the model revealed unique proximal interactions,
specifically, side chain hydrogen bonding with Y116 and van
der Waals contacts with V81 and F84, to accommodate the
substrate’s smaller size. In addition, this docking model revealed
the C-3′ aliphatic hydroxy of flavopiridol as the only aglycon
nucleophile in proximity to the critical H19. Importantly, this
collective analysis highlights common binding constraints of a
range of non-native substrates for this unique glycosyltransfer-
ase and, as such, serves as a basis for the application of virtual
screening in an attempt to rapidly identify new potential
substrates for enzyme-catalyzed glycoconjugation.46
In summary, this work is an important key follow-up to
studies that revealed the uniquely permissive nature of a range
of engineered/evolved OleD variants. Specifically, the full
characterization of product distributions deriving from OleD-
catalyzed reactions with the diverse set of non-native substrates
highlighted within this study provides important insights into
the regioselectivity of each target reaction and a critical basis for
the development of future predictive models for the substrate
specificity scope of this fascinating catalyst.
EXPERIMENTAL SECTION
General Experimental Procedures. All chemicals and reagents
were purchased from Sigma-Aldrich, unless otherwise stated. NMR
■
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1
spectra, including H, 13C, gCOSY, TOCSY, gHSQC, and gHMBC,
1H), 4.28 (d, J = 8.0 Hz, 1H), 3.71 (m, 1H), 3.67−3.57 (m, 3H), 3.54
(dd, J = 11.0, 3.0 Hz, 1H), 3.30−3.24 (m, 2H), 3.19 (dd, J = 11.0, 6.0
Hz, 1H), 3.06 (ddd, J = 9.5, 6.0, 3.0 Hz, 1H), 3.00 (dd, J = 9.5, 8.5 Hz,
1H), 2.99 (dd, J = 9.0, 8.0 Hz, 1H), 2.89 (s, 3H), 2.06 (m, 2H); 13C
NMR (CD3OD, 125 MHz) 184.0, 164.5, 163.0, 162.7, 162.2, 133.6,
133.4, 133.1, 132.4, 131.8, 129.0, 111.8, 106.8, 105.9, 101.7, 100.0,
77.9, 77.6, 75.3, 74.2, 72.3, 63.5, 58.8, 56.7, 44.2, 37.5, 24.5; HRESIMS
m/z 564.16438 [M + H]+ (calcd forC27H31ClNO10, 564.1631).
were recorded on a Varian UNITY INOVA 400 or 500 MHz
spectrometer. Mass spectra were acquired on a Bruker MaXis high-
resolution quadrupole time-of-flight mass spectrometer.
Protein Expression and Purification. A single colony of E. coli
BL21(DE3)pLysS (Stratagene) transformed with the pET28a-based
OleD expression vector (wt, ASP, AIP, TDP16, 3-1-H12)3 was used to
inoculate 3 mL of LB medium supplemented with 50 μg/mL
kanamycin and cultured overnight at 37 °C with shaking (250 rpm).
The starter culture was then transferred to 1 L of LB medium
supplemented with 50 μg/mL kanamycin and grown at 37 °C with
shaking (250 rpm) until the OD600 reached ∼0.7. Isopropyl β-D-
thiogalactoside was subsequently added to a final concentration of 0.4
mM, and the culture was incubated at 28 °C for approximately 18 h
with shaking at 250 rpm. Cell pellets were collected by centrifugation
at 10000g and 4 °C for 20 min, and the supernatant was discarded.
Cell pellets were resuspended in 10 mL of chilled lysis buffer (20 mM
phosphate buffer, 0.5 M NaCl, 10 mM imidazole, pH 7.4) and were
lysed by sonication (8 pulses of 40 s each) in an ice bath. The cell
debris was removed by centrifugation at 10000g and 4 °C for 20 min.
The cleared supernatant was immediately applied to 3 mL of nickel
nitrilotriacetic acid resin (QIAgen) pre-equilibrated with wash buffer
(20 mM phosphate buffer, pH 7.4, 0.3 M NaCl, 10 mM imidazole).
Protein was allowed to bind for 30 min at 4 °C with gentle agitation,
and the resin was subsequently washed with 4 × 50 mL wash buffer.
