Recognition properties of processing α-glucosidase I and α-glucosidase II

Article abstract of DOI:10.1081/CAR-120030022

All four possible monodeoxy derivatives of p-nitrophenyl α-D-glucopyranoside (PNP Glc) and 1-amino-2,6-anhydro-1-deoxy-D-glycero-D- ido-heptitol derivatives were prepared and used as substrates and inhibitors of rat liver processing α-glucosidases. α-Glucosidase II hydrolyzed the 2-deoxy derivative of PNP Glc (1); the hydrolysis of 1 was more rapid than that of PNP Glc. These results indicate that the presence of a C-2 hydroxyl group is not essential for the action of α-glucosidase II. In contrast, PNP Glc and all of the deoxy derivatives of PNP Glc 1-4 inhibited α-glucosidase I. These results indicate that α-glucosidase I does not necessarily need all of the hydroxyl groups of the glycon moiety for binding to the enzyme. 2,6-Anhydro-1-benzamide-D-glycero-D-ido-heptitol (11), with a terminal phenyl group, inhibited α-glucosidase I and α-glucosidase II. Both α-glucosidase I and II showed the same aglycon specificities. When probes 5-12 were assayed for their ability to inhibit processing by α-glucosidases at the cellular level, no effects on glycoprotein processing were observed.

Full text of DOI:10.1081/CAR-120030022

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Recognition Properties of Processing α‐Glucosidase I
and α‐Glucosidase II
Wataru Hakamata a c , Makoto Muroi a , Toshiyuki Nishio b , Tadatake Oku b & Akira
Takatsuki a
a Animal and Cellular Systems Laboratory , The Institute of Physical and Chemical
Research (RIKEN) , Saitama, Japan
b Department of Biological Chemistry, College of Bioresource Sciences , Nihon
University , Kanagawa, Japan
c Division of Organic Chemistry , National Institute of Health Sciences , 1‐18‐1 Kamiyoga,
Setagaya‐ku, Tokyo, 158‐8501, Japan
Published online: 18 Aug 2006.
To cite this article: Wataru Hakamata , Makoto Muroi , Toshiyuki Nishio , Tadatake Oku & Akira Takatsuki (2004)
Recognition Properties of Processing α‐Glucosidase I and α‐Glucosidase II, Journal of Carbohydrate Chemistry, 23:1, 27-39,
DOI: 10.1081/CAR-120030022
To link to this article: http://dx.doi.org/10.1081/CAR-120030022
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 23, No. 1, pp. 27–39, 2004
Recognition Properties of Processing a-Glucosidase I
and a-Glucosidase II
1
2
Wataru Hakamata,1, Makoto Muroi, Toshiyuki Nishio,
*
Tadatake Oku,2 and Akira Takatsuki1
1Animal and Cellular Systems Laboratory, The Institute of Physical and
Chemical Research (RIKEN), Saitama, Japan
2Department of Biological Chemistry, College of Bioresource Sciences,
Nihon University, Kanagawa, Japan
ABSTRACT
All four possible monodeoxy derivatives of p-nitrophenyl a-D-glucopyranoside (PNP
Glc) and 1-amino-2,6-anhydro-1-deoxy-D-glycero-D-ido-heptitol derivatives were
prepared and used as substrates and inhibitors of rat liver processing a-glucosidases.
a-Glucosidase II hydrolyzed the 2-deoxy derivative of PNP Glc (1); the hydrolysis of 1
was more rapid than that of PNP Glc. These results indicate that the presence of a C-2
hydroxyl group is not essential for the action of a-glucosidase II. In contrast, PNP Glc
and all of the deoxy derivatives of PNP Glc 1–4 inhibited a-glucosidase I. These results
indicate that a-glucosidase I does not necessarily need all of the hydroxyl groups of the
glycon moiety for binding to the enzyme. 2,6-Anhydro-1-benzamide-D-glycero-D-ido-
heptitol (11), with a terminal phenyl group, inhibited a-glucosidase I and a-glucosidase
II. Both a-glucosidase I and II showed the same aglycon specificities. When probes
5–12 were assayed for their ability to inhibit processing by a-glucosidases at the
cellular level, no effects on glycoprotein processing were observed.
Key Words: Processing glucosidase; Inhibitor; Substrate specificity; Deoxy glucoside.
*
Correspondence: Wataru Hakamata, Division of Organic Chemistry, National Institute of Health
Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; Fax: 81-3-3707-6950; E-mail:
hakamata@nihs.go.jp.
27
DOI: 10.1081/CAR-120030022
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

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Hakamata et al.
INTRODUCTION
Endoplasmic reticulum processing a-glucosidases are key enzymes in the
biosynthesis 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.[1–3] Substrate specificities are used
to categorize enzymes and are important in designing structure-based inhibitors. We
previously elucidated the glycon specificities of a-glucosidases (EC 3.2.1.20),[4–6]
a-galactosidases (EC 3.2.1.22),[5,7] and a-mannosidases (EC 3.2.1.24)[5,8] against all
possible monodeoxy and monomethyl derivatives of the corresponding p-nitrophenyl
a-D-glycopyranosides, and confirmed that some of them were good substrates for the
enzymes. Next, we planned to investigate the glycon and aglycon specificities of
the processing a-glucosidases I (EC 3.2.1.106), and a-glucosidases II (EC 3.2.1.84), in
rat liver microsomes using synthetic probes 1–12 (Fig. 1). Four probes 1–4 were
synthesized previously.[4,7,8] Thus, these studies required the efficient synthesis of
probes 5–12.
These latter eight probes included 1-amino-2,6-anhydro-1-deoxy-D-glycero-D-ido-
heptitol, which might mimic to a great extent the topography of a-D-glucopyranoside and
modified aglycon of a-glucopyranoside.
In this report, we first describe the synthesis of probes 5–12 with modified
aglycons. These compounds were evaluated with regard to their ability to inhibit
processing a-glucosidase I and a-glucosidase II. All of the monodeoxy analogs of
p-nitrophenyl a-D-glucopyranoside (PNP Glc) 1–4, available from previous studies,
were evaluated as substrates and inhibitors of both enzymes. Finally, probes 5–12 were
also tested in a cell culture system. We divided the recognition properties of a-
glucosidase I and a-glucosidase II into glycon-specific and aglycon-specific and discuss
our findings.
Figure 1. Chemical structure of probes 1–12.

