An efficient, scalable approach to hydrolyze flavonoid glucuronides via activation of glycoside bond

Article abstract of DOI:10.1016/j.tet.2017.02.039

Hydrolyzing flavonoid glucuronides into corresponding aglycones posed some significant challenges. To improve acid-catalyzed hydrolysis process of flavonoid glucuronide, structures of glucuronide, hydrolysis parameters and post-processing were optimized. The optimized condition was performed by hydrolysis flavonoid glycoside methyl ester in a mixed solvent consisting of 2?mol/L H₂SO₄/EtOH/H₂O (1/8/1, v/v/v) at 95?°C for 7?h and resulted in up to 90% aglycone yields, minimal byproduct formations and milder hydrolysis conditions. Furthermore, the optimized method avoids tedious purification steps and is easily conducted on a relatively large-scale using economical and commercially available reagents.

Full text of DOI:10.1016/j.tet.2017.02.039

Tetrahedron xxx (2017) 1e9
Contents lists available at ScienceDirect
Tetrahedron
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An efficient, scalable approach to hydrolyze flavonoid glucuronides via
activation of glycoside bond
,
, c, **
Xue-Yang Jiang a, Xin-Chen Li d, Wen-Yuan Liu b, Yun-Hui Xu a e, Feng Feng a
,
, c, *
Wei Qu a
a Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, 210009, China
b Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China
c Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing, 211198, China
d Anhui Normal University, Department of College of Life Sciences, Wuhu, 241000, China
e Marshall Institute for Interdisciplinary Research, Marshall University, Huntington, WV 25755, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrolyzing flavonoid glucuronides into corresponding aglycones posed some significant challenges. To
improve acid-catalyzed hydrolysis process of flavonoid glucuronide, structures of glucuronide, hydrolysis
parameters and post-processing were optimized. The optimized condition was performed by hydrolysis
flavonoid glycoside methyl ester in a mixed solvent consisting of 2 mol/L H2SO4/EtOH/H2O (1/8/1, v/v/v)
at 95 ꢀC for 7 h and resulted in up to 90% aglycone yields, minimal byproduct formations and milder
hydrolysis conditions. Furthermore, the optimized method avoids tedious purification steps and is easily
conducted on a relatively large-scale using economical and commercially available reagents.
© 2017 Elsevier Ltd. All rights reserved.
Received 27 December 2016
Received in revised form
15 February 2017
Accepted 20 February 2017
Available online xxx
Keywords:
Flavonoid glucuronide
Acid-catalyzed hydrolysis
Optimizations
Large-scale
1. Introduction
Except the differences on the structures of flavonoid aglycones
and their glycosides, the great bioactive differences are also avail-
Flavonoids, a broad class of polyphenolic secondary metabolites
from natural source, consist of flavonoid aglycones (FA) and flavo-
noid glycosides (FG). Variation in the molecular structures of FG
originates from the different type of aglycone, sugar and the
number of sugar groups, among which the fraction of glucuronide
is quite common.1 Flavonoids have gained much attention from
researchers and the food industry due to the fact that they are
natural antioxidants as an alternative to synthetic products for the
usage in food and cosmetics,2 as well as their important biological
roles in nitrogen fixation and chemical defense.3 Recent studies
also proved that flavonoids possess a broad range of pharmaco-
logical properties, including antioxidant, anticancer, and anti-
inflammatory activities4,5; hence received considerable therapeu-
tic importance.
able. FAs are superior to their corresponding glycosides in various
activities, due to the better solubility and oral bioavailability.6,7 In
addition, recent reports indicated that most FGs were hydrolyzed
into their corresponding aglycones by human intestinal microflora
after oral administration.8,9 Teruaki et al.10 found that baicalin could
be absorbed as its hydrolyzed products - baicalein but not baicalin
itself in the rat gastrointestinal tract. These previous studies show
that baicalein possesses more distinctive pharmacological effects
and metabolites safety than baicalin, so it is interesting to obtain FA
for further chemical modification and preclinical studies. Unfortu-
nately, natural FA is little in contrast to their respective glycosides.
Total syntheses of FAs, such as baicalein and scutellarein, have been
reported by Silvestri11 and Chen12 respectively, but they are un-
suitable for industrial production due to their lengthy processes
and low yields.
In the light of the natural abundance of FGs,13 the conversion
from FGs to their corresponding aglycones by bio- and chemical-
transformations is required urgently. Although bioconversion
seems more promising, their application has faced some diffi-
culties, including substrate specificity, enzyme purification,
* Corresponding author. Department of Natural Medicinal Chemistry, China
Pharmaceutical University, Nanjing, 210009, China.
** Corresponding author. Department of Natural Medicinal Chemistry, China
Pharmaceutical University, Nanjing, 210009, China.
E-mail addresses: fengfeng@cpu.edu.cn (F. Feng), popoqzh@126.com (W. Qu).
http://dx.doi.org/10.1016/j.tet.2017.02.039
0040-4020/© 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Jiang X-Y, et al., An efficient, scalable approach to hydrolyze flavonoid glucuronides via activation of glycoside
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
maintenance of enzyme activity and high cost.14e17 In comparison,
the acid catalyzed cleavage of the glycosidic bond has been
extensively used for glucosides hydrolysis in view of its universal
versatility and maneuverability.18 Different from glucosides, how-
ever, most studies have focused on the acid-catalyzed hydrolyses of
glucuronides, because the special structure of glucuronide moiety
is so stubborn that glucuronides are hard to be hydrolyzed under
mild acid hydrolysis conditions. Much effort has been devoted to
develop novel hydrolysis methods of glucuronide, but numerous
glucuronides, such as scutellarin and baicalin are still hardly hy-
drolyzed unless under vigorous acid hydrolysis conditions.