Finally, the enzyme was eluted from the resin via incubation with 2 mL
of wash buffer containing 250 mM imidazole for 15 min at 4 °C with
gentle agitation. The purified protein was applied to a PD-10 desalting
column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl
(pH 8.0) and eluted as described by the manufacturer. Protein aliquots
were immediately flash frozen in liquid nitrogen and stored at −80 °C.
Protein purity was confirmed by SDS-PAGE to be >95%, and protein
concentration for all studies was determined using the Bradford
protein assay kit from Bio-Rad.
General Pilot-Scale Reaction. Reactions were conducted in a
final volume of 100 μL under standard conditions (50 mM Tris HCl, 5
mM MgCl2, pH 8.0, 0.5 μg/μL purified enzyme, 2.5−5 equiv of UDP-
Glc, 1 equiv of aglycon, 25 °C). Two separate control reactions that
withheld either enzyme or UDP-Glc were performed in parallel.
Reactions were allowed to proceed at 25 °C for a specific time period,
quenched with 100 μL of cold MeOH, and centrifuged at 10000g for
10 min, and the supernatant was removed for analysis. The clarified
reaction mixtures were analyzed by analytical reverse-phase HPLC
[Phenomenex 250 mm × 4.6 mm Gemini 5 μ C18 column; 1 mL/min;
gradient of solvents A (0.1% TFA) and B (100% CH3CN): (a) 0−20
min, 10−75% B; (b) 20−21 min, 75−100% B; (c) 21−26 min, 100%
B; (d) 26−29 min, 100−10% B; and (e) 29−35 min, 10% B; A254
detection]. HPLC peak areas were integrated using the Star
Chromatography Workstation software (Varian), and the total percent
conversion was calculated as a percent of the total peak area of
substrate and products.
1
2-Methoxyestradiol 3-O-β-D-glucoside (17): white powder; H
NMR (CD3OD, 500 MHz) 6.91 (s, 1H), 6.88 (s, 1H), 4.84 (d, J = 7.5
Hz, 1H), 3.89 (m, 1H), 3.84 (s, 3H), 3.71 (m, 1H), 3.68 (m, 1H), 3.48
(m, 2H), 3.40 (m, 2H), 2.78 (m, 2H), 2.35 (dd, J = 14.0, 3.0 Hz, 1H),
2.20 (m, 1H), 2.06 (m, 1H), 2.00 (m, 1H), 1.90 (dd, J = 12.0, 2.5 Hz,
1H), 1.72 (m, 1H), 1.60−1.17 (m, 7H), 0.80 (s, 3H); 13C NMR
(CD3OD, 100 MHz) 148.8, 146.1, 136.2, 130.9, 118.8, 111.6, 103.3,
82.6, 78.4, 78.0, 75.1, 71.6, 62.7, 57.2, 51.4, 45.9, 44.5, 40.5, 38.2, 30.9,
30.3, 28.7, 27.8, 24.2, 11.8; HRESIMS m/z 487.2304 [M + Na]+ (calcd
for C25H36O8Na, 487.2302).
2-Methoxyestradiol 17-O-β-D-glucoside (18): white powder;
1H NMR (CD3OD, 500 MHz) 8.51 (s, 1H), 6.77 (s, 1H), 6.45 (s,
1H), 4.34 (d, J = 8.0 Hz, 1H), 3.83 (dd, J = 12.0, 2.0 Hz, 1H), 3.80 (d,
J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.65 (dd, J = 12.0, 6.0 Hz, 1H), 3.40−
3.15 (m, 4H), 2.68 (m, 2H), 2.63 (s, 1H), 2.17−2.10 (m, 3H), 1.66
(m, 2H), 1.54−1.15 (m, 7H), 0.86 (s, 3H); 13C NMR (CD3OD, 125
MHz) 147.1, 145.4, 132.8, 130.4, 116.6, 110.5, 104.9, 90.0, 78.3, 78.0,
75.6, 71.9, 63.0, 56.7, 51.3, 45.4, 44.4, 40.4, 39.0, 30.2, 30.1, 28.7, 28.0,
24.2, 12.3; HRESIMS m/z 487.2304 [M + Na]+ (calcd for
C25H36O8Na, 487.2302).
