Enzymatic synthesis of a selective inhibitor for α-glucosidases: α-acarviosinyl-(1→9)-3-α-D-glucopyranosylpropen

Article abstract of DOI:10.1021/jf703655k

Here, we describe the enzymatic synthesis of novel inhibitors using acarviosine-glucose as a donor and 3-α-D-glucopyranosylpropen (αGP) as an acceptor. Maltogenic amylase from Thermussp. (ThMA) catalyzed the transglycosylation of the acarviosine moiety to aGP. The two major reaction products were isolated using chromatographies. Structural analyses revealed that acarviosine was transferred to either C-7 or C-9 of the αGP, which correspond to C-4 and C-6 of glucose. Both inhibited rat intestine α-glucosidase competitively but displayed a mixed-type inhibition mode against human pancreatic α-amylase. The α-acarviosinyl-(1→7)-3- α-D-glucopyranosylpropen showed weaker inhibition potency than acarbose against both α-glycosidases. In contrast, the α-acarviosinyl- (1→9)-3-α-D-glucopyranosylpropen exhibited a 3.0-fold improved inhibition potency against rat intestine α-glucosidase with 0.3-fold inhibition potency against human pancreatic α-amylase relative to acarbose. In conclusion, α-acarviosinyl-(1→9)-3-α-D- glucopyranosylpropen is a novel α-glucosidase-selective inhibitor with 10-fold enhanced selectivity toward α-glucosidase over α-amylase relative to acarbose, and it could be applied as a potent hypoglycemic agent.

Full text of DOI:10.1021/jf703655k

5324 J. Agric. Food Chem. 2008, 56, 5324–5330
Enzymatic Synthesis of a Selective Inhibitor for
r-Glucosidases:
r-Acarviosinyl-(1f9)-3-r-D-glucopyranosylpropen
YOUNG-SU LEE,† MYOUNG-HEE LEE,† HEE-SEOB LEE,‡ SEUNG-JAE LEE,†
YOUNG-WAN KIM,§ RAN ZHANG,| STEPHEN G. WITHERS,| KWAN SOO KIM,⊥
SUNG-JOON LEE,# AND KWAN-HWA PARK*,†
Center for Agricultural Biomaterials and School of Agriculture and Biotechnology, Seoul National
University, Seoul 151-921, Korea, Department of Food and Nutrition, Pusan National University,
Pusan 609-735, Korea, Department of Food and Biotechnology, Korea University, Jochiwon 339-700,
Korea, Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z4, Canada,
Department of Chemistry, College of Science, Yonsei University, Seoul 120-749, Korea, and Division
of Food Bioscience and Technology, Korea University, Seoul 136-713, Korea
Here, we describe the enzymatic synthesis of novel inhibitors using acarviosine-glucose as a donor
and 3-R-D-glucopyranosylpropen (RGP) as an acceptor. Maltogenic amylase from Thermus sp. (ThMA)
catalyzed the transglycosylation of the acarviosine moiety to RGP. The two major reaction products
were isolated using chromatographies. Structural analyses revealed that acarviosine was transferred
to either C-7 or C-9 of the RGP, which correspond to C-4 and C-6 of glucose. Both inhibited rat
intestine R-glucosidase competitively but displayed a mixed-type inhibition mode against human
pancreatic R-amylase. The R-acarviosinyl-(1f7)-3-R-D-glucopyranosylpropen showed weaker inhibition
potency than acarbose against both R-glycosidases. In contrast, the R-acarviosinyl-(1f9)-3-R-D-
glucopyranosylpropen exhibited a 3.0-fold improved inhibition potency against rat intestine R-glu-
cosidase with 0.3-fold inhibition potency against human pancreatic R-amylase relative to acarbose.
In conclusion, R-acarviosinyl-(1f9)-3-R-D-glucopyranosylpropen is a novel R-glucosidase-selective
inhibitor with 10-fold enhanced selectivity toward R-glucosidase over R-amylase relative to acarbose,
and it could be applied as a potent hypoglycemic agent.
KEYWORDS: Maltogenic amylase from Thermus sp.; acarbose; 3-r-D-glucopyranosylpropen; inhibitor;
transglycosylation
INTRODUCTION
Acarbose, a pseudotetrasaccharide, consists of acarviosine and
maltose, which are linked via an R-(1f4)-glycosidic linkage
(Figure 1), and is a well-known as a potent inhibitor of
R-glycosidases (5, 6). Although acarbose is widely used as a
therapeutic agent for diabetes (7, 8), its inhibition mechanism
is somewhat obscure. In several three-dimensional structures
of different R-amylases cocrystallized with acarbose, a sub-
stantially rearranged product, pseudopentasaccharide, was found
in the subsites -3 through +2 (9, 12). The enzymatic modifica-
tion of acarbose presumably occurs as a result of a series of
enzymatic hydrolysis, condensation, and transglycosylation
events (13). Given these enzymatic modifications of acarbose,
there have been several attempts to synthesize elongated
acarbose derivatives as strong inhibitors through in situ extension
using an R-amylase (14, 15).
