Glycomonomeric and glycopolymeric inhibitors for β-Glucuronidase. π-π Stacking interaction, polymeric effect, and plausible conformation

Article abstract of DOI:10.1246/bcsj.20110043

N-p-Vinylbenzyl-6-D-glucaramic acid (VB-6-D-GlcaH, 1) and its corresponding polymer, P(VB-6-D-GlcaH-co-AAm) (2), were found to inhibit β-glucuronidase activity more efficiently than D-glucaro-6,3-lactone, while the inhibition ability of N-butyl-6-D-glucaramic acid (Butyl-6-D-GlcaH, 3) on the enzyme activity was seriously lower than that of the lactone. The π-π stacking interaction between the styryl part of 1 and Trp₅₈₇ of β-glucuronidase was confirmed by the blue shift detected on fluorescence spectrophotometry and the observation of the 3D motif for the active site with the glycomonomer, which must enhance the inhibition ability of the enzyme. In the case of 2, however, such a shift was not observed, which indicated that the effective inhibition of 2 was induced not by the π-π stacking interaction but a polymeric effect. Based on these results and our previous work, plausible conformations of 1 and 2 fitting in β-glucuronidase were proposed.

Full text of DOI:10.1246/bcsj.20110043

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Bull. Chem. Soc. Jpn. Vol. 84, No. 9, 912-917 (2011)
© 2011 The Chemical Society of Japan
Selected Papers
Glycomonomeric and Glycopolymeric Inhibitors for ¢-Glucuronidase.
³-³ Stacking Interaction, Polymeric Effect, and Plausible Conformation
Asei William Kawaguchi, Haruki Okawa, and Kazuhiko Hashimoto*
Department of Applied Chemistry, Faculty of Engineering, Kogakuin University, Nakano-cho, Hachioji, Tokyo 192-0015
Received February 9, 2011; E-mail: hashimot@cc.kogakuin.ac.jp
N-p-Vinylbenzyl-6-D-glucaramic acid (VB-6-D-GlcaH, 1) and its corresponding polymer, P(VB-6-D-GlcaH-co-
AAm) (2), were found to inhibit ¢-glucuronidase activity more efficiently than D-glucaro-6,3-lactone, while the inhibition
ability of N-butyl-6-D-glucaramic acid (Butyl-6-D-GlcaH, 3) on the enzyme activity was seriously lower than that of the
lactone. The ³-³ stacking interaction between the styryl part of 1 and Trp587 of ¢-glucuronidase was confirmed by the
blue shift detected on fluorescence spectrophotometry and the observation of the 3D motif for the active site with the
glycomonomer, which must enhance the inhibition ability of the enzyme. In the case of 2, however, such a shift was not
observed, which indicated that the effective inhibition of 2 was induced not by the ³-³ stacking interaction but
a polymeric effect. Based on these results and our previous work, plausible conformations of 1 and 2 fitting in
¢-glucuronidase were proposed.
When xenobiotics, such as medicines and toxins, are
and metabolized again.1 Such behavior is well-known as
enterohepatic circulation (Scheme 1). If the xenobiotics are
medicines, such circulation could be helpful because the
medicinal effects would be prolonged. In contrast, if they are
toxic, a serious condition could be caused by this circulation.
Indeed, irinotecan, an anticancer drug, has been reported to
cause serious diarrhea by its side effect as a result of this
circulation.3-7 Therefore, occasional inhibition of ¢-glucuroni-
dase activity could be beneficial for maintaining health.8-13
Several saccharic acids, metal ions, and amino acids have
been used as candidates for inhibitors of ¢-glucuronidase.14-21
absorbed in the human body, they are sent to the liver to be
oxidized and converted to their glucuronides. This metabolism
is referred to as detoxication.1 If the molecular weight of the
glucuronides is less than 500 « 50, they are sent to the kidneys
and excreted in urine. On the other hand, when their molecular
weight is more than 500 « 50, they are sent to the intestines
and discharged as feces.2 However, almost all glucuronides are
hydrolyzed to the free xenobiotics and glucuronic acid with
bacteria-derived ¢-glucuronidase in the intestine. Therefore the
free xenobiotics are sent back to the liver via the portal vein
Scheme 1. Xenobiotics in the human body and enterohepatic circulation.