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Processing a-Glucosidase I and II
29
RESULTS AND DISCUSSION
Synthesis of Heptitol Derivatives
The synthesis of the 1-amino-2,6-anhydro-1-deoxy-D-glycero-D-ido-heptitol deriva-
tives (5–12) that were used in the present study is presented in Sch. 1. 2,3,4,6-Tetra-
O-benzyl-a-D-glucopyranosyl cyanide (13)[9] was used as a starting material for
thesynthesis of 5–12. Compound 14[9] was coupled with acetoxyacetic acid, 3-hydroxy-
propionic acid, 4-hydroxybutyric acid, glutaric acid monomethyl ester, and adipic acid
monomethyl ester to give 15, 17–19, and 21, respectively.[10] Compound 15 was treated
with a base to afford 16. Compounds 19 and 21 were treated with lithium aluminum
hydride (LAH) in tetrahydrofuran (THF) to give 20 and 22, respectively. Compound 14
was acylated with acetic anhydride, benzoyl chloride, and pivaloyl chloride in pyridine to
give 23–25 in good yield, respectively. Treatment of the resulting amides (16–18, 20, and
22–25) with palladium hydroxide on carbon gave probes 5–12 in good yields. To the best
of our knowledge, there have been no previous reports on the synthesis of probes 5–12.
Specificities of Processing a-Glucosidases
D. M. Burns et al. have been reported that rat liver a-glucosidase II showed hydrolytic
activity against PNP Glc.[11] We investigated the specific activity of rat liver microsome
a-glucosidase II in the hydrolysis[11–14] of PNP Glc and its deoxy derivatives 1–4.
Clearly, of the four deoxy derivatives of PNP Glc, a-glucosidase II hydrolyzed only the
2-deoxy glucopyranoside (1); its activity with 1 appeared to be substantially higher than
that with PNP Glc. Kinetic studies of the hydrolysis of PNP Glc and 1 were also carried out
(Table 1). The Vmax/Km value of a-glucosidase II for 1 was 3.7-fold greater than for PNP
Glc, which indicated that probe 1 was a good substrate for the enzyme. These results
also indicated that the C-2 hydroxyl group of the a-glucopyranoside residue is not
indispensable for recognition between the substrate and a-glucosidase II.
In assays of the inhibitory effects of PNP Glc and probes 1–12 against processing
a-glucosidase I and a-glucosidase II in rat liver microsomes, we used [3H]glucose-labeled
Scheme 1. Synthesis of probes 5–12.

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Hakamata et al.
Table 1. Hydrolytic activity of processing a-glucosidase II.
Relative rate of
hydrolysis (%)
km
(mM)
Vmax (mmol/
min/unit)
Substrate
Vmax/km
PNP a-D-glucopyranoside
100
189
,1
,1
,1
0.92
0.76
—
1.12
3.44
—
1.23
4.53
—
PNP 2-deoxy-a-D-glucopyranoside (1)
PNP 3-deoxy-a-D-glucopyranoside (2)
PNP 4-deoxy-a-D-glucopyranoside (3)
PNP 6-deoxy-a-D-glucopyranoside (4)
—
—
—
—
—
—
Note: Relative rate of hydrolysis was expressed by comparison with the amount of p-nitrophenol
that was released from PNP Glc by a-glucosidase II, which was taken as 100%.
vesicular stomatitis virus glycoprotein (VSV G)[11–16] and PNP Glc as substrates, and the
results are summarized in Table 2. PNP Glc and probes 1–4 inhibited a-glucosidase I. At
5 mM, PNP Glc and probes 1–4 inhibited a-glucosidase I by 56.2%, 71.7%, 18.5%,
22.2%, and 32.3%, respectively. On the other hand, PNP Glc and probes 1–4 at 5 mM did
not inhibit a-glucosidase II. It has been reported that both PNP Glc and b-D-glucopyrano-
side inhibit purified mammary-gland a-glucosidase I, with 19% and p-nitrophenyl 14%
inhibition at 0.5 mM.[17] These findings indicate that the C-2, C-3, C-4, and C-6 hydroxyl
groups of the a-D-glucopyranoside residue are important for recognition of the substrate
by a-glucosidase I. However, the C-2 hydroxyl group of the a-D-glucopyranoside residue
is not critical for substrate recognition. The failure of the 3-, 4- and 6-deoxy derivatives
Table 2. Inhibitory activity against processing a-glucosi-
dases.
Enzyme
Inhibition (%)
Glucosidase I
Glucosidase II
PNP Glc
56.2
71.7
HD
HD
1
2
18.5
22.2
,1.0
,1.0
,1.0
,1.0
,1.0
,1.0
,1.0
,1.0
,1.0
5.9
3
4
32.3
5
,1.0
,1.0
,1.0
,1.0
,1.0
,1.0
18.2
6
7
8
9
10
11
12
,1.0
,1.0
Note: Probe concentrations: Probes 1–4: 5 mM; probes
5–12: 2 mM. Substrate of glucosidase I: [3H]Glucose
labeled VSV G. Substrate of glucosidase II: PNP Glc. HD:
Hydrolyzing activity was observed.

