Aglycon specificity profiling of α-glucosidases using synthetic probes

Article abstract of DOI:10.1016/j.bmcl.2004.12.086

We designed and synthesized hydrogen bond based probes 1-8 with the exception of known glycosidase inhibition mechanisms, and aglycon specificity of 11 different sources of α-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-d-glycero- d-ido-heptitol) showed a potent inhibition of S. cerevisiae α-glucosidase among all α-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae α-glucosidase with K_i 0.13 mM.

Full text of DOI:10.1016/j.bmcl.2004.12.086

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1489–1492
Aglycon specificity profiling of a-glucosidases using
synthetic probes
Wataru Hakamata,a,* Makoto Muroi,a Kazunari Kadokura,a Toshiyuki Nishio,b
Tadatake Oku,b Atsuo Kimura,c Seiya Chibac and Akira Takatsukia
aAnimal and Cellular Systems Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan
bDepartment of Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi,
Kanagawa 252-8510, Japan
cDivision of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Nishi-9, Kita-9, Kita-ku, Sapporo,
Hokkaido 060-8589, Japan
Received 9 November 2004; revised 27 December 2004; accepted 28 December 2004
Available online 25 January 2005
Abstract—We designed and synthesized hydrogen bond based probes 1–8 with the exception of known glycosidase inhibition mech-
anisms, and aglycon specificity of 11 different sources of a-glucosidases were investigated using their probes. Probe 4 (2,6-anhydro-1-
deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-D-ido-heptitol) showed a potent inhibition of S. cerevisiae a-glucosidase among
all a-glucosidases. Probe 4 was found to be a competitive inhibitor for S. cerevisiae a-glucosidase with Ki 0.13 mM.
Ó 2005 Elsevier Ltd. All rights reserved.
a-Glucosidases (EC 3.2.1.20) are also exo-acting carbo-
hydrases, catalyzing the release of a-D-glucopyranose
from the non-reducing ends of various substrates,1 and
on the basis of amino acid sequence similarities, a-gluco-
sidases are classified into two families, family 13 and
family 31.2 Endoplasmic reticulum (ER) processing a-
glucosidases, a-glucosidase I (EC 3.2.1.106), and a-glu-
cosidase II (EC 3.2.1.84), are key enzymes in the biosyn-
thesis of asparagine-linked oligosaccharides that
catalyze the first processing event after the transfer of
Glc3Man9GlcNAc2 to proteins. These enzymes are a
target for inhibition by anti-viral agents that interfere
with the formation of essential glycoproteins required
in viral assembly, secretion, and infectivity.3 Many pa-
pers reported that inhibitors of a-glucosidases are po-
tential therapeutics for the treatment of such diseases
as viral diseases, cancer, and diabetes.3a,4 However, lots
of screenings of a-glucosidase inhibitors were not used
enzymes from target tissues or organs. We think that
the substrate specificities of three kinds of a-glucosidases
(family 13, family 31, and ER processing a-glucosidases)
are different. Therefore, aglycon specificity profiling of
a-glucosidases were an important approach for the re-
search of a-glucosidase inhibitors. Chemically modified
substrates are effective methods in the study of substrate
specificity profiling. We have already applied this ap-
proach to family 13 and family 31 a-glucosidases,5–7
a-galactosidases,6,8 a-mannosidases,6,9 and ER process-
ing a-glucosidases10 using partially substituted monosac-
charide. We have reported that the presence of a C-2
hydroxyl group of glycon is not essential for the hydro-
lysis action of family 31 a-glucosidases and ER process-
ing a-glucosidase II, and family 13 a-glucosidases and
ER processing a-glucosidase I do necessarily need all
of the hydroxyl groups of the glycon for hydrolyzing
activity.
In a previous paper, we described the design and synthe-
sis of 1-amino-2,6-anhydro-1-deoxy-D-glycero-D-ido-
heptitol derivatives 1–8 as synthetic probes10 (Fig. 1).
These probes do not have the specific functional groups
for glycosidase inhibition, electrostatic interactions
(e.g., deoxynojirimycine), transition state mimetic struc-
ture (e.g., D-gluconolactone), and covalent bond forma-
tion with the enzyme catalytic site (e.g., conduritol B
Keywords: a-Glucosidase; ER processing a-glucosidase; Inhibitor;
Aglycon specificity.
*
Corresponding author at present address: Division of Organic
Chemistry, National Institute of Health Sciences (NIHS), 1-18-1
Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. Fax: +81 3 3707
6950; e-mail: hakamata@nihs.go.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.12.086