Although Li19 and Tang20 have independently examined the hy-
drolysis of scutellarin in aqueous mineral acid recently, both of their
methods needed high temperature, long reaction time and without
satisfied yields (mostly below 20%). In addition, the formation of
byproducts associated with this protocol might occur, which in-
volves an elaborate and tedious procedure; for example, the
isomerization of flavonoids is formed due to a reversible Wesse-
lyeMoser rearrangement reaction (Fig. 1).21 Consequently, alter-
native methods to hydrolyze FGs would be essential for the
industrial preparation of FAs, including important chemical con-
stituents in the pharmaceutical, cosmetic, and food industries. Until
now, to the best of our knowledge, no appropriate approach on
hydrolyzing flavonoid glucuronide into corresponding aglycone
with simple purification and high yield processing has been
reported.
glucuronide might obstruct the first two steps of acid hydrolysis of
glycoside based on theoretical calculations. Therefore, the protec-
tion for the carboxyl function in the chemical conversion of FGs to
FAs was required. The present study aims to improve dilute-acid
hydrolysis of the most FGs by protecting the carboxyl function
and to develop a new universally sustainable method to ensure a
convenient large-scale production of aglycones. The optimal C5-
substituent groups of glucuronides moiety (hereinafter denoted as
FGM) were first chosen based on both the physical properties of
glucuronides and the industrial feasibility by theoretical calculation
using Gaussian program. The synthetic FGM was then subjected to
dilute-acid hydrolysis for FA production. Additionally, the products
of the hydrolysis process of FGM were examined and compared
with that of glucuronide by the HPLC. The proportional amplifica-
tion and substrate applicability of this method were also evaluated
for the acid hydrolysis of scutellarin and other flavonoid
glucuronides.
2. Results and discussion
2.1. Cause of glucuronide hydrolysis reaction problems and their
solutions
2.1.1. The carboxyl of glucuronide is the “culprit”!
We chose scutellarin as the substrate and attempted to optimize
the hydrolysis process in order to improve the demonstrated pro-
ductivity of scutellarein. It should be noted that 90% EtOH was
chosen for economical reason, and H2SO4 was used as the acid
throughout our study because sulfuric acid can provide strong
acidity and stable proton concentration in the acid hydrolysis re-
action. Both of concentrations of sulfuric acid and reaction tem-
peratures were evaluated. Indeed, only a 15% conversion was
achieved using 0.5 mol/L H2SO4 at 120 ꢀC for 48 h (Table 1, entry 5),
which was consistent with the previous hydrolyses reaction per-
formed under similar conditions.20 Using a stronger acidic reaction
media could not increase the conversion ratio of scutellarin, while
scutellarein isomers might generate due to a reversible Wessely-
Moser rearrangement reaction under such hard condition.24 The
rearrangement reaction could happen following several steps: (A)
ring opening to the diketone, (B) bond rotation with the formation
of a favorable acetylacetone-like phenyl-ketone interaction and (C)
ring closure (Fig. 1).
We hypothesize that the proton of 5-carboxylic acid of flavonoid
Fig. 1. Mechanism of Wessley-Mosher rearrangement reaction for scutellarein.
Table 1
Optimization of hydrolysis conditions of scutellarin with sulfuric acid.a
Entry
H2SO4 (mol/L)
Solventb
T (ꢀC)
Time (h)
Conv. (%)c
1
2
3
4
5
6
1
2
3
3
3
4
90% Ethanol
90% Ethanol
90% Ethanol
90% Ethanol
90% Ethanol
90% Ethanol
90
90
90
100
120
120
24
24
24
48
48
48
4
5
8
10
15
16
a
Unless otherwise stated, all reactions were carried out with scutellarin (0.5 mmol) and H2SO4 (15 mmol, 30 equiv) under N2 atmosphere.
90% Ethanol represent EtOH/H2O ¼ 90/10 (v/v).
b
c
Conversion determined by HPLC analysis.
Please cite this article in press as: Jiang X-Y, et al., An efficient, scalable approach to hydrolyze flavonoid glucuronides via activation of glycoside
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
3
Fig. 2. The universally acknowledged hydrolysis mechanism of glycosides with acid catalyzed, in which the protonation of glycosidic oxygen atom (step 1) and cleavage of glycosidic
bond (step 2) are the two rate-determining steps in the acid hydrolysis of glucuronide.
In order to find the reason, firstly we have made a detailed
analysis of the acid hydrolysis mechanism of glycoside. As the
mechanism of acid hydrolysis reaction of glycoside showed in Fig. 2,
the first step is protonation at the exocyclic oxygen of the glycosidic
bond affording the conjugate acid and attaining equilibrium
rapidly, followed by loss of the aglycon with the formation of a
cyclic carbonium.22 The second step is rate-determining and shows
characteristics of an SN1-type substitution reaction which depends
on the stability of the oxo-carbenium ion formed upon the depar-
ture of the protonate leaving group as an alcohol.23 The conjugate
acid can advance to this rate-determining step. On the basis of
these characteristics, protonation of the glycosidic oxygen atom
(hereinafter denoted as Oe), viz. concentration of the conjugate acid,
and rate-limiting cleavage of the protonated glycosidic bond are the
practical rate-determining parameters during the acid hydrolysis
process.18 It has been accepted that this accessibility of glycosidic
bonds is hindered by the physical structure of glucuronide because
of a free carboxyl function in their glucuronide moiety.22 As a result,
protonation of the glycosidic oxygen atom and cleavage of the
glycosidic bond are the rate-determining steps in the acid hydro-
lysis of glycoside. The effect of carboxyl group on the key steps for
acid hydrolysis of glucuronide will be figured out.
