2,5-Dideoxy-2,5-imino-d-altritol as a new class of pharmacological chaperone for Fabry disease

Article abstract of DOI:10.1016/j.bmc.2010.04.048

Chromatographic separation of the extract from roots of Adenophora triphylla resulted in the isolation of two pyrrolidines, six piperidines, and two piperidine glycosides. The structures of new iminosugars were elucidated by spectroscopic methods as 2,5-d

Full text of DOI:10.1016/j.bmc.2010.04.048

Bioorganic & Medicinal Chemistry 18 (2010) 3790–3794
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2,5-Dideoxy-2,5-imino-D-altritol as a new class of pharmacological
chaperone for Fabry disease
Atsushi Kato a, , Yukiko Yamashita , Shinpei Nakagawa , Yuriko Koike , Isao Adachi , Jackie Hollinshead ,
a
a
a
a
b
*
b
c
c
Robert J. Nash , Kyoko Ikeda , Naoki Asano
Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan
Institute of Biological, Environmental and Rural Sciences/Phytoquest Limited, Plas Gogerddan, Aberystwyth, Ceredigion SY23 3EB, United Kingdom
Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Chromatographic separation of the extract from roots of Adenophora triphylla resulted in the isolation of
Received 26 March 2010
Revised 15 April 2010
Accepted 16 April 2010
Available online 21 April 2010
two pyrrolidines, six piperidines, and two piperidine glycosides. The structures of new iminosugars
were elucidated by spectroscopic methods as 2,5-dideoxy-2,5-imino-
nyl-1-deoxygalactonojirimycin (8), 2,3-dideoxy-b-1-C-ethyl-1-deoxygalactonojirimycin (9), and 6-O-b-
-glucopyranosyl-2,3-dideoxy-b-1-C-ethyl-1-deoxygalactonojirimycin (10). b-1-C-Butyl-1-deoxygalac-
tonojirimycin (7) and compound 8 were found to be better inhibitors of -galactosidase than
N-butyl-1-deoxygalactonojirimycin. The present work elucidated that DIA was a powerful competitive
inhibitor of human lysosome -galactosidase A ( -Gal A) with a K value of 0.5 M. Furthermore, DIA
improved the thermostability of -Gal A in vitro and increased intracellular -Gal A activity by 9.6-fold
D-altritol (DIA) (2), b-1-C-bute-
D
a
Keywords:
Adenophora triphylla
a
a
i
l
2
,5-Dideoxy-2,5-imino-D-altritol
a
a
a-Galactosidase A
in Fabry R301Q lymphoblasts after incubation for 3 days. These experimental results suggested that DIA
would act as a specific pharmacological chaperone to promote the smooth escape from the endoplasmic
reticulum (ER) quality control system and to accelerate transport and maturation of the mutant enzyme.
Ó 2010 Elsevier Ltd. All rights reserved.
Pharmacological chaperone
Glycosidase inhibitor
1
. Introduction
the mutant enzyme, thus allowing its maturation and trafficking
to the lysosome. To establish the concept of using competitive
5
Fabry disease is a lysosomal storage disorder caused by an
X-linked inherited deficiency of -galactosidase A ( -Gal A). Defi-
inhibitors as specific pharmacological chaperones, a number of
naturally occurring and chemically synthesized compounds were
a
a
ciency of the enzyme activity results in the progressive accumula-
tested for intracellular enhancement of the a-Gal A activity in
Fabry lymphoblasts. However, the initial studies using glycosidase
6
tion of globotriaosylceramide (Gb3) in the lysosomes of vascular
1
endothelial cells. More than 400 mutations have been identified
inhibitors as pharmacological chaperones have focused on six-
7
–9
in the
a-Gal A gene, and 57% of mutations are missense. Further-
membered sugar mimetics.
With respect to the five-membered
more, structural studies revealed that the majority of the muta-
tions do not directly contribute to the catalytic function of the
enzyme, but rather to the maintenance of the tertiary structure.