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W. Hakamata et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1489–1492
The values of % inhibition and IC50 were summarized in
Table 1. Probe 4 indicated specific inhibitions of S. cere-
visiae (IC50 = 55.5 lM) and B. stearothermophilus
(IC50 = 415 lM) a-glucosidases. Probe 7 inhibited a-glu-
cosidase from S. cerevisiae (IC50 = 449 lM). HBGase I
was inhibited by probe 1 (IC50 = 851 lM). Family 13
a-glucosidases and ER processing a-glucosidases were
inhibited by the specific probes. On the other hand, fam-
ily 31 a-glucosidases were broadly inhibited by probes
1–8. All probes did not inhibit b-glucosidase, a- and
b-mannosidases, and a- and b-galactosidases at a 5-fold
concentration. These facts indicated that aglycon speci-
ficities of a-glucosidases differed greatly among family
13 a-glucosidases, family 31 a-glucosidases, and ER
processing a-glucosidases. Moreover, each aglycon spec-
ificity of family 13 a-glucosidases is different in spite of
the highly conserved amino acid sequences in the cata-
lytic site.2f Therefore, it is better to use enzymes of target
tissues or organs for screening of agents for viral dis-
eases, cancer, and diabetes.
Figure 1. Chemical structure of probes 1–8.
epoxide). The structures of a-glucosidase inhibitors were
summarized in Figure 2. We think that with the elimina-
tion of the above functional groups from probes, the
hydrogen bond formations is expressed clearly. In this
paper, we report the inhibition of family 13 a-glucosi-
dases (Saccharomyces (S.) cerevisiae, Bacillus (B.) ste-
arothermophilus, and honeybee isozyme I, II, and III
(HBGase I, II, and III)), family 31 enzymes (rice, sugar
beet, flint corn, and Aspergillus (A.) niger), ER process-
ing a-glucosidase I and II from rat liver microsome, and
other glycosidases (almond b-glucosidase, jack beans
a-mannosidase, snail b-mannosidase, Moltierella (M.)
vinacea a-galactosidase, and jack beans b-galactosidase)
against the probes 1–8, and their aglycon specificity pro-
filing is discussed.
In the kinetic studies on the inhibitions of 4 and 7 and
hydrolysis of p-nitrophenyl a-D-glucopyranoside (PNP
a-Glc) by S. cerevisiae and B.stearothermophilus a-glu-
cosidases, the values of Ki and Km (mM) were calculated
Figure 2. Chemical structure of a-glucosidase inhibitors.
Table 1. Inhibitory activities of probes 1–8 against glycosidases11
Enzyme source
% Inhibition (IC50
)
1
2
3
4
5
6
7
8
Family 13 a-glucosidase
S. cerevisiae
<1.0
<1.0
21.1
<1.0
<1.0
<1.0
3.6
100 (55.5 lM)
100 (415 lM)
37.5
<1.0
<1.0
<1.0
4.4
<1.0
<1.0
10.4
<1.0
<1.0
67.4 (449 lM)
<1.0
4.6
6.1
B. stearothermophilus
HBGase I
HBGase II
<1.0
<1.0
2.7
<1.0
<1.0
<1.0
<1.0
52.3 (851 lM)
4.4
<1.0
21.4
<1.0
12.3
<1.0
HBGase III
3.2
<1.0
<1.0
Family 31 a-glucosidase
Rice
10.7
6.9
8.5
1.7
7.6
3.6
18.3
3.1
26.0
11.9
44.6
<1.0
21.8
8.8
16.0
9.8
3.8
3.2
5.6
1.2
Sugar beet
Flint corn
A. niger
29.1
6.6
14.1
2.6
18.5
<1.0
37.0
6.8
31.0
23.3
49.2
14.0
ER processing a-glucosidase
Glucosidase I
Glucosidase II
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
18.2
5.9
<1.0
<1.0
b-Glucosidase
a-Mannosidase
b-Mannosidase
a-Galactosidase
b-Galactosidase
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
<1.0
Probe concentrations (family 13 and 31 a-glucosidases: 1 lmol/mL, ER processing a-glucosidases: 2 lmol/mL, b-glucosidase, mannosidases, and
galactosidases:5 lmol/ml) Substrate (family 13 and 3l a-glucosidases, ER processing a-glucosidase II: PNP a-Glc, ER processing a-glucosidases I:
[3H] labeled vesicular stomatitis virus glycoprotein, b-glucosidase: PNP b-Glc, a-mannosidase: PNP a-Man, b-mannosidase: PNP b-Man,
a-galactosidase: PNP a-Gal, b-galactosidase: PNP b-Gal).