To understand the process of acid hydrolysis of glycosides, we
studied the protonation abilities of various types of sugar ligands.
As shown in Fig. 3AeC, 2-amino glucoside (A) have been reported
to be acid-hydrolyzed more rapidly than 2-deoxy-glucoside (B)
under the same reaction conditions.22 A possible explanation for
the slower hydrolysis rate of 2-amino glucoside (A) is that the
amino of sugar unit can not only compete with the exocyclic oxygen
of the glycosidic bond when it combined with a proton but also be
protonated to generate electron-withdrawing inductive effect,
which leads to a reduction of the electron cloud density at the
terminal carbon atom of sugar unit (hereinafter be denoted Ct) as
well as weakens the protonation ability of the Oe at the glycosidic
bond. In contrast, for 2-deoxy-glucoside (B), without the effect of
the amino at position 2 of glucoside, the electron cloud density of
the Ct became even higher, which could also explain that 2, 6-
dideoxy glucoside could be hydrolyzed just using 0.02e0.05 mol/
L HCl in EtOH-H2O system.25 Certainly, the aromatic aglycone has
such as daidzin, can be hydrolyzed to aglycone daidzein under
conventional acid hydrolysis conditions.26 Above all, we believe
that the difficulty of hydrolyzing the aromatic-aglycone-containing
glucuronide (C) might be attributed mainly to its 5’-carboxy group
for its steric hindrance and H-bonded to glycosidic oxygen,
impeded the proton attack of the Oe. Besides, 5’-carboxy group's
strong electron-withdrawing inductive effect could reduce the
stability of the oxo-carbenium ion formed upon the glycosidic bond
cleavage since protonation will facilitate the electron flow as
symbolized by the arrows in these formulas, which makes
aromatic-aglycone-containing glucuronides difficult to be hydro-
lyzed by dilute acid. Therefore, our study suggests that the proton of
5’-carboxylic acid might be the “culprit”!
2.1.2. Optimization of C5-substituent groups of aromatic-aglycone-
containing glucuronide
Next, we need to seek a solution in order to reduce the adverse
effects for the glucuronide hydrolysis caused by the carboxyl group.
Firstly, we tried to hydrogenate the 5-carboxyl of scutellarin into a
hydroxyl in order to eliminate the effect of the carboxyl proton. But,
this method was finally abandoned, considering the strict reaction
conditions required and high cost of some reducers, such as lithium
aluminum hydride or borane. Inspired by the application of pro-
tective groups in organic chemistry,27 we planned to accelerate the
hydrolysis reaction by protecting the 5-carboxyl with electron-
donating groups, which could inhibit the protonation of 5-
carboxyl and thereby might reduce its electron-withdrawing ef-
fect on the Ct (Fig. 3D).
The choice of the protecting groups mainly depended on their
molecular size, electronic effects and operability.28 On the base of
the advantage of alkanes on chemical stability, electron-donating
ability and modified difficulty, finally, ethyl or methyl was
selected as the protective group. The aim of chemical modification
with alkanes, which has methyl donor groups, is to weaken the
adverse effects from the 5-carboxyl and to obtain excellent yield of
aglycones with a mild acid hydrolysis conditions. We found that
methoxycarbonyl, an electron-donating group, is inductively less
electronegative than carboxy group and we proposed that the
introduction of the methoxycarbonyl group onto the 5-carboxyl,
through accumulating electron density at the terminal carbon, can
enhance both protonation of pre-equilibrium and rate-limiting
formation of oxo-carbenium ion intermediate. Therefore, we
p-electron conjugation system, which could also reduce the elec-
tron density of Oe. But, the effect of the conjugation on the Ct is
relatively weak. Most of aromatic-aglycone-containing glucosides,
Fig. 3. The protonated capability of different glycosides bond oxygen atoms of aromatic-aglycone-containing glucuronides.
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
hypothesize that the dual effects effectively accelerate the hydro-
lysis rate of glucuronide.
medical herb Erigeron breviscapus (vant.) Hand.-Mazz, and its
modified scutellarin: scutellarin methyl ester (SME) were repre-
sentatively selected for the subsequent experiments. The optimized
structures of scutellarin and SME were displayed in Fig. S3 (see ESI
for details).
To prove this hypothesis, the theoretical calculation studies
were used for the acid hydrolysis pathway and mechanism of
scutellarin, because it can provide valuable information to figure
out the key factors which affect the hydrolysis rate of scutellarin.
Density functional theory (DFT) has nowadays become general tool
to the understanding and predicting a broad range of chemical,
physical, and biological phenomena featuring the realm of coordi-
nation chemistry. It was noted that functional Becke's three-
parameter hybrid exchange functional and the LeeeYangeParr
non-local correlation functional (B3LYP) is the most widely used
and has been proven to yield satisfactory results on energy and
geometry, but with a low computational cost.29 More recent studies
on mechanism show that DFT method employing B3LYP success-
fully explains mechanistic features of the organic system as well.30
Therefore, to confirm this hypothesis and further understand the
acid hydrolysis reaction, Gaussian09 program with B3LYP and the
6-31 þ G* basis set was employed for the computational studies on
the acid hydrolysis mechanism of glucuronide.31 Scutellarin, a
glucuronide with the highest level from the traditional Chinese
2.1.2.1. The influence of methyl group on the protonation at Oe of
aromatic-aglycone-containing glucuronides. Atomic charges were
obtained by natural population analysis with Gaussian09.32 Table 2
presented the average atomic charge distribution of SME and less
positive charges of Ct in SME (0.13757 jej) than that of scutellarin
(0.35635jej), while in the 13C NMR spectrum (Table 2), the Ct signal
resonances of SME (dC 92.53) is in higher field region than that of
scutellarin (dC 93.55), which preliminarily suggested the higher
electron density of the Ct of SME than that of scutellarin, which
indicated that the glycosidic oxygen atom of SME protonated with
dilute acids more easily. Furthermore, mentioned the hydrolysis of
scutellarin, the calculation result revealed that the hydrogen-
bonding arrangement between the oxygen of glycoside bond and
the carboxyl hydrogen of glucuronide moiety at a distance of 2.05 Å,
which hampered the interaction between proton and Oe (Fig. 4,
INT-1). In contrast to scutellarin, the methoxycarbonyl group of
SME was too far away from the glycoside bond to interfere with the
protonation of Oe during the hydrolysis process (Fig. 4, INT-2).