Although enzyme replacement therapy has been approved for the
treatment of Fabry disease, it has no therapeutic benefit for the
treatment of stroke and fails to remove accumulated Gb3 from kid-
ney podocytes and blood vessel walls. In recent years, a remarkable
progress has been made in developing a molecular therapy for
sugar mimetics, there is little evaluation as pharmacological
chaperones. In this paper, we describe the isolation, structural
determination, and glycosidase inhibitory activity of three new
six-membered iminosugars with a 1-C-substitution and one five-
2
,3
membered iminosugar, 2,5-dideoxy-2,5-imino-D-altritol (DIA),
which is not a new compound but the first report as a natural prod-
uct, from Adenophora triphylla var. japonica (Campanulaceae). Fur-
thermore, we report for the first time that DIA is an excellent
pharmacological chaperone which effectively enhances intracellu-
Fabry disease. Residual
from Fabry patients and in tissues of R301Q
mice was enhanced by treatment with 1-deoxygalactonojirimycin
DGJ), a competitive inhibitor of the enzyme.4 The inhibitor ap-
a
-Gal A activity in lymphoblasts derived
a-Gal A transgenic
lar a-Gal A activity in Fabry lymphoblasts.
(
2
2
. Results and discussion
pears to act as a template that stabilizes the native folding state
in the endoplasmic reticulum (ER) by occupying the active site of
.1. Isolation and structural determination
The roots (10 kg) of A. triphylla were extracted with 50% aqueous
*
Corresponding author.
E-mail address: kato@med.u-toyama.ac.jp (A. Kato).
MeOH, and the extract was subjected a variety of chromatographic
0
968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.048

A. Kato et al. / Bioorg. Med. Chem. 18 (2010) 3790–3794
3791
steps with ion-exchange resins to give alkaloids 1 (1140 mg), 2
the carbon signals of d 12.6 and 29.7 are derived from the ethyl
side chain at C-1. The relatively high-field methine carbons at d
52.6 (C-1) and 59.3 (C-5) must be bonded to the nitrogen of the
piperidine ring. The strong NOE between H-1 and H-5 indicated
that these protons are 1,3-diaxial. The strong NOE between H-1
and H-5 and the coupling patterns of H-4 (ddd, J3a,4 = 3.3 Hz,
(
8
191 mg), 3 (130 mg), 4 (149 mg), 5 (32 mg), 6 (6 mg), 7 (103 mg),
(49 mg), 9 (14 mg), and 10 (125 mg). The H and C NMR spectra
1
13
of alkaloids 1 and 3–7 were in accord with those of 2,5-dideoxy-2,5-
imino- -mannitol (DMDP), 1-deoxynojirimycin, 1-deoxymannojir-
imycin, -1-C-ethyl-fagomine, 1-O-b- -glucopyranosyladenopho-
rine, and b-1-C-butyl-1-deoxygalactonojirimycin, respectively,
D
a
D
J3b,4 = 4.1 Hz, J4,5 = 1.5 Hz) indicated the equatorial orientation of
1
0,11
which have been isolated previously from A. triphylla.
The
H-4. Hence, the structure of 9 was determined to be 2,3-dideoxy-
b-1-C-ethyl-1-deoxygalactonojirimycin.
structural determination of the new alkaloids 2, 8, 9, and 10 is
described below.
OH
HO
OH
HO
OH
CH OH
HO
OH
3
4
3
2
1
4
5
2
5
6
1
6
HOH C
N
CH2OH
HO
H2
C
N
H
2
2
N
H
CH OH
H
2
1
2
3
OH
HO
4
OH
OH
3
2
5
6
8
HO
OH
OH
HOH C
O
O
CH3
3
4
2
2
1
N
H
8
1
7
5
6
H C
3
N
H
CH OH
N
H
CH OH
HO
OH
6
2
7
2
OH
4
5
OH
OH
HO
8
OH
HO
OH
OH
CH OR
3
2
1
4
1
0
5
6
H C
H C
3
H C
2
3
N
H
CH OH
N
H
CH OH
N
H
2
9
7
2
2
7
8
9
R = H
1
0 R = β-D-glucopyranoside
The H NMR and 13C NMR spectral data of 2 were superimpos-
able with those of 2,5-dideoxy-2,5-imino- -altritol, which has
been chemically synthesized.12 Furthermore, the optical rotation
+34.2 (c 0.83, H O)} of 2 was identical to that of the synthetic
compound {[ +34.8 (c 0.70, H O)}. Hence, compound 2 was
determined to be 2,5-dideoxy-2,5-imino- -altritol (DIA).