The development of R-glycosidase inhibitors is of particular
interest because of their therapeutic potential in the management
of diabetes (1–3). Selective inhibition against R-glucosidases
over R-amylases is important to allow polysaccharides in the
diet to be hydrolyzed by R-amylases, after which absorption of
the resulting oligosaccharides in the small intestine follows (4).
If undigested polysaccharides enter the colon because of the
strong inhibition of R-amylases by drugs, side effects including
flatulence are possible as a consequence of bacterial fermentation
(4).
* Corresponding author. Tel: 82-2-8804852. Fax: 82-2-8735095.
E-mail: parkkh@snu.ac.kr.
† Seoul National University.
Maltogenic amylases (MAases) are members of the glycoside
hydrolase family 13, which not only hydrolyzes cylcodextrins,
starch, and pullulan but also catalyzes transglycosylation to form
R-(1f6)-glycosidic linkages as well as R-(1f4)-linkages (16).
In addition, MAases can hydrolyze acarbose to acarviosine-
‡ Pusan National University.
§ Department of Food and Biotechnology, Korea University.
| University of British Columbia.
⊥ Yonsei University.
# Division of Food Bioscience and Technology, Korea University.
10.1021/jf703655k CCC: $40.75  2008 American Chemical Society
Published on Web 06/14/2008

Synthesis of an R-Glucosidase-Selective Inhibitor
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5325
Figure 1. (A) Scheme of the transglycosylation reaction catalyzed by ThMA with acarbiosine-glucose and RGP. The carbons of RGP are numbered. (B)
Chemical structures of compounds mentioned in this study: acarbose (Acb), isoacarbose (IAcb), and simmondsin.
in methanol. Detailed synthesis of RGP will be published elsewhere.
Enzymes used in this study [ThMA (18), HPA (13), and RIG (25)]
were prepared according to procedures published in the literature.
Preparation and Structural Analysis of Transfer Products. For
the transglycosylation reaction, ThMA (20 U per mg of Acv-G) was
used with 5% (w/v) Acv-G and 10% (w/v) RGP in 50 mM sodium
acetate buffer (pH 6.0) at 50 °C. The transglycosylation reaction was
monitored by TLC. After 24 h of incubation, the products were
separated by descending paper chromatography on Whatman No. 3
MM chromatography paper (Whatman, Maidstone, UK), developed with
isopropanol/ethyl acetate/water (3:1:1, v/v/v), and detected on the paper
using the alkaline AgNO3 dipping procedure (26). Each transfer product
was eluted from the paper with deionized water and concentrated using
a Speedvac (AES1010-120, SAVANT Instrument, Farmingdale, NY
USA). Each compound was dissolved in water and subjected to
recycling preparative HPLC (LC-910, JAI Ltd., Tokyo, Japan) equipped
with a GPC column (W-251, 20 × 500 mm, JAI Ltd.). The eluent was
monitored using a RI-50 refractive index detector (JAI Ltd.). Distilled
water was used as an eluent at a flow rate of 3.0 mL/min. The chemical
structures of the purified transfer products were determined using
matrix-assisted laser desorption/ionization-time-of-flight mass spec-
trometry (MALDI-TOF MS) using a Voyager-DE system (Applied
Biosystems, Foster City, CA, USA) and 13C nuclear magnetic resonance
(NMR) recorded with a JEOL LA-400 FT-NMR spectrometer (Jeol
Ltd., Tokyo, Japan). R-Cyano-4-hydroxy-cinnamic acid (Cat. No.
C-2620, Sigma Chemicals Co., St. Louis, MO, USA) was used as a
matrix for MALDI-TOF MS.
Thin Layer Chromatography. A silica gel K5F thin layer chro-
matography plate (Whatman, Maidstone, UK) was activated by placing
it for 30 min in an oven adjusted to 110 °C. Prepared samples were
spotted on a silica gel plate and placed in a TLC chamber containing
a solvent system of isopropyl alcohol, ethyl acetate, and water at a
ratio of 3:1:0.4 (v/v/v). The TLC plate was developed twice at room
temperature. The plate was dried thoroughly and developed by dipping
into a methanol solution containing 3 g of N-(1-naphthyl)-ethylenedi-
amine and 50 mL of concentrated H2SO4 per liter. The plate was dried
and then placed in an oven set at 110 °C for 10 min; purple-black
spots appeared on a white background.
glucose (Acv-G) and glucose, and subsequently transfer the
acarviosine-glucosyl moiety to other sugar acceptors (16–18).
Using the transglycosylation activity of MAases, novel acarvi-
osine-glucosyl derivatives have been synthesized, and their
inhibition potencies against R-glycosidases have been evalua-
ted (19, 20). A maltogenic amylase from Thermus sp. (ThMA)
has been characterized extensively in terms of its catalytic
properties and mechanism using mutagenesis, kinetic analysis,
and three-dimensional structure determination (18, 21–23). Of
the catalytic functions of ThMA, hydrolysis activity of Acv-G
is of particular interest because of the accompanying transferring
activity of the resulting acarviosine to other sugar acceptors.
Previously, we synthesized a novel compound with both
antiobesity and hypoglycemic activity in ViVo through transg-
lycosylation of simmondsin, a food intake-reducing molecule
found in the seeds of the jojoba plant (24).