Therefore, the increase of electron density on the Ct and easier
protonation of Oe, caused by the methyl esterification, accelerated
the first step of hydrolysis, which partially insinuated that SME was
much easier hydrolyzed than scutellarin under dilute acid
conditions.
Table 2
The average atomic charges of scutellarin and the modified SME at the B3LYP/6-
31 þ G(d) Level and their 13C NMR Data (75 MHz in DMSO-d6).
2.1.2.2. Effects of methyl group on the cleavage of the glycoside bond
of aromatic-aglycone-containing glucuronides. Analysis of the hy-
drolysis paths given in Fig. 2 indicates that this reaction takes place
through a stepwise mechanism. To shed further light on the acid
hydrolysis mechanism, the transition states (TSs) with reasonable
energy were identified. Thus, we attempted to optimize the
conformation of compounds and compare the activation energy
Atoms
Scutellarin
Charges
SME
barrier
DE from reactants (INT-1 and INT-2) with their corre-
dC
Charges
dC
sponding transition states (TS-1 and TS-2) to determine whether
the methyl group can also accelerate the second step of acid hy-
drolysis reaction. DFT calculations indicate that the TS-1 has an
activation energy barrier of 206.2 kcal/mol relative to the starting
material in ethanol, and the TS-2 has the lowest energy barrier of
only 128.6 kcal/mol, which is 77.6 kcal/mol lower in energy than
C1 (Ct)
O2
C3
C4
O5
0.35635
ꢁ0.41228
0.02401
0.69501
ꢁ0.326363
0.36953
93.55
e
0.13757
92.53
e
ꢁ0.31482
ꢁ0.13204
0.59146
75.45
170.04
e
73.19
169.70
e
ꢁ0.15769
C6
161.13
0.30161
151.39
Fig. 4. Energy profile for a simplified model of acid hydrolysis for scutellarin (red) and SME (blue) using B3LYP/6-31 þ G* methods.
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
5
that of TS-1 (Fig. 4). On the other hand, the activation enthalpy
associated with cleavage of the glycosidic bond of conjugate acid
INT-1 to form the corresponding cyclic cation intermediate PC-1 is
203.7 kcal/mol. The more stable the oxo-carbenium ion formed is,
the more easily the corresponding glycoside bond can be broken.33
Consistented with the theoretical prediction from the chemical
point of view, the conversion of INT-2 to PC-2 has an enthalpic
barrier height of only 76.4 kcal/mol (Table S1 in the ESI), which
suggested that the oxacarbenium-ion intermediate PC-2 is more
stable than PC-1, as a consequence of the electron-releasing char-
acter of the methyl group present in the glucuronide moiety of
intermediate INT-2.
Therefore, the designed alkyl ester groups might accelerate the
second step of acid hydrolysis reaction by reducing the activation
energy barrier DE and enthalpic barrier height DH in the hydrolysis
reaction of SME, which further indicated that the acid hydrolysis of
SME was faster than that of scutellarin and also verified the
designed rationality of SME. Finally, ethyl or methyl was selected as
the protective group.
Ethyl and methyl groups were connected to 5-carboxyl via ester
bond. The scutellarin ethyl (SE) was prepared by reacting scu-
tellarein with NaHCO3 to generate scutellarin sodium salt, followed
by adding stoichiometric amount of bromoethane in the presence
of potassium iodide in N, N-dimethylformamide (DMF), a high
boiling toxic solvent, at 120 ꢀC under nitrogen atmosphere (Scheme
1).34 Yellow powder of SE (21%) was obtained through tedious post-
processing steps. In contrast, the preparation of SME was easier and
faster in multiple aspects. We found that thionyl chloride dropped
in methanol under ice bath quickly generated chlorosulfurous acid
methyl ester (CAME), a methyl ester reagent, which could methyl
esterize carboxyl chemo-selectively but not methylate the phenolic
hydroxyl at room temperature. Considering this favorable property,
SME was obtained from scutellarin treated with excess CAME
(Scheme 2) in a quantitative yield without further purification
(Fig. 5A). The productivity and purity of SME were determined by
HPLC, 1H NMR (Fig. S2 in the ESI), 13C NMR (Fig. S3 in the ESI) and
Scheme 1. Synthesis of ethyl ester of scutellarin.
Scheme 2. Synthesis of methyl ester of glucuronide.
Fig. 5. HPLC chromatograms of scutellarin.a (A) Standards: a. Scutellarin; b. SME; c. Scutellarein; (B) scutellarin (1) and SME (2) hydrolyzed for 2 h; (C) scutellarin (1) and SME (2)
hydrolyzed for 4 h; (D) scutellarin (1) and SME (2) hydrolyzed for 6 h.
a
All reactions were carried out with scutellarin (0.5 mmol) or SME (0.5 mmol) and 2 mol/L H2SO4 (2.5 mmol) in 90% EtOH at 95 ꢀC under N2 atmosphere.