1
+
The HRFABMS (m/z 322.1874 [M+H] ) and 14 resonances in the
C NMR spectrum of 10 established that the molecular formula
1
3
D
was C14H27NO . The characteristic anomeric proton (d, J
8.2 Hz) and carbon (d 106.0) signals suggested that 10 was a glyco-
side of an alkaloid. After acid hydrolysis of this glycoside using
-glucose was detected in the filtrate
by the D-glucose oxidase peroxidase method. On the other hand,
the alkaloid part was displaced from the resin with 0.5 M NH OH,
7
1 ,2
=
0
0
{[a]
D
2
a
]
D
2
+
D
Dowex 50 W-X2 (H ) resin,
D
+
The HRFABMS (m/z 218.1393 [M+H] ) and 10 resonances in the
C NMR spectrum of 8 established that the molecular formula was
1
3
4
C
10
H
19NO
4
. The complete connectivity of the carbon and hydrogen
concentrated to dryness, and confirmed as 2,3-dideoxy-b-1-C-eth-
1
1
1
13
atoms was defined from H– H COSY, H– C COSY, and HMBC
spectroscopic data. The COSY and HMBC spectra elucidated that
the carbon signals of d 32.2, 33.0, 117.7, and 141.9 are derived from
the butenyl side chain at C-1. The remaining carbon signals (d 61.1,
yldeoxygalactonojirimycin (9) by direct comparison of its NMR
3
spectra with those of 9. The chemical shift (d 4.43) and J
H,H
cou-
pling (8.2 Hz) of the anomeric proton indicated that its glucosidic
linkage is b. Introduction of a b- -glucopyranosyl group into 9
produced a 9.3 ppm downfield shift for C-6 and a 1.6 ppm upfield
shift for C-5. From these results, the linkage site of the b- -gluco-
pyranosyl group is C-6 and the structure of 10 was determined
to be 6-O-b- -glucopyranosyl-2,3-dideoxy-b-1-C-ethyl-1-deoxyga-
D
6
1.5, 64.3, 71.7, 74.9, 77.8) were completely in accord with the
corresponding signals (d 61.1, 61.8, 64.2, 71.8, 74.9, 77.9 of b-1-
C-butyl-1-deoxygalactonojirimycin (7). The coupling patterns of
H-4 (dd, J3,4 = 3.2 Hz, J4,5 = 1.4 Hz) and H-3 (dd, J2,3 = 9.6 Hz,
D
D
J
3,4 = 3.2 Hz) indicated the equatorial orientation of H-4 and the
lactonojirimycin.
axial orientations of H-2 and H-3. Furthermore, the strong NOE be-
tween H-1 and H-5 indicated that these protons are 1,3-diaxial.
Hence, the structure of 8 was determined to be b-1-C-butenyl-1-
deoxygalactonojirimycin.
2.2. In vitro glycosidase inhibitory activities
The IC50 values of the alkaloids isolated from A. triphylla toward
various glycosidases are shown in Table 1. b-1-C-Butyl-DGJ (7) and
b-1-C-butenyl-DGJ (8) showed potent inhibitory activity toward
+
The HRFABMS (m/z 160.1338 [M+H] ) and eight resonances in
the 13C NMR spectrum of 9 established that the molecular formula
was C
8
H
17NO
4
. The complete connectivity of the carbon and hydro-
coffee beans a-galactosidase, with IC50 values of 1.2 and 2.0 lM,
respectively. We previously reported that b-1-C-butyl-DNJ and
b-1-C-ethyl-1-deoxymannojirimycin abolished their inhibition
1
1
1
13
gen atoms was defined from H– H COSY, H– C COSY, and HMBC
spectroscopic data. The COSY and HMBC spectra elucidated that

3
792
A. Kato et al. / Bioorg. Med. Chem. 18 (2010) 3790–3794
Table 1
Concentration of iminosugars giving 50% inhibition of various glycosidases
1
0
0
0
.9
.8
.7
IC50
2
(lM)
Enzyme
-Glucosidase
Yeast
Rice
7
8
9
10
DGJ
a
66
171
74
NIa
10
112
NI
45
239
NI
NI
NI
NI
670
NI
NI
NI
NI
0.6
0
0
.5
.4
Rat intestinal maltase
b-Glucosidase
Almond
C. saccharolyticum
Bovine liver
40
19
444
NI
152
NI
NI
1000
329
NI
NI
950
321
NI
NI
NI
367
NI
0.3
0
0
.2
.1
0
a
-Galactosidase
Coffee beans
Human lysosome
0.78
0.69
1.2
15
2.0
39
NI
NI
318
224
0.003
0.07
0
0.5
1
1.5
-
1
b-Galactosidase
Bovine liver
Rat intestinal lactase
1/[S] (mM)
190
7.2
370
NI
476
NI
332
NI
NI
NI
NI
72
Figure 1. Lineweaver–Burk plots of 2,5-dideoxy-2,5-imino-
lysosome
side were used to determine the K
versus 1/[S]. Concentrations of 2 were 0
triangle, 2.5
and 0.5 M, respectively.