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Processing a-Glucosidase I and II
31
(2, 3, and 4) to inhibit a-glucosidase II suggests that the C-3, C-4, and C-6 hydroxyl groups
of a-D-glucopyranoside are important for substrate recognition by a-glucosidase II.
Compound 11, with a terminal phenyl group, moderately inhibited both enzymes. At
2 mM compound 11 inhibited a-glucosidase I by 18% and a-glucosidase II by 6%. Both
a-glucosidase I and II showed the same aglycon specificities. Probes 5–12 were assayed
with regard to their ability to inhibit glycoprotein processing at the cellular level. The VSV
G was prepared from VSV-infected and probe-treated baby hamster kidney (BHK) cells.
Analyses of the N-glycan structure of obtained VSV G using endo H, which is known to
have hydrolytic activity against high-mannose type N-glycan, failed to confirm that these
compounds inhibited processing a-glucosidases (data not shown).
Asano et al. and Muroi et al. reported that fagomine (a 2-deoxy derivative of
1-deoxynojirimycin) and 1-azafagomine (in which the C-1 methylene group of fago-
mine is replaced by an –NH– group) inhibited processing a-glucosidases in rat liver
microsomes.[14,16] Based on these inhibitory activities and our data, fagomine and
1-azafagomine are considered to inhibit a-glucosidase II rather than a-glucosidase I.
We previously reported that the glycosyl hydrolase family 31 a-glucosidases from
rice, sugar beet, flint corn, and Aspergillus niger had 2-deoxyglucosidase activity; its
activity against 1 appeared to be substantially higher than that against PNP Glc.[4,6]
Therefore, rat liver microsome a-glucosidase II may have properties similar to those found
in glycosyl hydrolase family 31.
EXPERIMENTAL
General Methods
¨
Melting points were determined with a BUCHI 510 capillary apparatus and are
uncorrected. Optical rotations were measured with JASCO DIP-370 digital porlarimeter
at 258C. The NMR spectra were recorded with a JEOL a-400 spectrometer (400 MHz
1
for H and 100 MHz for 13C). Chemical shifts were expressed in ppm downfield shift
from Me4Si. Where appropriate, signal assignments were deduced by COSY and HMQC
NMR experiments. Low- and high-resolution mass spectra were obtained with a JEOL
JMS AX505WA instrument under positive ion FAB conditions. Column chromatog-
raphy was performed on silica gel 60 (0.063–0.200 mm, Merck, Tokyo, Japan). The
progress of all reactions was monitored by thin-layer chromatography on silica gel 60
F254 (0.25 mm, Merck, Tokyo, Japan).
Method A
To the solution of compound 13[9] (1.6 mmol), in THF (30 mL), a suspension of LAH
(3 mmol) in THF (5 mL) was slowly added. The mixture was stirred at room temperature
for 1 hr and then quenched with ethyl acetate (AcOEt). The resulting mixture was
diluted with satd Na2SO4aq and extracted with diethyl ether (Et2O). Evaporation of the
Et2O phase gave 1-amino-2,6-anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-D-glycero-D-ido-
heptitol (14) (1.51 g, 94.4%) as unstable syrup.

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Hakamata et al.
Method B
To the solution of compound 14 (0.63 mmol) in dichloromethane (CH2Cl2, 5 mL) and
the mixture was added triethylamine (NEt3, 1.9 mmol), carboxylic acid (0.94 mmol) in
CH2Cl2 (1 mL), and diphenylphosphoryl azide (0.94 mmol) in CH2Cl2 (1 mL). After
stirring the mixture at 08C for 8 hr. The resulting mixture was diluted with 1 M HClaq and
extracted with Et2O (2 ꢀ 80 mL). The organic extract was washed with satd NaHCO3aq,
and brine, and then concentrated after drying over Na2SO4.
Method C
To the solution of compound 14 (1.38 mmol) in N,N-dimethylformamide (DMF,
15 mL) and the mixture was added NEt3 (3.77 mmol), carboxylic acid (1.88 mmol)
in DMF (2 mL) and diethylphosphoryl cyanide (1.88 mmol) in DMF (2 mL) was
added.[10] After stirring the mixture at 08C for 18 hr. The resulting mixture was diluted
with 1 M HClaq and extracted with Et2O (3 ꢀ 50 mL). The organic extract was
washed with satd NaHCO3aq, and brine, and then concentrated after drying over
Na2SO4.
Method D
To the solution of methylester derivative (0.07 mmol), in THF (10 mL), a suspension
of LAH (0.14 mmol) in THF (2 mL) was slowly added. The mixture was stirred at room
temperature for 2 hr and then quenched with AcOEt. The resulting mixture was diluted
with satd Na2SO4aq and extracted with Et2O. The organic extract was washed with satd
NaHCO3aq, and brine, and then concentrated after drying over Na2SO4.
Method E
To the solution of compound 14 (1.0 mmol), in pyridine (20 mL) at room temperature
was added acyl halide (4.0 mmol). The mixture was stirred for overnight and poured into
water. The product was extracted with AcOEt (3 ꢀ 50 mL) and washed with water, 1 M
HClaq, satd NaHCO3aq, and brine, and then dried over Na2SO4. Removal of solvent
afforded the corresponding amide.
Method F
A mixture of benzyloxy derivative (0.3 mmol) in ethanol (EtOH, 10 mL) and 1 M
HClaq (50 mL) was hydrogenated under H2 with 20% palladium hydroxide on carbon
(20 mg). After stirring the mixture for 1 hr, the palladium charcoal was removed by
filtration through Celite and the solvent was concentrated.