Published on the web August 23, 2011; doi:10.1246/bcsj.20110043

A. W. Kawaguchi et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 9 (2011)
913
Chart 1.
Chart 2.
Among them, the saccharic acids were the most effective
inhibitors for the enzyme.15,19,20 If administrated orally how-
ever, most of them are absorbed as nutritive substances, before
they reach the intestine to work as inhibitors. Therefore, the
exclusive transportation of the inhibitors into the intestines is
important for clinical research on their activity in the human
body.
We have already reported that the styryl monomers
containing glucaric (VB-6-D-GlcaH, 1) (Chart 1), xylaric
(VB-D,L-XylaH), tartaric (VB-L-TartH), and mannaric
(VB-D-ManaH) moieties were synthesized, and subsequently
copolymerized with acrylamide to give the water-soluble
glycopolymers, respectively [P(VB-6-D-GlcaH-co-AAm) (2),
P(VB-D,L-XylaH-co-AAm), P(VB-L-TartH-co-AAm), and
P(VB-D-ManaH-co-AAm)].8-10,20,21 The all saccharic acid-
derived polymers prepared in our previous work were found
to inhibit ¢-glucuronidase activity in vitro much more
efficiently than not only the corresponding glycomonomers
but also the saccharic acids and lactones respectively, espe-
cially at lower concentration (<0.1 mol L¹1). Additionally, the
gluconic macromolecular analogs were also synthesized and
found not to inhibit the enzyme effectively. Therefore, the free
carboxy group in the saccharic moiety was revealed to be
essential for the inhibition.9
During our previous work,20,21 the following unexpected
phenomenon was observed. All glycomonomers bearing a free
carboxy group always inhibited the enzyme activity more
effectively than the corresponding saccharic acids or lactones.
Such behavior was observed especially in the cases of the
tartaric and mannaric series. Whether the polymerizable styryl
group exists or not is the biggest structural difference between
our glycomonomers and the corresponding saccharic acids or
lactones. Therefore, in this article, the effect of the styryl group
was first investigated by comparing the inhibition abilities and
the inhibition behavior of VB-6-D-GlcaH (1) with those of
N-butyl-6-D-glucaramic acid (Butyl-6-D-GlcaH (3)) (Chart 2)
bearing a hydrophobic but not aromatic group. After the effect
of the styryl group was confirmed more clearly by fluorescence
spectrophotometry, a plausible conformation of VB-6-D-GlcaH
(1) and P(VB-6-D-GlcaH-co-AAm) (2) fitting in the active site
of ¢-glucuronidase was proposed by computer simulation.
p-vinylbenzyl chloride, which was kindly supplied by AGC
Seimi Chemical Co., Ltd. (Chigasaki, Kanagawa, Japan).23
¢-Glucuronidase (bovine liver) was purchased from Sigma
(MO, USA) and used as-received. p-Nitrophenyl ¢-D-glucu-
ronide was purchased from Nacalai Tesque (Kyoto, Japan) and
used without further purification. VB-6-D-GlcaH (1) was
prepared through the reaction of D-glucaro-6,3-lactone with
p-vinylbenzylamine.8 Butyl-6-D-GlcaH (3) was prepared by a
similar method to that of VB-6-D-GlcaH (1) using butylamine
and D-glucaro-6,3-lactone (see Supporting Information for the
specific method and physical data). P(VB-6-D-GlcaH-co-AAm)
(2) was prepared by copolymerization of VB-6-D-GlcaH (1)
with acrylamide.8
Instruments. 1H NMR and 13C NMR spectra were taken
with a JEOL JNM-ECX-400 Fourier transform high-resolution
spectrometer. A Molecular devices SPECTRA·MAX 190
microplate spectrophotometer was used for the determination
of p-nitrophenol generated during the hydrolysis test. Elemen-
tal analysis was carried out with a CHN/O Analyzer 2400II
(Perkin-Elmer). The amino-acid sequence of bovine ¢-glucu-
ronidase by BLAST was obtained from UniPort and the 3D
motif of the human enzyme was obtained from Protein Data
Bank Japan. The Fluorescence spectrometry was measured
with F-3010 Fluorescence Spectrophotometer (Hitachi Co.,
Ltd.).