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
ESI-MS (Fig. S4 in the ESI). Compared with SE, the synthesis of SME
exhibited advantages in four areas: solvent, temperature, post-
treatment and yield. Further, the acid hydrolysis of SME has a
higher conversion than that of SE under the same reaction condi-
tions (the data not shown). As a consequence, methyl was
confirmed to be the most promising protective group, and the hy-
drolysis process of SME could be conscientiously evaluated.
for 7 h.
With the optimum conditions, we further investigated the hy-
drolysis procedure of SME compared with scutellarin. The trans-
formation of scutellarin and SME could be readily monitored,
comparing the peak area of the hydrolysates with that of the
standards (Fig. 5A). The results are shown in Fig. 5B D. For the
conversion of scutellarin, only 6.9% yield of the desired product was
detected at 6 h (Fig. 5B and D). Excitingly, the same condition could
successfully convert SME into scutellarein within 6 h and no isomer
was detected (Fig. 5D). The cleavage of ester bond of SME under
acidic hydrolysis conditions did not happen, which was confirmed
by HPLC. Eventually, the yield of scutellarein could reach 96% with
only traces of SME remaining by HPLC (Fig. 5A and b) and 1H NMR
(Fig. S5 in the ESI). Therefore, no further purification was required.
2.2. Successful hydrolysis of scutellarin and its excellent
applicability
2.2.1. Optimization of the hydrolysis parameters of SME
Initially, the acid hydrolysis of SME was performed under mild
conditions. It was cheerful to find that the hydrolysis reaction of
SME could proceed in the presence of 0.5 mol/L H2SO4 at 90 ꢀC for
12 h, yielded 61% of scutellarein (Table 3, entry 1), which indicated
that it was crucial to protect the carboxyl for an efficient acid hy-
drolysis of scutellarin. Encouraged by this result, we optimized the
top two most important hydrolysis parameters35 - the concentra-
tion of sulfuric acid (0.5e3 mol/L) and the reaction temperature
(90e100 ꢀC) (Table 3). To our delight, among the different condi-
tions explored in the optimization, scutellarein was obtained in a
95% yield when the concentration of H2SO4 was elevated to 2 mol/L
at 100 ꢀC (Table 3, entry 6). However, the conversion was not
further increased at higher concentration of H2SO4 (95%, entry 7),
so rather harsh acidic conditions have not been evaluated. Inter-
estingly, lowering the temperature to 95 ꢀC resulted in a slightly
higher yield (96%, entry 8), which may be due to the instability of
the ester bond at high temperature. Finally, the optimum hydrolytic
reaction of SME was performed in 2 mol/L H2SO4/EtOH: H2O (9:1)
2.2.2. The proportional amplification and substrate applicability of
this method
Notably, both methyl esterification of scutellarin and its hy-
drolysis are scaled up which proved to be a practical route for the
preparation of scutellarin. In our study, scutellarin in 10-g scale
(10.01 g) was subjected to react with CAME to obtain SME, which
was further hydrolyzed under the optimized conditions for 9 h
(Scheme 3), then fine yellow powder of scutellarein (5.57 g, 90%
yield) was obtained after a facile purification procedure.
To efficiently obtain other pharmaceutically valuable aglycones,
we applied these selected conditions to the corresponding flavo-
noid glucuronides which are widely distributed in plants and
readily available, such as baicalin and wogonoside.36 Gratifyingly,
baicalein was obtained by hydrolyzing baicalin methyl ester (BME)
in an excellent yield of 97%, and the selected conditions were also
Table 3
Optimization of hydrolysis conditions of SME with sulfuric acid.a
Entry
H2SO4 (mol/L, equiv.)
T (ꢀC)
Time (h)
Conv. (%)b
Yield (%)c
1
2
3
4
5
6
7
8
9
10
0.5, 5
0.5, 10
1, 10
1, 10
1, 10
2, 10
3, 10
2, 10
2, 5
90
90
90
100
100
100
100
95
12
12
10
10
15
10
10
7
69
71
82
86
88
98
99
98
98
94
61
66
75
80
83
95
95
96
96
91
95
90
7
7
2, 5
a
Unless otherwise stated, all reactions were carried out with scutellarin (0.5 mmol) in 90% ethanol under N2 atmosphere.
Conversion determined by HPLC analysis.
Isolated yield.
b
c
Scheme 3. 10-Gram scale acid hydrolysis of scutellarin.
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
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Scheme 4. Acid hydrolysis of BME and WME.
applied for the preparation of wogonin from wogonoside without
any difficulties (Scheme 4).
can be readily scaled up and represents a practical route to prepare
scutellarein, as well as successfully prepare other biologically
important bioactive flavone aglycones.
For the aliphatic-aglycone-containing glucuronides, such as
glycyrrhizic acid, the glycosidic bond without
p-electron conjuga-
tion system is less stable and easily hydrolyzed at room tempera-
ture. But, we speculate that the cleavage of the glycosidic bond to
produce glycyrrhetinic acid is more likely occurred by alcoholysis
rather than hydrolysis reaction in the presence of HCl/MeOH at
room temperature.37 In order to verify this conjecture, we operated
four sets of comparative experiments (Table 4).
As shown in Table 4, the hydrolytic yield of glycyrrhizic acid was
relatively low, only a 14% conversion was achieved under 6 M HCl at
20 ꢀC (entry 1). The methanolysis reaction of glycyrrhizic acid went
very smoothly in the presence of hydrogen chloride which was
furnished via methanol and thionyl chloride/acetyl chloride (90%
and 92%, entry 2 and 3). Glycyrrhizic acid can also be subjected to
ethanolysis at a lower yield compared with the methanolysis
because of the weaker reactivity of ethanol than methanol (71%,
entry 4).