D
-altritol (2) of human
a
-galactosidase A. Increasing concentration of PNP-
a-
D
-galactopyrano-
values and the data were plotted as 1/V
M (J): closed circle, 1.0 M (H): closed
a
-Mannosidase
m
and K
i
Jack beans
64
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
l
l
b-Mannosidase
Rat epididymis
i
lM (H): closed diamond. The calculated Km and K values were 16 mM
NI
l
a-L-Fucosidase
Bovine epididymis
503
631
NI
1
00
Trehalase
Porcine kidney
80
Amyloglucosidase
Aspergillus niger
6
0
NI
a
NI: no inhibition (less than 50% inhibition at 1000
lM).
40
20
toward
a-glucosidases and a
-mannosidases, respectively,13,14
while the present study revealed that b-1-C-butyl-DGJ (7) and
b-1-C-butenyl-DGJ (8) retained their inhibitory potential toward
0
0
20
40
60
a
-galactosidase. Furthermore, both compounds were more potent
inhibitors of the enzyme than N-butyl-DGJ with an IC50 value of
M. New alkaloids 2,3-dideoxy-b-1-C-ethyl-DGJ (9) and 6-O-
-glucopyranosyl-2,3-dideoxy-b-1-C-ethyl-DGJ (10) markedly
incubation time (min)
Figure 2. Effect of DIA (2) on the in vitro thermostability. Human lysosome
galactosidase A ( -Gal A) was incubated at 48 °C in 100 mM sodium citrate buffer
pH 4.6) containing 1 mg/mL of BSA at various incubation time with or without 2,5-
dideoxy-2,5-imino- -altritol (2). Concentrations of 2 were 0 M (J): closed circle,
10 M (Ç).
M (}), 100
a-
14 l
b-
a
D
(
lowered their inhibition toward this enzyme. DMDP (1) is known
to be a potent inhibitor of yeast5a-glucosidase, mammalian b-glu-
cosidase, and b-galactosidase.1 In contrast, DIA (2) showed a
significant inhibition against coffee bean and human lysosome
D
l
l
l
was determined with p-nitrophenyl-a-D-galactopyranoside as
substrate. The enzyme activity was lost within 60 min under
incubation without DIA, while the incubation with DIA effectively
a
-galactosidases, with IC50 values of 0.78 and 0.69
tively. DIA is the most potent inhibitor of -galactosidase among
five-membered iminosugars reported to date. Fabry disease is a
lysosomal storage disorder caused by deficiency of lysosomal
galactosidase A ( -Gal A), an enzyme responsible for hydrolysis
of the terminal -galactosyl residue in glycosphingolipids. Studies
on residual -Gal A activity of mutant enzymes in many Fabry pa-
tients revealed that some had kinetic properties similar to those of
lM, respec-
a
stabilized
ity remained over 70% even under the incubation for 60 min in the
presence of 100 M DIA. Thus, DIA occupied the catalytic center of
-Gal A and enhanced the thermostability. DIA that showed strong
competitive inhibition and improvement of thermostability was
further tested for enhancement of intracellular -Gal A activity
in Fabry R301Q lymphoblasts. Treatment with DIA for 3 days
dose-dependently increased intracellular -Gal A activity, with
maximal increase of 9.6-fold at 500 M (Fig. 3). In conclusion, this
a-Gal A in a dose-dependent manner. The enzyme activ-
a-
l
a
a
a
a
a
normal a
-Gal A, but were significantly less stable.1 We have pre-
viously reported that a potent competitive inhibitor appears to act
as a template that stabilizes the native folding state by occupying
the active site of the unstable enzyme. We therefore focused on a
strong competitive inhibitor, DIA (2), and studied whether this
compound could act as pharmacological chaperone. We first deter-
6
a
l
4
study indicates that the five-membered iminosugar DIA could as a
specific pharmacological chaperone to effectively enhance intra-
cellular enzyme activity in Fabry R301Q lymphoblasts.