In this article, we describe the enzymatic synthesis of novel
acarviosinyl glycosides using Acv-G as a donor and a C-glucosyl
compound, 3-R-D-glucopyranosylpropen (RGP) as an acceptor
(Figure 1A). ThMA produced two stereoisomers containing
either an R-(1f7)- or an R-(1f9)-glycosidic linkage between
acarviosine and RGP. We investigated inhibition potencies
against human pancreatic R-amylase (HPA) and rat intestine
R-glucosidase (RIG) relative to acarbose. Conclusively, the
R-(1f9)-linked transfer product showed a highly enhanced
selectivity to RIG over HPA compared to acarbose.
MATERIALS AND METHODS
Reagents and Enzymes. Acarbose was extracted from Glucobay
tablets and was purified through gel permeation chromatography (GPC)
using a Bio-Gel P2 column (1 × 90 cm). In order to prepare Acv-G,
5% (w/v) acarbose in 50 mM sodium acetate buffer (pH 6.0) was
incubated with 10 units of ThMA per milligram of acarbose for 24 h
at 60 °C. The products were purified using GPC. Acarbose and Acv-G
from GPC were desalted by preparative high performance liquid
chromatography (HPLC, Waters, USA), and their purities were
confirmed by high performance anion-exchange chromatography
(HPAEC) analysis and thin layer chromatography (TLC). RGP was
achieved via replacement of the trichloroimidate group of per-O-
benzoyl-R-D-glycosyltrichloroimidate with a propenyl group using
allyltrimethylsilane followed by deprotection using sodium methoxide
HPAEC Analysis. The reaction mixture treated by ThMA was boiled
for 10 min and centrifuged at 12,000 × g for 10 min and filtered using
a membrane filter kit (0.45 µm, Millipore). The products were analyzed
by HPAEC with a pulsed amperometric detector (ED40, Sunnyvale,
CA, DIONEX). A CarboPac PA-1 column (0.4 × 25 cm, DIONEX)
was used. A sample (200 µL) was injected and eluted with a gradient

5326 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Lee et al.
Scheme 1
of 1 M sodium acetate (0-2 mM; 2-37 min, increased from 50 mM
to 350 mM, and 37-45 min, increased from 350 mM to 850 mM) in
150 mM NaOH with a flow rate of 1 mL/min.
Enzymatic Analysis of the Structures of Transfer Products. The
pure transfer products [final 0.01% (w/v)] were incubated with either
isoamylase (final 1 unit/mL, Hayashibara Biochemical Laboratories Inc.,
Okayama, Japan) in 50 mM sodium acetate buffer (pH 5.0) at 30 °C
or ꢀ-amylase (final 10 mg/mL, Cat. No. A7005, Sigma-Aldrich Co.,
St. Louis, MO, USA) in 50 mM sodium acetate (pH 4.5) at 50 °C. The
reactions were carried out 72 h, and the reaction mixtures were analyzed
by HPAEC.
Assay of Rat Intestine r-Glucosidase Activity. Assay of R-glu-
cosidase activity was carried out at 37 °C according to the glucose
oxidase/peroxidase method as described previously (19). The glucose-E
kit (Youngdong Pharmaceutical Co., Yongin, Korea) was used accord-
ing to a manual supplied by the manufacturer. Maltose (Sigma-Aldrich
Co.) was used as a substrate, and the amount of glucose in the sample
was calculated using a glucose calibration curve. One unit (U) of
enzyme activity was defined as the amount of enzyme that splits 1
µmol of maltose in 1 min.
Assay of Human Pancreatic r-Amylase Activity. All reactions
for HPA were carried out in 50 mM sodium phosphate buffer (pH 7.0)
containing 6 mM NaCl at 37 °C. Soluble starch (Showa Chemical Co.,
Tokyo, Japan) was used as a substrate, and the amount of reducing
sugars produced by HPA was measured using the copper-bicinchoninate
reducing-value method (27).
Enzyme Inhibition Kinetics of Acarbose and Its Derivatives. The
assays for RIG and HPA were conducted in the presence of the proper
concentration of inhibitors. The inhibition types of the inhibitors were
determined using Lineweaver-Burk plots (28), and kinetic parameters
such as Vmax, Km, Ki, and Ki′ were evaluated by fitting data into the
following equation using the SigmaPlot program (SPSS Inc., Chicago,
IL, USA):
Figure 2. Thin layer chromatography analysis of the reaction catalyzed
by ThMA. Lane 1: maltodextrin standards (G1-G5); lane 2: acarbose
standard [Acv-G, acarbose (Acb), and isoacarbose (IAcb)]; lane 3: before
reaction; lane 4: after reaction; lane 5: isolated compound 2; lane 6:
isolated compound 1.
determine their molecular masses. The peak appeared at m/z
530, which corresponds to the molecular mass of a sodium
adduct of R-D-acarviosinylglucopyranosylpropen (Acv-RGP,
calculated molecular mass ) 507 Da). The mass spectrum also
suggested that compound 2 is an isomer with the same molecular
mass as that of compound 1. Given the regioselectivity of
transglycosylation by ThMA and other MAases (16–18), the
expected chemical structures of these compounds were either R-
(1f7)-linked Acv-RGP (a compound corresponding to R-(1f4)-
linked glycoside transfer products by ThMA) or R-(1f9)-linked
Acv-RGP (a compound corresponding to R-(1f6)-linked gly-
coside transfer products by ThMA).