4. Experimental section
4.1. Materials
Baicalin (purity > 99%), scutellarin (purity > 99%), wogonoside
(purity > 98%) and glycyrrhizinate (purity > 99%) were purchased
from Aladdin Reagent Company. Acetonitrile (HPLC grade) was
purchased from Tedia Company, Inc. (USA). Unless otherwise
specified, all the solvents and reagents were analytical grade and
obtained from commercial suppliers. Solvents were purified as per
the procedures given in the “Text book of practical organic chemis-
try” by Vogel (6th Edition). Water was purified with a Millipore
Milli Q-Plus system (Millipore, MA, USA). All the reactions were
performed under a nitrogen atmosphere unless otherwise noted.
4.2. Computational details
3. Conclusions
The structures corresponding to the reactants, transition states
[TSs], intermediates, and the products for the hydrolysis reaction
were optimized, using the GAUSSIAN 09 computational package
with DFT methods as implemented in the computational package.31
All calculations were carried out at the B3LYP level using the 6-31G
(d,p) basis set. The compounds were also optimized using the same
We have developed an efficient approach to obtain scutellarein
by hydrolyzing the methyl-esterification product of scutellarin,
which has been successfully applied in our laboratory. This protocol
has possessed the advantages of mild condition, simple operation
and excellent yield. It is worth noting that the present methodology
Table 4
The alcoholysis reaction of glycyrrhizic acid.a
Entry
Acid
Solvent
T (ꢀC)
Time (h)
Yield (%)f
1
2
3
4
6 M HClb
HClc
Ethanol
20
20
20
20
8
8
8
8
14
90
92
71
Methanol
Methanol
Ethanol
HCld
HCle
a
Reaction conditions: all of the reactions were performed with glycyrrhizic acid (82.2 mg, 0.1 mmol) and acid in solvent (2 mL).
5mol/L HCl (3 mmol, 30 equiv).
b
c
d
e
f
Thionyl chloride (1.2 mmol, 90 m
L) was slowly added to anhydrous methanol (2 ml) at 0 ꢀC for 1 h.
Acetyl chloride (1.2 mmol, 86
Acetyl chloride (1.2 mmol, 86
Yield of isolated product.
m
m
L) was slowly added to anhydrous methanol (2 ml) at 0 ꢀC for 1 h.
L) was slowly added to anhydrous ethanol (2 ml) at 0 ꢀC for 1 h.
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8
X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
functional and basis set in EtOH as the solvent based on the
Polarizable Continuum Model (PCM) which is developed by Tomasi
et al.38 The vibration frequencies were calculated at the same level
to confirm that whether the structures correspond to energy
minima or transition states (zero or one imaginary frequencies,
respectively).
4.5.1. Scutellarin methyl ester (SME)
Following the general procedure with scutellarin (1 mmol,
462 mg), the mixture was stirred at room temperature for 9 h.
Methanol was removed and SME (455 mg, 95.6%) was obtained
without further purification as a yellow powder; mp 251e252 ꢀC;
1H NMR (DMSO-d6, 300 MHz,
d ppm) d 12.85 (s, 1H, 5-OH), 10.39 (s,
1H, 40eOH), 7.93 (d, J ¼ 9.0 Hz, 2H, H-20, H-60), 7.00 (s, 1H, H-8), 6.94
(d, J ¼ 9.0 Hz, 2H, H-40, H-50), 6.81 (s, 1H, H-3), 5.28 (d, J ¼ 7.0 Hz,1H,
H-1’0), 4.20 (d, J ¼ 6.0 Hz, 1H, H-200), 3.70e3.90 (3H, m, other sugar
4.3. Analytical methods
1H and 13C NMR spectra were recorded with a Bruker Avance
300 MHz spectrometer at 300 K, using dimethyl sulfoxide (DMSO-
d6) as solvent and tetramethylsilane (TMS) as an internal standard.
Mass spectra were measured on Waters Q-TOF Micro™ using
electrospray ionization (ESI). Melting points were determined on a
RY-1 micro melting point apparatus (Tianjin Analytical Instrument
Corp., Tianjin, China). Analytical thin-layer chromatography (TLC)
were performed on silica gel GF/UV 254 and visualized under UV
light at 254 and 365 nm.
Analysis of hydrolysis was performed with a Shimadzu LC-2010/
CHT system equipped with quaternary gradient pump, auto
sampler, column oven and UV detector. The data were processed by
LC-solution software which was used for instrument control and
data acquisition. The separation was carried out on a phenomenex
protons), 3.68 (3H, s, eOCH3); 13C NMR (DMSO-d6, 75 MHz,
d ppm):
182.8, 169.7, 164.5, 161.6, 151.3, 149.4, 147.3, 130.8, 128.9, 121.7, 116.4,
106.3, 102.9, 100.2, 93.9, 75.7, 75.4, 73.1, 71.8, 52.4 (eOCH3); MS
(ESI) m/z ¼ 477.1 [MþH]þ, 475.1 [M - H]-; HRMS (ESI): m/z calcd for
C
22H19O12: 475.0882; found: 475.0868 [MꢁH]ꢁ.
4.5.2. Baicalin methyl ester (BME)
Following the general procedure with baicalin (1 mmol,
446 mg), the mixture was stirred at room temperature for 8.5 h.