mined the inhibition constant (K
DIA by the Lineweaver–Burk plots, and found to inhibit
in a competitive manner, with K value of 0.5 M (Fig. 1).
i
) and the mode of inhibition of
a-Gal A
3. Experimental section
i
l
3
.1. General experimental procedures
2
.3. In vitro stabilization of
a-Gal A by DIA and effect of DIA on
a
-Gal A activity in Fabry lymphoblasts
Optical rotations were measured with a JASCO DIP-370 digital
1
13
polarimeter (Tokyo, Japan). H NMR (500 MHz) and C NMR
(125 MHz) spectra were recorded on a Bruker DRX500 spectrome-
ter. Chemical shifts are expressed in ppm downfield from sodium
The effect of DIA on the stabilization of
studied in vitro (Fig. 2). The enzyme -Gal A was incubated in
00 mM sodium citrate buffer (pH 4.6) containing 1 mg/mL of
BSA for 0, 20, 40, and 60 min and the remaining enzyme activity
a-Gal A at 48 °C was
a
1
2
3-(trimethylsilyl)-propionate (TSP) in D O as internal standard.
The assignment of proton and carbon NMR signals was determined

A. Kato et al. / Bioorg. Med. Chem. 18 (2010) 3790–3794
3793
5
4
4
3
3
2
2
1
1
0
0
.0
.5
.0
.5
.0
.5
.0
.5
.0
.5
.0
(1H, dd, J = 6.6, 11.2 Hz, H-1a), 3.78 (1H, dd, J = 3.9, 11.7 Hz, H-
6
8
b), 3.82 (1H, dd, J = 6.6, 11.2 Hz, H-1b), 4.03 (1H, dd, J = 4.2,
13
.5 Hz, H-4), 4.21 (1H, dd, J = 4.1, 4.2 Hz, H-3);
C NMR
(
7
C
125 MHz, D
2
O) d 62.8 (C-2), 63.2 (C-1), 64.3 (C-5), 64.6 (C-6),
+
4.7 (C-3), 76.3 (C-4); HRFABMS m/z 164.0925 [M+H] (calcd for
164.0923).
6 4
H14NO
3
.3.2. b-1-C-Butenyl-1-deoxy-1-galactonojirimycin (8)
1
Colorless powder; [
O) d 1.51 (1H, m, H-7a), 1.93 (1H, m, H-7b), 2.15 (1H, m, H-8a),
a
]
D
ꢀ10.8 (c 0.10, H
2
O); H NMR (500 MHz,
D
2
2
.24 (1H, m, H-8b), 2.51 (1H, ddd, J = 3.2, 8.3, 9.6 Hz, H-1), 2.80 (1H,
ddd, J = 1.4, 6.4, 6.9 Hz, H-5), 3.42 (1H, t, J = 9.6 Hz, H-2), 3.51 (1H,
dd, J = 3.2, 9.6 Hz, H-3), 3.64 (1H, dd, J = 6.9, 11.0 Hz, H-6a), 3.68
(1H, dd, J = 6.4, 11.0 Hz, H-6b), 3.99 (1H, dd, J = 1.4, 3.2 Hz, H-4),
5
.04 (1H, dd, J = 1.8, 10.5 Hz, H-10a), 5.14 (1H, dd, J = 1.8, 17.4 Hz,
13
H-10b), 5.94 (1H, dd t, J = 6.4, 10.5, 17.4 Hz, H-9); C NMR
(125 MHz, D O) d 32.2 (C-7), 33.0 (C-8), 61.1 (C-5), 61.5 (C-1),
64.3 (C-6), 71.7 (C-4), 74.9 (C-2), 77.8 (C-3), 117.7 (C-10), 141.9
2
non treat
50
100
250
500
+
(
C-9); HRFABMS m/z 218.1393 [M+H] (calcd for C10
H20NO
4
2
,5-dideoxy-2,5-imino-D-altritol (μM)
218.1392).