13C NMR analyses of the isolated transfer products were
carried out, and chemical shifts of Acv-RGPs were compared
to those of RGP and Acv-G in the 13C NMR spectrum (Table
1). The chemical shifts of Acv-G were identified using the
previous studies (20, 24). In the case of compound 1, there was
a large chemical shift from 74.1 to 79.5 ppm of C-7 of RGP
(ring C in Table 1), indicating that the acarviosine group was
linked to C-7 of RGP. From these results, the chemical structure
of compound 1 was defined as R-acarviosinyl-(1f7)-3-R-D-
glucopyranosylpropen (Acv-GP1). As shown in Table 1, there
was a large chemical shift of C-9 of RGP from 61.8 to 69.6
ppm, suggesting that the acarviosine group was linked to C-9
of RGP. From these results, the chemical structure of compound
2 was determined as R-acarviosinyl-(1f9)-3-R-D-glucopyra-
nosylpropen (Acv-GP2). The overall reaction mechanism of
transglycosylation by ThMA in this study is summarized in
Figure 1A.
In order to confirm our NMR analysis, enzymatic hydrolysis
of two transfer products was carried out using ꢀ-amylase and
isoamylase, and the reaction mixtures were analyzed by using
HPAEC. The preliminary hydrolysis test of acarbose using these
enzymes, ꢀ-amylase was able to hydrolyze acarbose, resulting
in the production of acarviosine and maltose (Supporting
Information). However, isoamylase, which can hydrolyze only
R-(1f6)-glycosidic linkages in amylopectin or glycogen, did
not hydrolyze acarbose. Acv-GP1 was hydrolyzed by only
ꢀ-amylase into acarviosine and RGP, but isoamlyase did not
cleave Acv-GP2 (Supporting Information). The results of
HPAEC analysis, therefore, revealed that the acarviosine residue
of Acv-GP1 is linked to C-7 of RGP, corresponding to C-4 of
Vmax × S
υi )
(1)
I
Ki
I
Ki′
Km 1 +
+ S 1 +
(
)
(
)
where Vi is the initial velocity in the absence and presence of the
inhibitor; S and I are the concentration of substrate and inhibitor,
respectively; Vmax is the maximum velocity, Km is the Michaelis-Menten
constant, Ki is the inhibitor constant, defined as [E][I]/[EI] in Scheme
1, and Ki′ corresponds to the inhibition constant defined as [ES][I]/
[ESI].
RESULTS AND DISCUSSION
Preparation of Transfer Products. Acarviosine is unstable
and rearranges itself to form a tricyclic compound (5). After
self-rearrangement of acarviosine, it was not detectable by the
general carbohydrate visualization methods using the N-(1-
napthyl) ethylenediamine-sulfuric acid reagent or the sulfuric
acid procedure. However, we could monitor the reaction
progress using the production of glucose by ThMA (Figure 2,
lanes 3 and 4). The transglycosylation reaction yielded two
transfer products (product 1 and 2) with less than 10% yields
based on the TLC result. These compounds were purified
homogeneously by paper chromatography and recycling pre-
parative-HPLC (Figure 2, lanes 5 and 6).
Structural Analysis of Transfer Products. The purified
compound 1 and 2 were analyzed by MALDI-TOF MS to

Synthesis of an R-Glucosidase-Selective Inhibitor
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5327
Table 1. 13C NMR Chemical Shifts of Acarbose, RGP, Acv-GP1, and Acv-GP2
(unit: ppm)
Acv-GP2d
Acv-GP1c
chemical shift
carbon atoms
ring A
acarbosea
RGPb
∆δ
chemical shift
∆δ
1
2
3
4
5
6
7
56.0
123.8
139.0
71.2
74.0
72.7
56.1
123.9
139.0
70.9
74.4
72.7
+0.1
+0.1
0
+0.3
0
56.0
123.8
139.0
71.0
73.5
71.7
0
0
0
-0.2
-0.5
-1.0
-1.2
0
61.6
60.8
-0.8
60.4
ring B
1
2
3
4
5
6
99.8
70.8
73.0
65.0
69.6
17.3
99.8
70.8
73.0
65.0
69.7
17.4
0
0
0
0
97.7
70.8
72.5
65.2
70.0
17.3
-2.1
0
-0.5
+0.2
+0.4
0
+0.1
+0.1
ring C
1
2
3
4
5
6
7
8
9
118.3
135.5
29.7
73.3
76.2
71.1
74.1
72.0
61.8
117.6
134.6
29.5
73.0
77.7
71.2
79.5
71.8
61.7
-0.7
+0.1
-0.2
-0.3
+1.5
+0.1
+5.4
-0.2
-0.1
117.6
134.6
28.7
73.0
75.6
71.2
74.2
71.0
69.6
-0.7
--0.9
-1.0
-0.3
-0.6
+0.1
+0.1
+1.0
+7.8
b
a Acarbose, 13C NMR data of acarviosine residue of acarbose in this table. RGP, 3-R-D-glucopyranosylpropen. c Acv-GP1, d Acv-GP2.