Methanol was removed and BME (442 mg, 96.1%) was obtained
without further purification as a yellow powder; mp 197e199 ꢀC;
1H NMR (DMSO-d6, 300 MHz,
d ppm) d 12.60 (s, 1H, 5-OH), 8.69 (s,
1H, 6-OH), 8.07 (m, 2H, H-20, H-60), 7.62 (m, 3H, H-30, H-40, H-50),
7.06 (s, 1H, H-3), 7.01 (s, 1H, H-8), 5.52 (m, 2H, sugar hydroxy), 5.30
(d, J ¼ 6.3 Hz, 1H, H-100), 4.22 (d, J ¼ 6.0 Hz, 1H, H-200), 3.68 (3H, s,
eOCH3), 3.40e3.43 (3H, m, other sugar protons); 13C NMR (DMSO-
Gemini C18 column (4.6 mm ꢂ 250 mm, 5
mm). The column tem-
perature was set at 25 ꢀC. The mobile phase consisted of acetonitrile
and 1% formic acid water in the ratio of 76:24 (v/v) with isocratic
elution. The flow rate was 1.0 mL/min. Analytes were monitored at
d6, 75 MHz,
d ppm): 182.5, 169.1, 163.5, 151.2, 149.1, 146.7, 132.0,
130.8, 130.5, 129.1, 126.3, 106.1, 104.7, 99.7, 93.6, 75.5, 75.0, 72.7,
71.3, 51.9 (eOCH3); MS (ESI) m/z ¼ 459.1 [M - H]-; HRMS (ESI): m/z
calcd for C22H19O11: 459.0933; found: 459.0917 [MꢁH]ꢁ.
254 nm. The injection volume was 10 mL.
4.4. Preparation of scutellarin ethyl ester (SE)
4.5.3. Wogonoside methyl ester (WME)
Scutellarin (2 mmol, 924 mg) was dissolved in 10 mL water, then
sodium bicarbonate (2 mmol, 168 mg) was added. The solution was
stirred 0.5 h at room temperature, then stirred at 50 ꢀC for 0.5 h.
After the completion of the reaction, water was evaporated under
reduced pressure. Scutellarin salt was obtained. A mixture of scu-
Following the general procedure with wogonoside (1 mmol,
460 mg), the mixture was stirred at room temperature for 8.5 h.
Methanol was removed and WME (443 mg, 96.4%) was obtained
without further purification as a yellow powder; mp 241e243 ꢀC;
1H NMR (DMSO-d6, 300 MHz,
d ppm) d 12.56 (s, 1H, 5-OH), 8.04 (m,
tellarin salt (1 mmol, 484 mg), bromoethane (3 mmol, 224
mL) and
2H, H-20, H-60), 7.59 (m, 3H, H-30, H-40, H-50), 7.02 (s, 1H, H-6), 6.72
(s, 1H, H-3), 5.35 (d, J ¼ 6.1 Hz, 1H, H-100), 4.23 (d, J ¼ 7.3 Hz, 1H, H-
200), 3.40e3.46 (3H, m, other sugar protons), 3.89 (s, 3H, 8-OCH3),
KI (0.1 mmol, 16.6 mg) in DMF (3 mL) was stirred at 120 ꢀC for 3 h
under a N2 atmosphere. Then the reaction mixture was poured into
water (30 mL) and extracted with ethyl acetate (3 ꢂ 15 mL). The
organic layer was separated, washed with water (2 ꢂ 10 mL) and
dried over anhydrous sodium sulfate. Organic solvent was
concentrated under vacuum, recrystallization with ethyl acetate to
yield SE as a yellow powder. Yield 21%; mp 259e261 ꢀC; 1H NMR
3.68 (s, 3H, 500eOCH3); 13C NMR (DMSO-d6, 75 MHz,
d ppm): 182.7,
169.7, 163.9, 157.8, 156.4,150.1, 132.6, 131.2, 129.7, 128.1, 126.8, 105.8,
105.4, 99.9, 98.9, 76.0, 75.5, 73.3, 71.7, 61.8, 52.5 (eOCH3); MS (ESI)
m/z ¼ 473.1 [MꢁH]ꢁ; HRMS (ESI): m/z calcd for C23H21O11
:
473.1089; found: 473.1102 [MꢁH]ꢁ.
(DMSO-d6, 300 MHz,
d ppm): 12.79 (1H, s, 5-OH), 10.37 (1H, s, 6-
OH), 8.63 (1H, s, 40eOH), 7.91 (m, 2H, 20eH, 60-H), 7.10 (1H, s, 8-
H), 6.92 (2H, d, J ¼ 7.4 Hz, 30eH, 50-H), 6.81 (1H, s, 3-H), 5.46 (2H,
m, 100eH, 500-H), 5.28 (2H, m, eCH2e),4.13 (3H, m, 200eH, 300eH, 400-
H), 2.50e3.46 (3H, m, other sugar protons), 1.22 (3H, t, J ¼ 4.4 Hz,
4.6. Acid hydrolysis of scutellarin
In a 10 mL round-bottomed flask, 300 mg (0.65 mmol) of scu-
tellarin was dissolved in 5 mL of specific ethanol. To the mixture
was added 1 mL of sulfuric acid with a specific concentration
(Table 1), and the solution was stirred and heated to a specific
temperature (Table 1). The reaction was timed just after the addi-
tion of H2SO4. After completion of the reaction, aliquots of the re-
action mixture were subjected to HPLC analysis. The mobile phase
for HPLC consisted of solvent (A) i.e., and (B), solvent i.e. acetoni-
trile. The mobile phase consisted of acetonitrile and 0.1% (v/v)
formic acid in filtered MilliQ water in the ratio of 24: 76 (v/v) with
isocratic elution. The flow rate was 1.0 mL/min. Analytes were
eCH3); 13C NMR (DMSO-d6, 75 MHz,
d ppm): 183.0, 168.9, 163.8,
160.5, 152.1, 150.3, 148.7, 129.9, 129.2, 121.4, 115.9, 106.0, 103.4,
100.7, 93.1, 74.9, 74.5, 73.2, 71.3, 60.5, 15.3; MS (ESI) m/z ¼ 489.1
[MꢁH]ꢁ. The melting points and 1H NMR were in good agreement
with the reported.33
4.5. General procedure: preparation of glucuronide methyl ester
To a stirred solution of methanol (19 ml) in an ice both was
added thionyl chloride (900
mL) drop wise over a period of 1 min.
monitored at 254 nm. The injection volume was 10 mL.