2-Methoxyestradiol 3,17-di-O-β-D-glucoside (19): white pow-
der; 1H NMR (CD3OD, 500 MHz) 6.86 (s, 1H), 6.83 (s, 1H), 4.80 (d,
J = 7.0 Hz, 1H), 4.34 (d, J = 7.5 Hz, 1H), 3.83 (m, 1H), 3.79 (s, 3H),
3.66 (d, J = 5.0 Hz, 1H), 3.64 (d, J = 5.0 Hz, 1H), 3.73−3.63 (m, 3H),
3.54 (dd, J = 12.0, 4.0 Hz, 1H), 3.45 (m, 2H), 3.38 (m, 2H), 3.30 (m,
1H), 3.26 (m, 1H), 3.19 (m, 1H), 2.79 (m, 2H), 2.35 (dd, J = 14.0, 3.0
Hz, 1H), 2.21 (m, 1H), 2.06 (m, 1H), 2.00 (m, 1H), 1.90 (dd, J =
12.0, 2.0 Hz, 1H), 1.72 (m, 1H), 1.60−1.17 (m, 7H), 0.81 (s, 3H); 13C
NMR (CD3OD, 125 MHz) 148.7, 145.3, 136.1, 130.8, 118.7, 111.5,
104.9, 103.2, 89.9, 78.36, 78.32, 78.04, 78.00, 75.6, 75.1, 71.8, 71.6,
62.9, 62.7, 57.1, 51.3, 45.9, 44.7, 40.5, 40.2, 30.3, 30.1, 28.6, 27.9, 24.1,
12.3; HRESIMS m/z 649.2838 [M + Na]+ (calcd for C31H46O13Na,
649.2831).
2-Methoxyestradiol 3-(O-β-D-glucopyranosyl)-(1″→ 2′)-(O-β-
1
D-glucoside) (20): white powder; H NMR (CD3OD, 500 MHz)
6.91 (s, 1H), 6.85 (s, 1H), 5.13 (d, J = 7.5 Hz, 1H), 4.78 (d, J = 8.0
Hz, 1H), 3.90 (m, 1H), 3.82 (s, 3H), 3.78 (dd, J = 9.0, 8.0 Hz, 1H),
3.73−3.63 (m, 3H), 3.54 (dd, J = 12.0, 4.0 Hz, 1H), 3.45 (m, 2H),
3.38 (m, 2H), 3.30 (m, 1H), 3.26 (m, 1H), 3.19 (m, 1H), 2.79 (m,
2H), 2.35 (dd, J = 14.0, 3.0 Hz, 1H), 2.21 (m, 1H), 2.06 (m, 1H), 2.00
(m, 1H), 1.90 (dd, J = 12.0, 2.0 Hz, 1H), 1.72 (m, 1H), 1.60−1.17 (m,
7H), 0.81 (s, 3H); 13C NMR (CD3OD, 125 MHz) 148.0, 145.8, 135.6,
130.7, 117.0, 111.4, 104.2, 100.9, 82.6, 82.5, 78.0, 77.93, 77.88, 77.78,
75.7, 71.2, 71.0, 62.7, 62.0, 57.1, 51.4, 45.9, 44.5, 40.5, 38.3, 30.9, 30.4,
28.7, 28.0, 24.2, 11.9; HRESIMS m/z 649.2835 [M + Na]+ (cacld for
C31H46O13Na, 649.2831).
Molecular Dynamics Simulations. Multiple conformations of
each ligand were generated using OMEGA (Open Eye Scientific
Software). The conformations of each ligand were fitted in the binding
cavity of OleD (PDBID 2IYF)1f using Sabre and Fred software
packages.47 The docking strategy exhaustively explored all possible
positions of each ligand in the binding site and generally focused upon
two parameters: shape and optimization. During the shape fitting, the
ligand was placed into a grid box encompassing all active-site atoms
(including hydrogen atoms) using smooth Gaussian potential.48 Two
optimization filters were subsequently processed: rigid-body opti-
mization and optimization of the ligand pose in the dihedral angle
space. The pose ensemble was then filtered to first reject poses that did
not have sufficient shape complementarity with the active site of the
protein followed by rejection of those lacking at least one heavy atom
hydrogen bond with the His19 imidazole.