Inhibition Test for ¢-Glucuronidase Activity. A model
compound for the ¢-D-glucuronide conjugates of xenobiotics,
p-nitrophenyl ¢-D-glucuronide, was hydrolyzed with ¢-glucu-
ronidase in the absence or presence of the resulting inhibitors
(Scheme 2). The amount of p-nitrophenol liberated from the
glucuronide was determined by spectroscopy. The inhibition
value (%) was calculated from the hydrolytic rates of the
substrate in the absence and presence of the inhibitors. A
Lineweaver-Burk plot was constructed using data determined
in 0.4-1.4 mM of p-nitrophenyl ¢-glucuronide in the presence
of different amounts of the inhibitors.24,25
3D Computer Graphical Observation of the ¢-Glucu-
ronidase Active Site. Discovery Studio Visualizer ver. 2.0
(DS Visualizer, Accelrysμ) was used to observe the 3D motif of
the human ¢-glucuronidase active site, which was reported by
Jain.26 The 3D motif of the human ¢-glucuronidase was
obtained from Protein Data Bank Japan (PDBj). Since
Discovery Studio Visualizer ver. 2.0 is the viewer software
for the protein, plausible structures were illustrated manually.
Measurement of the Tryptophanyl Fluorescence Spectra.
Tryptophanyl fluorescence spectra were measured in the range
of 300-400 nm employing an excitation wavelength of 285 nm
Experimental
Materials. D-Glucose, butylamine, and acetic anhydride
were purchased from Kanto Kagaku Co. (Tokyo, Japan).
D-Glucaro-6,3-lactone was prepared from D-glucose according
to the literature.22 p-Vinylbenzylamine was prepared from

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Bull. Chem. Soc. Jpn. Vol. 84, No. 9 (2011)
The Glyco-Inhibitors for ¢-Glucuronidase
Scheme 2. Inhibition test for ¢-glucuronidase activity by inhibitors.
Figure 1. The Inhibition of ¢-glucuronidase activity by
different glucaric inhibitors at 37 °C (p-nitrophenyl ¢-D-
glucuronide, 7 mmol L¹1; ¢-glucuronidase, 14 IU L¹1).
P(VB-6-D-GlcaH-co-AAm) (2):9 ; VB-6-D-GlcaH (1):
D-Glucaro-6,3-lactone: ; Butyl-6-D-GlcaH (3).
;
;
Figure 2. A Lineweaver-Burk plot for kinetic data of the
hydrolysis of p-nitrophenyl ¢-D-glucuronide by ¢-glucu-
ronidase in the presence of Butyl-6-D-GlcaH (3).
and slit widths of 5 nm with F-3010 Fluorescence Spectropho-
tometer (Hitachi Co., Ltd.). The ¢-glucuronidase solutions
mixed with VB-6-D-GlcaH (1), P(VB-6-D-GlcaH-co-AAm) (2),
and Butyl-6-D-GlcaH (3) were prepared by adding the enzyme
(80 mg) with the corresponding inhibitors (0.12 mmol) in
10 mL of acetic buffer, and compared with the blank solution
(the enzyme 80 mg in 10 mL).
corresponding glycopolymer 2. In contrast, the inhibition
ability of Butyl-6-D-GlcaH (3) was much lower than those of
VB-6-D-GlcaH (1) and D-glucaro-6,3-lactone, especially in the
range of 1.37-6.67 mmol L¹1. Additionally, the inhibition
behavior of Butyl-6-D-GlcaH (3) was confirmed to be
competitive from the Lineweaver-Burk plot in which all
extrapolating lines intersected on the vertical axis (Figure 2).
Therefore, the inhibition type was found not to change from
competitive to the any other else by introducing the n-butyl
group instead of the styryl group to the glucaric moiety. These
two results suggest that the styryl moiety assists the inhibition
indirectly as the aromatic part rather than as the hydrophobic
part.