Table 2. Analysis of Inhibition against Α-Amylase and Α-Glucosidase by Acarbose and Transfer Productsa
c
Kib (µM)
Ki′ (µM)
enzyme
HPAe
substrate
starch
inhibitors
type of inhibition
inhibitor potencyd
acarbose
Acv-GP1
Acv-GP2
mixed
mixed
mixed
2.3
2.6
6.7
5.0
8.4
77
1.0
0.9
0.3
RIGf
maltose
acarbose
Acv-GP1
Acv-GP2
competitive
competitive
competitive
0.3
0.6
0.1
1.0
0.5
3.0
a Error ranges in this table are 5-10%. b Ki is the inhibition constant, defined as [E][I]/[EI]. c Ki′ is the inhibition constant, defined as [EI][S]/[ESI]. d Inhibitor potencies
for R-glucosidase and R-amylase are calculated on the basis of assigning the value of acarbose as 1 and dividing the Ki value of acarbose by the Ki value of the inhibitor.
e HPA, human pancreatic R-amylase. f RIG, rat intestine R-glucosidase.
glucose (Figure 1). By contrast, isoamylase only hydrolyzed
Acv-GP2 into acarviosine and RGP, but Acv-GP1 was not
hydrolyzed (Supporting Information). The isoamylase treatment
of the transfer products allowed us to confirm that Acv-GP2
has a R-(1f9)-glycosidic linkage between acarviosine and RGP,
which corresponds to the R-(1f6)-glycosidic linkage of amy-
lopectin and glycogen.
Inhibition of Acarbose and Its Derivatives against HPA.
The inhibitory activities of acarbose and two derivatives
(Acv-GP1 and Acv-GP2) against HPA were investigated
(Table 2). All inhibitors showed mixed type inhibition
behaviors against HPA, and this result is consistent with our
previous studies of other acarbose derivatives against porcine
pancreatic R-amylase (19, 24). The values of Ki of acarbose,
Acv-GP1, and Acv-GP2 were determined to be 2.3, 2.6, and
6.7 µM, respectively (Table 2). Acv-GP1, which differs from
the parent molecule (Acv-G) only in the group at the
anomeric center of the reducing end, where a propenyl group
replaces a hydroxyl group (Figure 1), showed inhibition
potency (0.9-fold of acarbose inhibition) very similar to that
of Acv-G (1.2-fold of acarbose inhibition; from ref 19). In
contrast, the counterpart (Acv-GP2) exhibited relatively low
inhibition potency (0.3-fold of acarbose inhibition).
Acarbose is rearranged by HPA, and the resulting pseudo-
pentasaccharide is found in the active site of HPA (13).
However, isoacarbose, whose glucose moiety at the reducing
end is linked to Acv-G via an R-(1f6)-glycosidic linkage
(Figure 1B), is resistant to the enzymatic rearrangement (14).
Likewise, Acv-G and Acv-GP1, with inhibition potency
similar to that of acarbose, would bind to HPA at subsites

5328 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Lee et al.
Figure 3. Electrostatic potential of the surface of HPA active site with acarviosine-glucose. Part B represents the model structure in A rotated
in a counterclockwise direction by 90°. A coordinate for the structures used in this figure was 1XCW (14) obtained from the Protein Data Bank.
Acv-G (yellow color for carbon atoms) and isomaltose (IM, gray color for carbon atoms) in the active site are presented as sticks. The ring of
Acv-G bound in the +1 subsite was used as the basis for superposition with the glucose ring of IM. A coordinate for the structures of IM was
obtained from isoacarbose bound in HPA [PDB file 1XCX (14)]. The distances between the amino acid residues of HPA and Acv-G are indicated
with yellow dashed lines in A. The distances between amino acid residues of HPA and IM are represented as white dashed lines in A. The
subsites -1 through +2 of HPA are indicated in the B. The figure was made using PyMol v.0.99.
-1 through +2 without the enzymatic rearrangement. Acv-
GP2, which is also expected to bind to HPA without
rearrangement, showed lower inhibition potency than other
R-(1f4)-linked pseudotrisaccharides (Acv-G and Acv-GP1).
Therefore, these inhibition patterns of RGPs are consistent
with the investigation of acarviosine-R-(1f6)-simmondsin
(Acv-6-Sim) by Baek et al. (24) and suggest that subsites
+1 and +2 of HPA are oriented for the binding of linear
R-(1f4)-linked maltooligosaccharides or their derivatives
rather than R-(1f6)-linked counterparts.