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Processing a-Glucosidase I and II
33
Preparation of Probes
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-oxoethyl-2-acetoxy)
amino]-D-glycero-D-ido-heptitol (15)
According to methods A and B, compound 15 was prepared from 13 (1.14 g,
2.08 mmol). The product was purified by column chromatography on silica gel (1 : 1
hexane–AcOEt) to afford 0.44 g (32.6%) of 15, which was recrystallized from hexane–
1
AcOEt: [a]D þ27.88 (c 3.6, CHCl3); mp 86–878C; H NMR (CDCl3) d 2.02 (s, 3H,
–OCOCH3), 3.42–3.81 (m, 8H, H-1, H-3, H-4, H-5, H-6, and H-7), 4.09 (m, 1H, H-2),
4.46–4.92 (4 AB system, 8H, –CH2-Ph), 4.51 (s, 2H, –COCH2O–), 6.37 (t, 1H, NH),
7.13–7.36 (m, 20H, aromatic H); MS: 654 (MHþ).
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-oxoethyl-2-hydroxy)
amino]-D-glycero-D-ido-heptitol (16)
A stirred solution of methanol (MeOH) : NEt3 : H2O (5 : 1 : 1, 14 mL) was added acetyl
derivative 15 (0.18 g, 0.28 mmol). After the mixture was stirred for 3 hr at room
temperature, solvent was evaporated. The product was purified by column chromatog-
raphy on silica gel (AcOEt) to afford 0.16 g (96.7%) of 16, which was recrystallized from
hexane–AcOEt: [a]D þ 56.58 (c 2.3, CHCl3); mp 144–1458C; 1H NMR (CDCl3) d 3.47–
3.81 (m, 8H, H-1, H-3, H-4, H-5, H-6, and H-7), 3.96 (s, 2H, –CH2OH), 4.10 (m, 1H,
H-2), 4.44–4.90 (4 AB system, 8H, –CH2-Ph), 6.62 (t, 1H, JNH, 1 5.2 Hz, NH), 7.12–7.35
(m, 20H, aromatic H); MS: 612 (MHþ).
2,6-Anhydro-1-deoxy-1-[(1-oxoethyl-2-hydroxy)amino]-D-glycero-
D-ido-heptitol (5)
According to method F, compound 5 was prepared from 16 (80.0 mg, 0.13 mmol), the
product was purified by column chromatography on silica gel (3 : 1 CH2Cl2–MeOH) to
afford 32.4 mg (98.6%) of 5: [a]D þ 20.08 (c 0.6, MeOH); 1H NMR (CD3OD) d 3.28 (dd,
1H, J4,5 ¼ J5,6 9.2 Hz, H-5), 3.54 (dd, 1H, J3,4 8.8 Hz, H-4), 3.56–3.68 (m, 4H, H-1, H-6,
and H-7a), 3.66 (dd, 1H, J2,3 5.6 Hz, H-3), 3.78 (dd, 1H, J6,7b 2.4 Hz, J7a,7b 11.6 Hz, H-7b),
3.99 (s, 2H, H-20), 4.03 (ddd, 1H, J2,3 4.0 Hz, H-2); 13C NMR (CD3OD) d 36.02 (C-1), 62.64
(C-20), 62.87 (C-7), 71.87 (C-5), 72.38 (C-3), 75þ.16 (C-6), 75.31 (C-4), 75.82 (C-2), 175.30
(C-10); HRMS: Anal. calcd for C9H18NO7 þ H ; 252.1083. Found; 252.1095.
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-oxopropyl-3-hydroxy)amino]-
D-glycero-D-ido-heptitol (17)
According to methods A and C, compound 17 was prepared from 13 (0.61 g,
1.11 mmol). The product was purified by column chromatography on silica gel (AcOEt) to
afford 0.23 g (37.3%) of 17, which was recrystallized from hexane–AcOEt: [a]D þ 55.08
1
0
0
0
(c 1.2, CHCl3); mp 113–1158C; H NMR (CDCl3) d 2.26 (td, 2H, J2 ,3 5.8 Hz, J3 ,OH
1.6 Hz, H-30), 3.42–3.81 (m, 10H, H-1, H-3, H-4, H-5, H-6, H-7, and H-20), 4.08 (m, 1H,
H-2), 4.45–4.91 (4 AB system, 8H, –CH2-Ph), 6.09 (bs, 1H, NH), 7.14–7.35 (m, 20H,
aromatic H); MS: 626 (MHþ).