Results and Discussion
Whether the polymerizable styryl group exists or not was the
biggest structural difference between our glycomonomers and
the corresponding saccharic acids or lactones. Therefore, a new
inhibitor with an aliphatic moiety, Butyl-6-D-GlcaH (3), was
synthesized in order to compare its inhibition ability of the
¢-glucuronidase activity with that of VB-6-D-GlcaH (1) having
the styryl group which is a typical competitive inhibitor.10
A
model compound for the ¢-D-glucuronide conjugates of
xenobiotics, p-nitrophenyl ¢-D-glucuronide, was hydrolyzed
with ¢-glucuronidase in the absence or presence of VB-6-D-
GlcaH (1), Butyl-6-D-GlcaH (3), or D-glucaro-6,3-lactone, that
was kinetically traced by spectroscopy. The inhibition value
(%) was calculated from the hydrolytic rates of the substrate in
the absence and presence of the above-mentioned saccharic
derivatives (v0 and v, respectively), as shown in eq 1.
³-³ stacking interaction is a well-known aromatic inter-
action. In addition, some researchers have reported that this
interaction enhances the ability of inhibitors.27-29 Moreover,
aromatic amino acid residues in enzymes such as tryptophan
and phenylalanine have been confirmed to interact with the
aromatic groups in the inhibitors by this interaction. Therefore,
if this interaction assisted the inhibition ability of VB-6-D-
GlcaH (1) for ¢-glucuronidase activity, the corresponding
amino acid residue should exist around the active site in the
enzyme, because VB-6-D-GlcaH (1) is a typical competitive
inhibitor.
Inhibition value ð%Þ ¼ ½ðv0 ꢀ vÞ=v0ꢁ ꢂ 100
ð1Þ
As shown in Figure 1, the inhibition ability of VB-6-D-GlcaH
¹1
(1) increased to 95% at the 6.67 mmol L saccharic concen-
Since only the human ¢-glucuronidase 3D motif has been
reported, the active site of human enzyme was used for our
discussion instead of that of the bovine ¢-glucuronidase. The
tration and was higher than that of D-glucaro-6,3-lactone at
all concentrations, although it was lower than that of the

A. W. Kawaguchi et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 9 (2011)
915
Figure 3. A stereo view of the active center of ¢-glucuronidase.
Table 1. The Wavelength of Maximal Fluorescence Emis-
siona) of ¢-Glucuronidase in the Presence of the Inhibitors
System
-max/nm Relative intensity
¢-Glucuronidase (E)
E + Butyl-6-D-GlcaH (3)
E + P(VB-6-D-GlcaH-co-AAm) (2) 337.0
E + VB-6-D-GlcaH (1)
337.2
337.8
313
311
983
373
321.2
a) Excitation -, 285 nm.
tryptophan environment in the enzyme becomes hydrophobic
in the presence of VB-6-D-GlcaH (1). Therefore, this is
consistent with the Trp587 residue interacting with the styryl
group through ³-³ stacking and the VB-6-D-GlcaH (1) being
localized around the active site. Consequently, the inhibition
ability by VB-6-D-GlcaH (1) was inferred to be higher than
those of the corresponding saccharic acid or lactones.
Figure 4. The fluorescence emission spectra of ¢-glucu-
ronidase (black chain line), ¢-glucuronidase with Butyl-6-
D-GlcaH (3) (blue line), ¢-glucuronidase with P(VB-6-D-
GlcaH-co-AAm) (2) (green line), and ¢-glucuronidase
with VB-6-D-GlcaH (1) (red line).