Figure 3. Influence of DIA (2) on
was added to the culture medium of R301Q cells at a concentration of 50–500
Cells were subsequently incubated for 3 days and cell growth was not affected by
the inclusion of DIA.
a
-Gal A activity in Fabry R301Q lymphoblasts. DIA
3
.3.3. 2,3-Dideoxy-b-1-C-ethyl-1-deoxygalactonojirimycin (9)
l
M.
1
Colorless powder; [ +11.8 (c 1.89, H
a
]
D
2
O); H NMR (500 MHz,
D
2
O) d 93Ht J = 73 Hz CH ), 1.27 (1H, m, H-2a), 1.42–1.48 (2H, m,
3
H-7a, H-7b), 1.52 (1H, m, H-2b), 1.80–1.92 (2H, m, H-3a, H-3b),
2.81 (1H, m, H-5), 3.19 (1H, m, H-1), 3.74 (1H, dd, J = 4.6,
11.5 Hz, H-6a), 3.87 (1H, dd, J = 9.6, 11.5 Hz, H-6b), 3.98 (1H,
from extensive homonuclear decoupling experiments, and the
1
13
DEPT, H– C COSY, HMQC, and HMBC spectroscopic data. FABMS
were measured using glycerol as a matrix on a JEOL JMS-700 spec-
trometer. The purity of samples was checked by HPTLC on Silica
1
3
DDD, J = 1.5, 3.3, 4.1 Hz, H-4); C NMR (125 MHz, D
2
O) d 12.6
(C-8), 29.7 (C-2, 3, 7), 52.6 (C-1), 59.3 (C-5), 60.2 (C-6), 70.5 (C-
+
Gel 60F254 (E. Merck) using the solvent system PrOH/AcOH/H
4:1:1), and a chlorine-o-tolidine reagent or iodine vapor was used
for detection.
2
O
8 2
4); HRFABMS m/z 160.1338 [M+H] (calcd for C H18NO 160.1335).
(
3.3.4. 6-O-b-
D-Glucopyranosyl-2,3-dideoxy-b-1-C-ethyl-1-deoxy-
galactonojirimycin (10)
3
.2. Plant material
Colorless powder; [
22.1874 [M+H] (C14
a
H
]
D
ꢀ1.0 (c 3.15, H
requires 322.1866);
), 1.28 (1H, m, H-2a),
2
O); HRFABMS m/z
+
1
3
28NO
7
H NMR
Roots of A. triphylla var. japonica were purchased at a herbal
medicine shop (Uchida Wakanyaku Co., Tokyo, Japan) in May
008. A voucher specimen (no. RJN2008012) is deposited at the
(500 MHz, D
2
O) d 0.86 (3H, t, J = 7.3 Hz, CH
3
1.45 (2H, H-7a, H-7b), 1.53 (1H, m, H-2b), 1.80 (1H, m, H-3a),
2
1.86 (1H, m, H-3b), 2.87 (1H, m, H-1), 3.25 (1H, dd, J = 8.2,
0
0
herbarium of the Institute of Biological, Environmental and Rural
Sciences, Aberystwyth, UK.
9.2 Hz, H-2 ), 3.34 (1H, t, J = 9.2 Hz), 3.37–3.44 (2H, m, H-5, H-5 ),
0
0
3.45 (1H, t, J = 9.2 Hz, H-3 ), 3.63 (1H, dd, J = 5.5, 12.4 Hz, H-6 a),
.87 (1H, dd, J = 2.3, 12.4 Hz, H-6 b), 3.91 (1H, dd, J = 10.1,
0
3
3
.3. Extraction and isolation
11.0 Hz, H-6a), 3.95 (1H, m, H-4), 4.03 (1H, dd, J = 3.7, 11.0 Hz, H-
0
13
6
b), 4.43 (1H, d, J = 8.2 Hz, H-1 ); C NMR (125 MHz, D
2
O) d 12.7
The bulbs (10 kg) of A. triphylla were extracted with 50% aque-
(C-8), 28.4 (C-7), 29.2 (C-2, C-3), 53.7 (C-1), 57.7 (C-5), 63.5 (C-
0
0
0
0
ous MeOH. The filtrate was applied to a column of Amberlite IR-
6 ), 69.4 (C-4), 69.5 (C-6), 72.4 (C-4 ), 76.0 (C-2 ), 78.4 (C-3 ), 78.8
+
0
0
+
1
20B (2000 mL, H form). The 0.5 M NH
4
OH eluate was concen-
(C-5 ), 106.0 (C-1 ); HRFABMS m/z 322.1874 [M+H] (calcd for
14 7
C H28NO 322.1866).