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Hakamata et al.
2,6-Anhydro-1-deoxy-1-[(1-oxopropyl-3-hydroxy)amino]-D-glycero-
D-ido-heptitol (6)
According to method F, compound 6 was prepared from 17 (60.1 mg, 96 mmol), the
product was purified by column chromatography on silica gel (10 : 4 : 2 : 1 CH2Cl2–
1
MeOH–EtOH–H2O) to afford 20.1 mg (78.9%) of 6: [a]D þ 66.78 (c 1.0, MeOH); H
NMR (CD3OD) d 2.43 (t, 2H, J2 ,3 6.4 Hz, H-30), 3.23 (dd, 1H, J4,5 ¼ J5,6 9.0 Hz, H-5),
3.46–3.56 (m, 2H, H-1a and H-6), 3.52 (dd, 1H, J3,4 8.8 Hz, H-4), 3.59–3.66 (m, 3H,
H-1b, H-3, and H-7a), 3.79–3.82 (dd, 1H, H-7b), 3.80 (t, 2H, H-20), 4.00 (ddd, 1H, J1,2
0
0
13
4.2 Hz, J1,2 10.2 Hz, J2,36.0 Hz, H-2); C NMR (CD3OD) d 36.25 (C-1), 40.10 (C-30),
59.43 (C-20), 63.12 (C-7), 72.04 (C-5), 72.35 (C-3), 75.11 (C-6), 75.26 (C-4), 75.90 (C-2),
174.37 (C-10); HRMS: Anal. calcd for C10H20NO7 þ Hþ; 266.1240. Found; 266.1236.
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-oxobutyl-4-hydroxy)amino]-
D-glycero-D-ido-heptitol (18)
According to methods A and C, compound 18 was prepared from 13 (0.52 g,
0.95 mmol). The product was purified by column chromatography on silica gel (AcOEt) to
afford 0.23 g (41.3%) of 18, which was recrystallized from hexane–AcOEt: [a]D þ 62.08
1
0
0
0
0
(c 1.0, CHCl3); mp 138–1398C; H NMR (CDCl3) d 1.78 (tt, 2H, J2 ,3 12.4 Hz, J3 ,4
6.2 Hz, H-30), 2.23 (td, 2H, J4 , OH 1.4 Hz, H-40), 3.40–3.80 (m, 10H, H-1, H-3, H-4, H-5,
H-6, H-7, and H-20), 4.09 (m, 1H, H-2), 4.45–4.91 (4 AB system, 8H, –CH2-Ph), 6.21
(m, 1H, NH), 7.13–7.36 (m, 20H, aromatic H); MS: 640 (MHþ).
0
2,6-Anhydro-1-deoxy-1-[(1-oxobutyl-4-hydroxy)amino]-D-glycero-
D-ido-heptitol (7)
According to method F, compound 7 was prepared from 18 (168 mg, 0.26 mmol), the
product was purified by column chromatography on silica gel (10 : 4 : 2 : 1 CH2Cl2–
1
MeOH–EtOH–H2O) to afford 72.1 mg (98.5%) of 7: [a]D þ 70.08 (c 0.8, MeOH); H
0
0
0
0
0
0
NMR (CD3OD) d 1.81 (tt, 2H, J2 ,3 ¼ J3 ,4 6.8 Hz, H-3 ), 2.30 (t, 2H, H-3 ), 3.24 (dd, 1H,
J4,5 ¼ J5,6 8.8 Hz, H-5), 3.43–3.53 (m, 3H, H-1a, H-4, and H-6), 3.57 (t, 2H, H-20), 3.59–
3.66 (m, 3H, H-1b, H-3, and H-7a), 3.80 (dd, 1H, J6,7b 2.4 Hz, J7a,7b 11.6 Hz, H-7b), 3.98
(ddd, 1H, J1,2 4.0 Hz, J1,2 10.8 Hz, J2,3 5.6 Hz, H-2); 13C NMR (CD3OD) d 29.80 (C-30),
33.63 (C-40), 36.22 (C-1), 62.26 (C-20), 63.10 (C-7), 72.04 (C-5), 72.37 (C-3), 75.15 (C-6),
75.21 (C-4), 76.01 (C-2), 176.13 (C-10); HRMS: Anal. calcd for C11H22NO7 þ Hþ;
280.1396. Found; 280.1393.
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-methoxycarbonyl)
propylcarbonylamino]-D-glycero-D-ido-heptitol (19)
According to methods A and B, compound 19 was prepared from 13 (0.35 g,
0.63 mmol). The product was purified by column chromatography on silica gel (1 : 1
hexane–AcOEt) to afford 0.27 g (63.2%) of 19, which was recrystallized from hexane–
1
AcOEt: [a]D þ 38.78 (c 1.5, CHCl3); mp 113–1158C; H NMR (CDCl3) d 2.11 (tt, 2H,
0
0
0
0
0
0
J2 ,3 ¼ J3 ,4 7.4 Hz, H-3 ), 2.31 (t, 2H, H-2 ), 3.44–3.78 (m, 10H, H-1, H-3, H-4, H-5, H-6,