After complete addition, the solution was stirred 1 h at room
temperature to give chlorosulfurous acid methyl ester. Then
glucuronide (0.1 eq) was added. The mixture was stirred at room
temperature for the indicated time. After the completion of the
reaction, solvents were removed under reduced pressure and the
desired products were obtained.
4.7. General procedure: acid hydrolysis of glucuronide methyl ester
to obtain corresponding aglycones
To a stirring mixture of glucuronide methyl ester (0.5 mmol) and
sulfuric acid (1.1 mL) in ethanol (8 mL) was added water (1.1 mL),
Please cite this article in press as: Jiang X-Y, et al., An efficient, scalable approach to hydrolyze flavonoid glucuronides via activation of glycoside
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X.-Y. Jiang et al. / Tetrahedron xxx (2017) 1e9
9
the reaction mixture was stirred at 95 ꢀC for the indicated time
Acknowledgments
under a N2 atmosphere. After cooled down to the room tempera-
ture, the mixture was poured into water. Then the precipitate was
filtered off, washed with water and recrystallized from methanol to
obtain the desired products.
This research work was financially supported by the National
Natural Science Foundation of China (Nos. 81673567, 81373956,
81573557 and 81502950), the Fundamental Research Funds for the
Central Universities (2015ZD010), and the Priority Academic Pro-
gram Development of Jiangsu Higher Education Institutions.
4.7.1. Scutellarein
137 mg, 96% yield; yellow powder; mp: 325e328 ꢀC; 1H NMR
(300 MHz, DMSO-d6,
d
ppm)
d
12.79 (s, 1H, 5-OH), 10.47 (s, 1H, 7-
Appendix A. Supplementary data
OH), 10.32 (s, 1H, 40eOH), 8.75 (s, 1H, 6-OH), 7.89 (d, J ¼ 8.5 Hz,
2H, H-20, H-60), 6.90 (d, J ¼ 8.5 Hz, 2H, H-30, H-50), 6.73 (s, 1H, H-8),
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2017.02.039.
6.56 (s, 1H, H-3); 13C NMR (DMSO-d6, 75 MHz,
d ppm): 182.2, 163.7,
161.2, 153.4, 149.9, 147.2, 129.3, 128.5, 121.6, 116.2, 104.2, 102.4, 94.1;
MS (ESI) m/z ¼ 285.1 [MꢁH]ꢁ.
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131 mg, 97% yield; yellow powder; mp: 263e265 ꢀC; 1H NMR
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(300 MHz, DMSO-d6,
d ppm) d 12.65 (s, 1H, 5-OH), 10.57 (s, 1H, 7-
OH), 8.82 (s, 1H, 6-OH), 8.05 (d, J ¼ 8.2 Hz, 2H, H-20, H-60),
7.55e7.58 (m, 3H, H-30, H-40, H-50), 6.93 (s, 1H, H-8), 6.62 (s, 1H, H-
3); 13C NMR (DMSO-d6, 75 MHz,
d ppm): 182.0, 162.7, 153.5, 149.7,
146.9, 136.6, 130.8, 129.2, 128.9, 126.1, 104.3, 104.2, 93.9; MS (ESI) m/
z ¼ 269.1 [MꢁH]ꢁ.
4.7.3. Wogonin
136 mg, 96% yield; yellow powder; mp: 204e205 ꢀC; 1H NMR
(300 MHz, DMSO-d6,
d ppm) d 12.48 (s, 1H, 5-OH), 10.82 (s, 1H, 7-
OH), 8.02e8.04 (m, 2H, H-20, H-60), 7.57e7.58 (m, 3H, H-30, H-40,
H-50), 6.94 (s, 1H, H-6), 6.29 (s, 1H, H-3), 3.84 (s, 3H, 8-OCH3); 13C
NMR (DMSO-d6, 75 MHz,
d ppm): 181.9, 162.8, 157.3, 156.2, 149.5,
131.9, 130.7, 129.1, 127.6, 126.1, 104.9, 103.7, 99.1, 60.9; MS (ESI) m/
z ¼ 283.1 [MꢁH]ꢁ.
4.8. Alcoholysis of glycyrrhizinate
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90%). mp 263e265 ꢀC; 1H NMR (300 MHz, DMSO-d6,
d ppm) d 5.31
(d, 1H, J ¼ 7.2 Hz), 3.00 (m, 1H), 1.87 (m, 1H), 1.34, 1.26, 1.25, 1.22,
1.14, 1.08 and 0.95 (s, seven methyl groups); 13C NMR (DMSO-d6,
75 MHz, d ppm): 199.1,177.7, 170.0,127.3, 71.58, 61.1, 54.3, 48.1, 44.9,
43.1, 42.9, 37.6, 36.4, 32.1, 31.6, 30.4, 28.4, 28.2, 28.1, 27.8, 27.1, 26.1,
25.8, 23.1, 23.0, 18.4, 17.0, 16.2, 16.1, 15.9; MS (ESI) m/z ¼ 269.1
[MꢁH]-.
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Please cite this article in press as: Jiang X-Y, et al., An efficient, scalable approach to hydrolyze flavonoid glucuronides via activation of glycoside
bond, Tetrahedron (2017), http://dx.doi.org/10.1016/j.tet.2017.02.039