To investigate the specificity and mechanism of inhibition
by acarviosine derivatives, a model structure of HPA with
an isomaltose (IM) bound in subsites +1 and +2 was
established using the enzyme-inhibitor complex of HPA with
Acv-G (14). Upon using the ring of Acv-G (yellow) bound
in the +1 subsite as the basis for superposition with the
nonreducing end-glucose ring of IM (gray), it is clear that
O-3′ of the glucose residue of IM in the +2 subsite would
clash with Tyr151 (2.5 Å away from O-3 of the glucose
residue of IM, Figure 3). In addition, hydrophobic interaction
between Ile235 of HPA and the glucose ring at the reducing
end of Acv-G would be removed because of the distortion
of the sugar chain by the R-(1f6)-glycosidic linkages
compared to Acv-G. Upon binding of Acv-GP2, the RGP
residue is expected to locate at the position of the reducing
end of IM. Therefore, the architecture of the active site of
HPA around the +2 subsite interacting with the glucose
residue of Acv-G may not allow proper binding of the RGP
residue of Acv-GP2 in the +2 subsite of HPA.
Inhibition of Acarbose and Its Derivatives against RIG.
Next, we explored the inhibition potencies of the inhibitors against
RIG. Lineweaver-Burk plots for inhibition against RIG clearly
show that their inhibition modes are competitive. As in the case
of HPA, Acv-GP1 showed inhibition potency against RIG similar
to that of Acv-G; the Ki value of Acv-GP1 was 0.6 µM,
corresponding to 0.5-fold inhibition by acarbose (Table 2). Acv-G
showed inhibition 0.4-fold of that of acarbose (19). Interestingly,
a value of 0.1 µM was determined for the Ki value of Acv-GP2,
which translates to 6-fold and 3-fold enhanced inhibition potency
relative to that of Acv-GP1 and acarbose, respectively (Table 2).
This result suggests that R-(1f9)-linked RGP of Acv-GP2 may
lead to a tighter binding of Acv-GP2 to the active site of RIG.
Overall, the selectivity of Acv-GP2 against R-glucosidase over
R-amylase is enhanced 10-fold relative to that of acarbose.
Very recently, the three-dimensional structure of the
N-terminal catalytic subunit of human intestine maltase-
glucoamylase (NtMGAM) was solved (29). In the complex
structure of the human enzyme with acarbose, acarviosine
clearly binds in the -1 and +1 subsites, but there was no
evidence for the presence of +2 and +3 subsites for binding
of the maltose residue, which is linked via linear R-(1f4)-
glycosidic linkages (29). The 3-D structural information has
not been released yet, but we are expecting that the structure
of RIG and the binding mode of the inhibitor in the active
site of RIG may be similar to those of NtMGAM. Therefore,
the structural information on NtMGAM and the inhibition
kinetics of Acv-GP2 in this study allow us to anticipate that
the acarviosine residue of Acv-GP2 binds in the -1 and +1
subsites of RIG, and additional interactions would be possible
between the enzyme and RGP of Acv-GP2 that were not
present in the complex structure, NtMGAM:acarbose. How-
ever, Acv-6-Sim, which is another R-(1f6)-linked acarvi-
osinyl glycoside, has worse inhibition potency than that of
Acv-G (24). Probably, steric hindrances caused by a ꢀ-linked
bulky 2-(cyanomethylene)-3-hydroxy-4,5-dimethoxycyclo-
hexyl group in simmondsin (see Figure 1B) are responsible
for weakening the affinity of Acv-6-Sim to RIG. The smaller
and R-glycosidic-linked propenyl group in RGP would reduce
such steric hindrances in the Acv-6-Sim case.
CONCLUSIONS
Here, we synthesized two novel Acv-G derivatives through
enzymatic transglycosylation catalyzed by ThMA using
Acv-G as a donor and RGP as an acceptor. Of these products,
Acv-GP2 with a structure corresponding to the R-(1f6)-
linked transfer product showed a 10-fold enhanced selectivity
toward R-glucosidase over R-amylase compared to acarbose.
On the basis of the model structure of the HPA-inhibitor
complex, Acv-GP2 seems to be unfitted into the active site
of HPA, resulting in a lower inhibition potency than those
of acarbose and Acv-GP1. A higher R-glucosidase inhibition
potency of Acv-GP2 than those of acarbose and Acv-GP1
implies that additional interaction would be generated by the
binding of Acv-GP2 in the active site of RIG. Conclusively,
this R-glucosidase-specific inhibition by Acv-GP2 would not
only allow sufficient hydrolysis of carbohydrate in the diet
by R-amylases but also decrease blood glucose by strong
inhibition against R-glucosidases.

Synthesis of an R-Glucosidase-Selective Inhibitor
J. Agric. Food Chem., Vol. 56, No. 13, 2008 5329
ABBREVIATIONS USED
chem. 1996, 238, 561–569.
(12) Qian, M. N. V.; Bonicel, J.; Bischoff, H.; Henrissat, B.; Payan,
F. Enzyme-catalyzed condensation reaction in a mammalian
R-amylase. High-resolution structural analysis of an enzyme-
inhibitor complex. Biochemistry 2001, 40, 7700–7709.
(13) Brayer, G. D.; Sidhu, G.; Maurus, R.; Rydberg, E. H.; Braun, C.;
Wang, Y.; Nguyen, N. T.; Overall, C. M.; Withers, S. G. Subsite
mapping of the human pancreatic R-amylase active site through
structural, kinetic, and mutagenesis techniques. Biochemistry 2000,
39, 4778–4791.