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Processing a-Glucosidase I and II
35
H-7, and H-40), 3.65 (s, 3H, –OMe) 4.06 (m, 1H, H-2), 4.46–4.91 (4 AB system, 8H,
–CH2-Ph), 5.79 (m, 1H, NH), 7.13–7.36 (m, 20H, aromatic H); MS: 682 (MHþ).
2,6-Anhydro-3, 4, 5, 7-tetra-O-benzyl-1-deoxy-1-[(1-oxopentyl-
5-hydroxy) amino]-D-glycero-D-ido-heptitol (20)
According to method D, compound 20 was prepared from 19 (0.55 g, 0.81 mmol), the
product was purified by column chromatography on silica gel (1 : 6 hexane–AcOEt) to
afford 0.38 g (72.5%) of 20, which was recrystallized from hexane–AcOEt: [a]D þ 45.38
1
0
0
0
0
(c 1.5, CHCl3); mp 130–1318C; H NMR (CDCl3) d 1.53 (tt, 2H, J3 ,4 ¼ J4 ,5 8.0 Hz,
H-40), 1.67 (tt, 2H, J2 ,3 8.0 Hz, H-30), 2.14 (t, 2H, H-50), 3.41–3.80 (m, 10H, H-1, H-3,
H-4, H-5, H-6, H-7, and H-20), 4.09 (m, 1H, H-2), 4.45–4.91 (4 AB system, 8H, –CH2-
Ph), 6.04 (m, 1H, NH), 7.13–7.35 (m, 20H, aromatic H); MS: 654 (MHþ).
0
0
2,6-Anhydro-1-deoxy-1-[(1-oxopentyl-5-hydroxy)amino]-D-glycero-
D-ido-heptitol (8)
According to method F, compound 8 was prepared from 19 (269.8 mg, 0.41 mmol),
the product was purified by column chromatography on silica gel (10 : 4 : 2 : 1 CH2Cl2–
1
MeOH–EtOH–H2O) to afford 92.6 mg (76.5%) of 8: [a]D þ 36.48 (c 1.1, MeOH); H
NMR (CD3OD) d 1.55 (tt, 2H, H-30), 1.67 (tt, 2H, H-40), 2.24 (t, 2H, J4 ,5 7.4 Hz, H-50),
0
0
3.24 (dd, 1H, J4,5 ¼ J5,6 9.2 Hz, H-5), 3.43–3.53 (m, 2H, H-1a and H-6), 3.56 (dd, 1H, J3,4
0
0
0
9.0 Hz, H-4), 3.56 (t, 2H, J2 ,3 6.4 Hz, H-2 ), 3.59–3.67 (m, 3H, H-1b, H-3, and H-7a), 3.80
(dd, 1H, J6,7b 2.2 Hz, J7a,7b 11.8 Hz, H-7b), 3.98 (ddd, 1H, J1,2 3.6 Hz, J1,2 10.8 Hz, J2,3
5.8 Hz, H-2); 13C NMR (CD3OD) d 23.41 (C-40), 33.06 (C-30), 36.17 (C-1), 36.76 (C-50),
62.53 (C-20), 63.09 (C-7), 72.02 (C-5), 72.37 (C-3), 75.15 (C-6), 75.20 (C-4), 76.07 (C-2),
176.32 (C-10); HRMS: Anal. calcd for C12H24NO7 þ Hþ; 294.1553. Found; 294.1556.
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-methoxycarbonyl)
butylcarbonylamino]-D-glycero-D-ido-heptitol (21)
According to methods A and B, compound 21 was prepared from 13 (0.45 g,
0.81 mmol). The product was purified by column chromatography on silica gel (1 : 1
hexane–AcOEt) to afford 0.12 g (21.3%) of 21, which was recrystallized from hexane–
1
AcOEt: [a]D þ 39.08 (c 2.0, CHCl3); mp 104–1058C; H NMR (CDCl3) d 1.60 (tt, 2H,
J2 ,3 ¼ J3 ,4 7.2 Hz, H-30), 2.06 (tt, 2H, J4 ,5 6.8 Hz, H-40), 2.28 (t, 2H, H-20), 3.43–3.79
(m, 10H, H-1, H-3, H-4, H-5, H-6, H-7, and H-50), 3.65 (s, 3H, –OMe), 4.07 (m, 1H, H-2),
4.46–4.91 (4 AB system, 8H, –CH2-Ph), 5.78 (m, 1H, NH), 7.13–7.32 (m, 20H, aromatic
H); MS: 696 (MHþ).
0
0
0
0
0
0
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-deoxy-1-[(1-oxohexyl-
6-hydroxy)amino]-D-glycero-D-ido-heptitol (22)
According to method D, compound 22 was prepared from 21 (0.214 g, 0.31 mmol),
the product was recrystallized from hexane–AcOE to afford 0.184 g (89.6%) of 22:
[a]D þ 50.18 (c 1.7, CHCl3); mp 152–1538C; 1H NMR (CDCl3) d 1.33 (tt, 2H,
0
0
0
0
0
0
0
0
0
0
0
J3 ,4 ¼ J4 ,5 7.6 Hz, H-4 ), 1.52 (tt, 2H, J5 ,6 7.4 Hz, H-5 ), 1.60 (tt, 2H, J2 ,3 7.6 Hz, H-3 ),