W. Hakamata et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1489–1492
1491
Figure 3. Dixon plots of the inhibition of S. cerevisiae a-glucosidase by probes 4 and 7.
from Dixon plots (Figs. 3 and 4) and Michaelis–Menten
plots, respectively, and these values and inhibition types
were summarized in Table 2. Probes 4 and 7 were com-
petitive type inhibitors of S. cerevisiae enzyme (Ki = 0.13
and 0.50 mM). Probe 4 was a mixed type inhibitor of B.
stearothermophilus enzyme (Ki = 0.58 mM). The affini-
ties of 4 against both enzymes were higher than PNP
a-Glc as substrate. These results indicated that probe 4
formed a specific hydrogen bond between the primary
hydroxyl group of aglycon moiety and S. cerevisiae en-
zyme, and that probe 7, with a terminal phenyl group,
formed a hydrophobic interaction with S. cerevisiae en-
zyme. These results were compared with other typical
a-glucosidase inhibitors. The Ki values of deoxynoji-
rimycine, castanospermine (see Fig. 2), D-gluconolac-
tone, conduritol B epoxide, and probe 4 against S.
cerevisiae a-glucosidase were 0.013 mM,17 >1.5 mM,17
2.0 mM,18 ꢀ25 mM,17 and 0.13 mM, respectively. From
this comparison, the combination of the designed hydro-
gen bond formation and the other static binding factors
(electrostatic interaction, transition state mimetic con-
formation, and covalent bond formation), it may be
possible to become a potent and specific a-glucosidase
inhibitor. Further studies using new probes (in which
the –C–O–C– group of glycon moiety is replaced by a
–C–NH–C– group) should be done to develop new po-
tent and specific a-glucosidase inhibitors. The discovery
of a-glucosidase inhibitors may help us to understand
the roles of oligosaccharides of glycoproteins and glyco-
lipids in cellular functions, and pharmaceutical
applications.
Figure 4. Dixon plot of the inhibition of B. stearothermophilus
a-glucosidase by probe 4.
Acknowledgements
This work was preformed at Special Postdoctoral
Researchers Program (RIKEN).
Table 2. Kinetic studies of the inhibition of a-glucosidases
S. cerevisiae
B. stearothermophilus
Probe
Ki (mM) Inhibition
type
Ki (mM)
Inhibition
type
References and notes
4
0.13
0.50
0.35a
Competitive
Competitive
—
0.58
—
1.16a
Mixed
—
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7
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