Our previous and present work has so far elucidated that 1)
the free carboxy group in the saccharic moiety is essential for
the inhibition of ¢-glucuronidase activity,9 2) the inhibition
behaviors of VB-6-D-GlcaH (1) and P(VB-6-D-GlcaH-co-
AAm) (2) are competitive, and 3) the styryl group interacts
with Trp587 residue through ³-³ stacking. Additionally,
although no crystal structure of the substrates and/or inhibitors
in ¢-glucuronidase has been reported, Asn450 was predicted to
interact with the hydroxy group of saccharide moiety.34,35
Based on these results, plausible conformations of VB-6-D-
GlcaH (1) and P(VB-6-D-GlcaH-co-AAm) (2) in the enzyme
are illustrated in Figure 5. Since the styryl group overlaps with
Trp587 by ³-³ stacking interaction and the glucaric moiety
occupies the active site, VB-6-D-GlcaH (1) (as illustrated in
yellow in Figure 5A) seems to be consequently bent in the
enzyme. However, in the case of P(VB-6-D-GlcaH-co-AAm)
(2), the styryl moiety would not be located near the active cite
in the enzyme because of steric hindrance. Hence, it is easily
supposed that P(VB-6-D-GlcaH-co-AAm) (2) inhibits the
¢-glucuronidase activity by the simple insertion of the glucaric
moiety in the active site (Figure 5B). This inference suggests
also that the efficient inhibition ability of P(VB-6-D-GlcaH-co-
AAm) (2) is induced only by a polymeric effect. Since glucaric
units are linked as pendant groups along the polymer chain, the
inhibitor concentration may become higher locally, even at
lower concentration. As a side note, these plausible conforma-
tions were illustrated manually, so the uncertain interaction
between the hydroxy group and amino acid residues will not be
discussed here.
amino-acid sequence homologies of the human and bovine
¢-glucuronidase were confirmed to ca. 80% and the active sites
were almost completely the same (Figure S1). Islam et al.
reported that Glu451 and Glu540 were essential catalytic residues
(illustrated in light blue) in ¢-glucuronidase as shown in
Figure 3.30 As illustrated in pink in Figure 3, there was Trp587
on the straight line of the catalytic residues. So, we expected
that Trp587 interacts with the styryl group in the glycomono-
meric inhibitor by ³-³ stacking. It is well-known that the -max
of tryptophan residual fluorescence emission becomes shorter
with increasing hydrophobicity of the tryptophan environ-
ment.31-33 Thus, tryptophanyl fluorescence emission spectra of
four systems (¢-glucuronidase only, the enzyme with VB-6-D-
GlcaH (1), the enzyme with Butyl-6-D-GlcaH (3), and the
enzyme with P(VB-6-D-GlcaH-co-AAm) (2)) were investigated
and the results are illustrated in Figure 4 and each -max is
summarized in Table 1. The -max in the systems of the enzyme
with Butyl-6-D-GlcaH (3) and P(VB-6-D-GlcaH-co-AAm) (2)
were hardly changed from that of the enzyme only. However,
the intensity increased in the latter system with 2, which was
caused by the styryl moiety of the polymer side chain.
Therefore, these results revealed that the ³-³ stacking
interaction did not occur between Trp587 and both inhibitors.
In contrast, in the case of the enzyme with VB-6-D-GlcaH (1),
the -max was found to be 16 nm shorter, which indicates that the

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Bull. Chem. Soc. Jpn. Vol. 84, No. 9 (2011)
The Glyco-Inhibitors for ¢-Glucuronidase
(A)
(B)
Figure 5. Stereo views for plausible structures of (A) VB-6-D-GlcaH (1) and (B) P(VB-6-D-GlcaH-co-AAm) (2) in
¢-glucuronidase.
Summary
Supporting Information
In summary, the comparison of the inhibition abilities of
VB-6-D-GlcaH (1) for ¢-glucuronidase with that of Butyl-6-D-
GlcaH (3) and their tryptophanyl fluorescence spectra revealed
the ³-³ stacking interaction between the styryl group in the
inhibitor and Trp587 in the enzyme, which assisted indirectly
the inhibition of the ¢-glucuronidase activity in the case of
monomeric inhibitors. On the other hand, in the case of
polymeric inhibitor, the ³-³ stacking interaction does not
contribute to the inhibition of the enzyme because the polymer
chain itself creates steric hindrance. Plausible structures of
VB-6-D-GlcaH (1) and P(VB-6-D-GlcaH-co-AAm) (2) fitting
in the enzyme were proposed, which had not yet been
confirmed by X-ray crystallography. Investigation and con-
firmation of the structures would be helpful for the design of
new inhibitors.
This file contains as follows: A) the synthesis method and
the physical data of Butyl-6-D-GlcaH (3), B) the figure of the
amino-acid sequence homologies of the human and bovine
¢-glucuronidase. This material is available free of charge on
the web at http://www.csj.jp/journals/bcsj/.
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