ORDER
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36
Hakamata et al.
2.09 (t, 2H, H-60), 3.37–3.78 (m, 10H, H-1, H-3, H-4, H-5, H-6, H-7, and H-20), 4.08
(m, 1H, H-2), 4.46–4.94 (4 AB system, 8H, –CH2-Ph), 5.92 (m, 1H, NH), 7.14–7.34
(m, 20H, aromatic H); MS: 668 (MHþ).
2,6-Anhydro-1-deoxy-1-[(1-oxohexyl-6-hydroxy)amino]-D-glycero-
D-ido-heptitol (9)
According to method F, compound 9 was prepared from 22 (132 mg, 0.20 mmol), the
product was purified by column chromatography on silica gel (10 : 4 : 2 : 1 CH2Cl2–
MeOH–EtOH–H2O) to afford 60.4 mg (99.3%) of 9: [a]D þ 88.68 (c 0.7, MeOH);
1H NMR (CD3OD) d 1.39 (tt, 2H, H-40), 1.54 (tt, 2H, H-30), 1.64 (tt, 2H, H-50), 2.24 (t, 2H,
J5 ,6 7.6 Hz, H-60), 3.25 (dd, 1H, J4,5 ¼ J5,6 9.2 Hz, H-5), 3.43–3.53 (m, 3H, H-1a, H-4,
0
0
and H-6), 3.55 (t, 2H, J2 ,3 6.4 Hz, H-20), 3.56–3.67 (m, 3H, H-1b, H-3, and H-7a), 3.78
(dd, 1H, J6,7b 2.0 Hz, J7a,7b 12.0 Hz, H-7b), 3.98 (ddd, 1H, J1,2 3.6 Hz, J1,2 11.0 Hz, J2,3
5.8 Hz, H-2); 13C NMR (CD3OD) d 26.54 (C-40), 26.84 (C-50), 33.32 (C-30), 36.17 (C-7),
37.04 (C-60). 62.77 (C-20), 63.07 (C-1), 72.02 (C-5), 72.34 (C-3), 75.1þ3 (C-6), 75.18 (C-4),
76.03 (C-2), 176.41 (C-10); HRMS: Anal. calcd for C13H26NO7 þ H ; 308.1709. Found;
308.1702.
0
0
1-Acetamide-2,6-anhydro-D-glycero-D-ido-heptitol (10)
According to method F, compound 10 was prepared from 23 (0.16 mg, 0.27 mmol),[9]
the product was purified by column chromatography on silica gel (10 : 4 : 2 : 1 CH2Cl2–
1
MeOH-EtOH–H2O) to afford 63.2 mg (98.9%) of 6: [a]D þ 26.08 (c 1.0, MeOH); H
NMR (CD3OD) d 1.96 (s, 3H, COCH3), 3.24 (dd, 1H, J4,5 ¼ J5,6 9.0 Hz, H-5), 3.45 (dd,
1H, J6,7a 11.2 Hz, J7a,7b 14.4, H-7a), 3.51 (dd, 1H, J3,4 9.0 Hz, H-4), 3.54 (m, 1H, H-6),
3.59–3.64 (m, 3H, H-1 and H-7a), 3.65 (dd, 1H, J2,3 6.2 Hz, H-3), 3.80 (dd, 1H, J7a,7b
2.4 Hz, J6,7b 11.6 Hz, H-7b), 3.98 (ddd, 1H, J1a,2 3.6 Hz, H-2); 13C NMR (CD3OD) d 22.56
(CH3), 36.26 (C-1), 63.09 (C-7), 72.02 (C-5), 72.30 (C-3), 75.08 (C-4), 75.20 (C-6), 76.00
(C-2), 173.75 (C55O); HRMS: Anal. calcd for C9H18NO6 þ Hþ; 236.1134. Found;
236.1135.
2,6-Anhydro-1-benzamide-3,4,5,7-tetra-O-benzyl-D-glycero-D-ido-heptitol (24)
According to methods A and E, compound 24 was prepared from 13 (0.54 g,
0.98 mmol), the product was purified by column chromatography on silica gel (AcOEt)
to afford 0.38 mg (70.4%) of 24, which was recrystallized from hexane–AcOEt:
[a]D þ 68.38 (c 1.2, CHCl3); mp 141–1438C; 1H NMR (CDCl3) d 3.51 (dd, 1H, J4,5 ¼ J5,6
9.0 Hz, H-5), 3.59 (dd, 1H, J6,7a 5.4 Hz, J7a,7b 10.2 Hz, H-7a), 3.67 (dd, 1H, J6,7b 2.0 Hz,
H-7b), 3.68–3.87 (m, 4H, H-1a, H-3, H-4, and H-6), 3.97 (ddd, 1H, J1a,1b 14.8 Hz, J1b,2
6.4 Hz, J1b,NH 5.0 Hz, H-1b), 4.21 (ddd, 1H, H-2), 4.47–4.92 (4 AB system, 8H,
–CH2-Ph), 6.55 (bs, 1H, NH), 7.14–7.71 (m, 24H, aromatic H); MS: 658 (MHþ).
2,6-Anhydro-1-benzamide-D-glycero-D-ido-heptitol (11)
According to method F, compound 11 was prepared from 24 (0.20 g, 0.30 mmol), the
product was purified by column chromatography on silica gel (5 : 1 CH2Cl2–MeOH) to