(14) Li, C.; Begum, A.; Numao, S.; Park, K. H.; Withers, S. G.; Brayer,
G. D. Acarbose rearrangement mechanism by the kinetic and
structural analysis of human pancreatic R-amylase in complex with
analogues and their elongated counterparts. Biochemistry 2005,
44, 3347–4457.
ThMA, maltogenic amylase from Thermus sp.; MAases,
maltogenic amylases; HPA, human pancreatic R-amylase;
RIG, rat intestine R-glucosidase; GPC, gel permeation
chromatography; HPLC, high performance liquid chroma-
tography; HPAEC, high performance anion-exchange chro-
matography; TLC, thin layer chromatography; MALDI-TOF
MS, matrix-assisted laser desorption/ionization-time-of-flight
mass spectrometry; NMR, nuclear magnetic resonance; Acv-
G, acavosine-glucose; RGP, 3-R-D-glucopyranosylpropen;
Acv-RGP, R-D-acarviosinylglucopyranosylpropen; Acv-6-
Sim, acarviosine-R-(1f6)-simmondsin; Acv-GP1, R-acarvi-
osinyl-(1f7)-3-R-D-glucopyranosylpropen; Acv-GP2, R-ac-
arviosinyl-(1f9)-3-R-D-glucopyranosylpropen; IM, isomaltose;
NtMGAM, N-terminal catalytic subunit of human intestine
maltase-glucoamylase.
(15) Numao, S.; Damager, I.; Li, C.; Wrodnigg, T. M.; Begum, A.;
Overall, C. M.; Brayer, G. D.; Withers, S. G. In situ extension as
an approach for identifying novel R-amylase inhibitors. J. Biol.
Chem. 2004, 279, 48282–48291.
Supporting Information Available: HPAED chromatograms
of the reaction mixtures of acarbose by ꢀ-amylase and
isoamylase, HPAED chromatograms of the reaction mixtures
of Acv-PG1 by ꢀ-amylase and isoamylase, HPAED chro-
matograms of the reaction mixtures of Acv-GP2 by R-amy-
lase and isoamylase, Lineweaver-Burk plots of the reactions
of human pancreatic R-amylase inhibition in the presence of
acarbose, Acv-GP1, and Acv-GP2, and Lineweaver-Burk
plots of the reactions of rat intestinal R-glucosidase inhibition
in the presence of acarbose, Acv-GP1, and Acv-GP2. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(16) Park, K. H.; Kim, T. J.; Cheong, T. K.; Kim, J. W.; Oh, B. H.;
Svensson, B. Structure, specificity and function of cyclomalto-
dextrinase, a multispecific enzyme of the R-amylase family.
Biochim. Biophys. Acta 2000, 1478, 165–185.
(17) Cha, H. J.; Yoon, H. G.; Kim, Y. W.; Lee, H. S.; Kim, J. W.;
Kweon, K. S.; Oh, B. H.; Park, K. H. Molecular and enzymatic
characterization of a maltogenic amylase that hydrolyzes and
transglycosylates acarbose. Eur. J. Biochem. 1998, 253, 251–
262.
(18) Kim, T. J.; Kim, M. J.; Kim, B. C.; Kim, J. C.; Cheong, T. K.;
Kim, J. W.; Park, K. H. Modes of action of acarbose hydrolysis
and transglycosylation catalyzed by a thermostable maltogenic
amylase, the gene for which was cloned from a Thermus strain.
Appl. EnViron. Microbiol. 1999, 65, 1644–1651.
LITERATURE CITED
(19) Kim, M. J.; Lee, S. B.; Lee, H. S.; Baek, J. S.; Kim, D.; Moon,
T. W.; Robyt, J. F.; Park, K. H. Comparative study of the
inhibition of R-glucosidase, R-amylase, and cyclodextrin glu-
canosyltransferase by acarbose, isoacarbose, and acarviosine-
glucose. Arch. Biochem. Biophys. 1999, 317, 277–283.
(20) Bae, H. K.; Lee, S. B.; Park, C. S.; Shim, J. H.; Lee, H. Y.; Kim,
M. J.; Baek, J. S.; Roh, H. J.; Choi, J. H.; Choe, E. O.; Ahn,
D. U.; Park, K. H. Modification of ascorbic acid using transgly-
cosylation activity of Bacillus stearothermophilus maltogenic
amylase to enhance its oxidative stability. J. Agric. Food Chem.
2002, 50 (11), 3309–3326.
(21) Kim, J. S.; Cha, S. S.; Kim, H. J.; Kim, T. J.; Ha, N. C.; Oh,
S. T.; Cho, H. S.; Cho, M. J.; Kim, M. J.; Lee, H. S.; Kim, J. W.;
Choi, K. Y.; Park, K. H.; Oh, B. H. Crystal structure of a
maltogenic amylase provides insights into a catalytic versatility.
J. Biol. Chem. 1999, 274, 26279–26286.
(22) Kim, T. J.; Park, C. S.; Cho, H. Y.; Cha, S. S.; Kim, J. S.; Lee,
S. B.; Moon, T. W.; Kim, J. W.; Oh, B. H.; Park, K. H. Role of
the glutamate 332 residue in the transglycosylation activity of
Thermus maltogenic amylase. Biochemistry 2000, 39, 6773–6780.