General Procedure for Preparative-Scale OleD-Catalyzed
Glycosylation Reaction. Aglycon was dissolved in 5% DMSO and
transferred to pH 8 buffer solution (50 mM Tris HCl, 5 mM MgCl2).
UDP-Glc was added followed by OleD catalyst. After the designated
time of agitation at rt, the reaction was subsequently frozen and
lyophilized, and the debris was resuspended in 2 mL of ice-cold
MeOH and filtered. Product O-glucosides were isolated by collecting
fractions from semipreparative reverse-phase HPLC [Phenomenex 250
mm × 10 mm Gemini 5 μ C18 column; 4.5 mL/min; solvent A (0.1%
trifluoroacetic acid) and B (100% CH3CN) using the gradient 0−20
min, 10−75% B; 20−21 min, 75−100% B; 21−26 min, 100% B; 26−
29 min, 100−10% B; and 29−35 min, 10% B; A254 detection]. The
product-containing fractions were subsequently collected and
lyophilized to provide the corresponding O-glucosides. The compound
was then characterized using 1D and 2D NMR, including H, 13C,
gCOSY, TOCSY, gHSQC, and gHMBC.
1
1
Flavopiridol 3′-O-β-D-glucoside (13): white powder; H NMR
(CD3OD, 500 MHz) 7.81 (dd, J = 7.5, 2.0 Hz, 1H), 7.63 (dd, J = 7.0,
2.0 Hz, 1H), 7.60−7.50 (m, 2H), 6.48 (s, 1H), 6.37 (s, 1H), 4.33 (m,
284
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Journal of Natural Products
Article
In separate docking runs, the binding poses of the ligand structure
were refined by MD simulations followed by MM-GBSA calculations
using the Sander module from the Amber11 package49 as previously
described.46 Briefly, the OleD−ligand binding complex was neutralized
by adding appropriate counterions and was solvated in a rectangular
box of TIP3P H2O molecules with a minimum solute−wall distance of
10 Å.50 The partial atomic charges used for the ligand were the
electrostatic potential-fitted atomic charges, calculated at the ab initio
HF/6-31G* level using the Gaussian03 program.51 The solvated
systems were energy-minimized and carefully equilibrated. These
systems were gradually heated from T = 10 K to T = 298.15 K in 100
ps before running the MD simulation. The MD simulations were
performed with a periodic boundary condition in the NPT ensemble at
T = 298.15 K with Berendsen temperature coupling and constant
pressure (P = 1 atm) with isotropic molecule-based scaling.52 A time
step of 2.0 fs was used, with a cutoff of 12 Å for the nonbonded
interactions, and the SHAKE algorithm was employed to keep all
bonds involving hydrogen atoms rigid.53 Long-range interactions were
handled using the particle mesh Ewald algorithm.54 As we did in our
previous work,4b only the ligand and residue side chains in the binding
pocket were permitted to move during the energy minimization and
MD simulations. We used the constraint to prevent any changes in the
OleD structure due to the presence of residues in the loops on the top
of the protein active site. MD simulations were then carried out for 4.0
ns. During the simulations, the coordinates of the system were
collected every 10 ps.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C spectroscopic data, molecular docking data, and
individual docking illustrations. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(16) (a) Kopp, P. Eur. J. Endocrinol. 1998, 138, 619−620.
Corresponding Author
*E-mail: jsthorson@uky.edu.
Notes
The authors declare the following competing financial interest:
J.S.T. is a co-founder of Centrose (Madison, WI, USA).
̀
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ACKNOWLEDGMENTS
■
This work was supported by a NIH MERIT Award R37
AI52218 (J.S.T.) and the National Center for Advancing
Translational Sciences (UL1TR000117). We thank the
University of Wisconsin College of Pharmacy Analytical
Instrumentation Center for analytical support and Prof. G.
Williams (North Carolina State University), Dr. S. Singh
(University of Kentucky) and Dr. R. W. Gantt (Dupont) for
materials and helpful discussions.
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