ORDER
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Processing a-Glucosidase I and II
37
1
afford 77.6 mg (87.9%) of 11: [a]D þ 107.58 (c 0.8, MeOH); H NMR (CD3OD) d 3.23
(dd, 1H, J4,5 ¼ J5,6 9.4 Hz, H-5), 3.53 (dd, 1H, J3,4 9.0 Hz, H-4), 3.58–3.65 (m, 3H, H-3,
H-6, and H-7a), 3.66 (dd, 1H, J1a,1b ¼ J1a,2 10.8 Hz, H-1a), 3.74–3.79 (m, 2H, H-1b and
H-7b), 4.10 (ddd, 1H, J1,2 4.0 Hz, J2,3 6.5 Hz, H-2), 7.38–7.79 (m, 4H, aromatic H); 13C
NMR (CD3OD) d 36.88 (C-1), 63.04 (C-7), 72.06 (C-5), 72.42 (C-3), 75.25 (C-4 and C-6),
76.06 (C-2), 128.34, 129.52, 132.62, and 135.78 (aromatic C), 170.64 (C55O); HRMS:
Anal. calcd for C14H20NO6 þ Hþ; 298.1291. Found; 298.1287.
2,6-Anhydro-3,4,5,7-tetra-O-benzyl-1-trimethylacetamide-D-glycero-
D-ido-heptitol (25)
According to methods A and E, compound 25 was prepared from 13 (0.61 g,
1.10 mmol), the product was purified by column chromatography on silica gel (AcOEt) to
afford 0.65 g (72.3%) of 25: [a]D þ 35.68 (c 1.8, CHCl3); 1H NMR (CDCl3) d 1.12 (s, 9H,
C(CH3) ), 3.42–3.83 (m, 8H, H-1, H-3, H-4, H-5, H-6 and H-7), 4.11 (ddd, 1H, J1a,2
¼
3
J2, 3 4.8 Hz, J1b, 2 9.6 Hz, H-2), 4.46–4.91 (4 AB system, 8H, –CH2–Ph), 5.98 (bs, 1H,
NH), 7.14–7.35 (m, 20H, aromatic H); MS: 638 (MHþ).
2,6-Anhydro-1-trimethylacetamide-D-glycero-D-ido-heptitol (12)
According to method F, compound 12 was prepared from 25 (0.17 g, 0.26 mmol), the
product was purified by column chromatography on silica gel (4 : 1 CH2Cl2–MeOH) to
1
afford 64.8 mg (89.1%) of 12: [a]D þ 105.08 (c 0.8, MeOH); H NMR (CD3OD) d 1.18
(s, 9H, 3 ꢀ CH3), 3.25 (dd, 1H, J4, 5 9.6 Hz, J5, 6 8.8 Hz, H-5), 3.50–3.56 (m, 4H, H-4,
H-6, and H-1), 3.63 (dd, 1H, J2, 3 6.0 Hz, J3, 4 9.6 Hz, H-3), 3.65 (dd, 1H, J7a, 7b 2.0 Hz, J6,
7a 6.0 Hz, H-7a), 3.77 (dd, J6, 7b 11.8 Hz, H-7b), 4.00 (ddd, 1H, H-2); 13C NMR (CD3OD) d
27.82 (3 ꢀ CH3), 36.69 (C-1), 39.74 (C(CH3)3), 62.95 (C-7), 72.00 (C-5), 72.55 (C-3),
75.28 (C-4 and C-6), 75.71 (C-2), 181.59 (C55O); HRMS: Anal. calcd for
C12H24NO6 þ Hþ; 278.1604. Found; 278.1601.
Enzymatic Assays
p-Nitrophenyl a-D-glucopyranoside was purchased from Sigma–Aldrich Inc.
[3H]Glucose labeled VSV G was prepared as previously reported methods.[15,18,19] Rat
liver microsomes were prepared following the previously reported methods, and membranes
were collected by centrifugation at 12,000g for 1 hr and resuspended in 100 mM potassium
phosphate buffer (pH 6.8) containing 10 mM glycerol, 2 mM MgCl2, 0.5mM DTT.[15]
Activities of processing a-glucosidase I and a-glucosidase II were assayed using rat liver
microsomes as an enzyme source. In assays of the inhibitory effects of PNP Glc and probes
1–12 against processing a-glucosidase I, we used [3H]glucose labeled VSV G as substrate.
The hydrolytic activities of the enzyme was assayed by measuring the release of [3H]glucose
under 100mM potassium phosphate buffer (pH 6.8) containing 20mM EDTA, 0.02% NaN3,
0.05% Triton X-100, and protease inhibitor cocktails at 378C. The processing a-glucosidase
II assays were performed by using PNP Glc as substrate and were assayed the previously
reported methods,[11–14] the assay condition was 100 mM potassium phosphate buffer
containing 0.05% Triton X-100 (pH 6.8) at 378C. One unit of enzyme activity was defined as

ORDER
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38
Hakamata et al.
the amount of enzyme required to liberate 1mmol of p-nitrophenol/min at each assay
condition. Kinetic study on the hydrolysis of PNP Glc and 1 by a-glucosidase II was
performed at each concentration, and values of Km (mM) and Vmax (mmol/min/unit) were
calculated from reciprocal plots of their reaction curves.
Inhibition Assays at Cellular Level
Confluent monolayer of BHK cells are infected with the VSV, and nectrisine
(a final concentration 10 mg/mL, Nectrisine was kindly supplied from Fujisawa
Pharmaceutical Co., Tokyo, Japan), probes 5–10 and 12 (final concentrations 100 mg/
mL), and probe 11 (a final concentration 500 mg/mL) are added to infected cultures as
described.[18–20] The obtained VSV G proteins were digested with endo H by reported
method.[18] The treated VSV G proteins were subjected to SDS-polyacrylamide gel
electrophoresis using 10% acryl amide gel, followed by electrophoretic transfer onto
nitrocellulose membrane. The protein replica was incubated with rabbit polyclonal
antibodies raised against VSV G.[18] Antigen–antibody complexes were detected with a
goat anti-rabbit IgG antibody–alkaline phosphatase conjugate by staining with 5-bromo-
4-chloro-3-indolyl phosphate.
ACKOWLEDGMENTS
This work was preformed at Special Postdoctoral Researchers Program (RIKEN).
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Received June 19, 2003
Revised July 4, 2003
Accepted August 7, 2003

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