(23) Kim, T. J.; Nguyen, V. D.; Lee, H. S.; Kim, M. J.; Cho, H. Y.;
Kim, Y. W.; Moon, T. W.; Park, C. S.; Kim, J. W.; Oh, B. H.;
Lee, S. B.; Svensson, B.; Park, K. H. Modulation of the
multisubstrate specificity of Thermus maltogenic amylase by
truncation of the N-terminal domain and by a salt-induced shift
of the monomer/dimer equilibrium. Biochemistry 2001, 40, 14182–
14190.
(24) Baek, J. S.; Kim, H. Y.; Abbott, T. P.; Moon, T. W.; Lee, S. B.;
Park, C. S.; Park, K. H. Acarviosine-simmondsin, a novel
compound obtained from acarviosine-glucose and simmondsin by
Thermus maltogenic amylase and its in vivo effect on food intake
and hyperglycemia. Biosci. Biotechnol. Biochem. 2003, 67, 532–
539.
(1) Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J. Sugar-
mimic glycosidase inhibitors: natural occurrence, biological
activity and prospects for therapeutic application. Tetrahedron:
Asymmetry 2000, 11, 1645–1680.
(2) Bols, M. 1-aza sugars, apparent transition state analogues of
equatorial glycoside formation/cleavage. Acc. Chem. Res. 1998,
31, 1–8.
(3) Winchester, B.; Fleet, G. W. J. Amino-sugar glycosidase inhibitors:
Versatile tools for glycobiologists. Glycobiology 1992, 2, 199–
210.
(4) Bischoff, H. The mechanism of R-glucosidase inhibition in the
management of diabetes. Clin. InVest. Med. 1995, 18, 303–311.
(5) Junge, B.; Boshagen, H.; Stoltefu, J.; Mu¨ller, L. DeriVatiVes of
Acarbose and Their Inhibitory Effects on R-Glucosidase; Verlag
Chemie: Weinheim, Germany, 1981.
(6) Truscheit, E.; Frommer, W.; Junge, B.; Mu¨ller, L.; Schmidt, D. D.;
Wingender, W. Chemistry and biochemistry of microbial R-glu-
cosidase inhibitors. Angrew. Chem., Int. Ed. Engl. 1981, 20, 774–
761.
(7) Chiasson, J. L.; Josse, R. G.; Gomis, R.; Hanefeld, M.; Karasik,
A.; Laakso, M. Acarbose for prevention of type 2 diabetes
mellitus: the STOPNIDDM randomised trial. Lancet 2002, 359,
2072–2077.
(8) Balfour JA, M. D. Acarbose. An update of its pharmacology and
therapeutic use in diabetes mellitus. Drugs 1993, 46, 1025–1054.
(9) Brzozowski, A. M.; Davies, G. J. Structure of the Aspergillus
oryzae R-amylase complexed with the inhibitor acarbose at 2.0
Å resolution. Biochemistry 1997, 36, 10837–10845.
(10) Strokopytov, B.; Penninga, D.; Rozeboom, H. J.; Kalk, K. H.;
Dijkhuizen, L.; Dijkstra, B. W. X-ray structure of cyclodextrin
glycosyltransferase complexed with acarbose. Implications for the
catalytic mechanism of glycosidases. Biochemistry 1997, 34,
2234–2240.
(25) Rhinehart, B. L.; Robinson, K. M.; Liu, P. S.; Payne, A. J.;
Wheatley, M. E.; Wagener, S. R. Inhibition of intestinal disac-
charidases and suppression of blood glucose by a new R-gluco-
(11) Gilles, C.; Astier, J. P.; Marchis-Mouren, G.; Cambillau, C.; Payan,
F. Crystal structure of pig pancreatic R-amylase isoenzyme II, in
complex with the carbohydrate inhibitor acarbose. Eur. J. Bio-

5330 J. Agric. Food Chem., Vol. 56, No. 13, 2008
Lee et al.
hydrolase inhibitor. J. Pharmacol. Exp. Ther. 1987, 241, 915–
920.
(26) Han, N. S.; Robyt, J. F. Separation and detection of sugars and
alditols on thin layer chromatograms. Carbohydr. Res. 1998, 313,
135–137.
(29) Sim, L.; Quezada-Calvillo, R.; Sterchi, E. E.; Nichols, B. L.; Rose,
D. R. Human intestinal maltase-glucoamylase: Crystal structure
of the N-terminal catalytic subunit and basis of inhibition and
substrate specificity. J. Mol. Biol. 2008, 375, 782–792.
(27) Fox, J. D.; Robyt, J. F. Miniaturization of three carbohydrate
analyses using a microsample plate reader. Anal. Bbiochem. 1991,
195, 93–96.
(28) Segel, I. H., Simple Inhibition Systems in Enzyme Kinetics:
BehaVior and Analysis of Rapid Equilibrium and Steady-State
Enzyme System; Wiley: New York, 1975..
Received for review December 16, 2007. Revised manuscript received
March 17, 2008. Accepted April 24, 2008. This work was supported by
a grant from the Korea Health 21 R&D project, Ministry of Health
and Welfare, Republic of Korea (AD050376).
JF703655K