trated to give a brown oil (89.3 g). The oil was further applied to
ꢀ
Amberlite IRA-400J (OH form) to remove amino acids and pig-
ments, and eluted with H
2
O. This eluate was concentrated and
3.4. Preparation of N-butyl-1-deoxygalactonojirimycin
chromatographed over an Amberlite CG-50 column (2.0 ꢁ 55 cm,
þ
(
NH
form) with H
2
O as eluant (fraction size 8 mL). Fractions
1-Deoxygalactonojirimycin was synthesized in a highly stereo-
4
were divided into three pools: I (fractions 18–50, 28.5 g), II (frac-
tions 51–150, 2.7 g), and III (fractions 32–41, 1.4 g). Each pool
was further chromatographed with Dowex 1-X2 (OH form) with
controlled mode from cis-4,5-oriented dioxanylpiperidene as a chi-
1
7
ral building block according to the literature. The N-butylation of
DGJ was prepared by treated with the butyl bromide and K CO in
ꢀ
2
3
þ
H
H
2
O as eluant and/or Amberlite CG-50 column (NH4 form) with
dimethylformamide. The reaction mixture was evaporated in va-
2
O and 0.5 M NH
-1-deoxygalactonojirimycin (103 mg), DNJ (130 mg),
-glucopyranosyladenophorine (6 mg) from pool I, 1-
deoxymannojirimycin (149 mg), DMDP (1140 mg), -1-C-ethyl-
fagomine (32 mg), and 10 (125 mg) from pool II, 9 (14 mg), and 2
191 mg) from pool III.
4
OH as eluents to give alkaloids 8 (49 mg), b-
cuo, and the residual syrup was resolved in MeOH and applied to
+
1
-C-butyl-
D
an Amberlist 15 column (H form), eluted with 0.5 M NH
4
OH and
and 1-O-b-
D
concentrated. The eluate was finally purified by Dowex 1-X2
ꢀ
þ
a
(OH form) and Amberlite CG-50 column (NH4 form) chromatog-
raphy with water as eluent.
(
3
.4.1. N-Butyl-1-deoxygalactonojirimycin (NB-DGJ)
+
3
.3.1. 2,5-Dideoxy-2,5-imino-
Colorless powder; [ +34.2 (c 0.83, H
O) d 3.19 (1H, ddd, J = 3.9, 4.2, 5.9 Hz, H-5), 3.36 (1H, ddd,
D
-altritol (DIA) (2)
Colorless powder; HRFABMS m/z 220.1547 [M+H] (C10H22NO
4
1
13
a]
D
2
O); H NMR (500 MHz,
2
requires 220.1549); C NMR (125 MHz, D O) d 16.1, 23.0, 27.9,
D
2
55.0 (N-butyl), 58.6 (C-1), 63.3 (C-6), 65.5 (C-5), 69.9 (C-2), 73.0
J = 4.1, 6.6, 6.6 Hz, H-2), 3.67 (1H, dd, J = 5.9, 11.7 Hz, H-6a), 3.67
(C-4), 77.9 (C-3).

3
794
A. Kato et al. / Bioorg. Med. Chem. 18 (2010) 3790–3794
3
.5. Biological assays
3.6. Thermostability of a-Gal A
The enzymes
yeast, assayed at pH 6.8), b-glucosidase (from almond, pH 5.0; from
C. saccharolyticum, pH 5.0; bovine liver, pH 6.8), -galactosidase
from coffee bean, pH 6.5), b-galactosidase (from bovine liver, pH
.8), -mannosidase (from jack beans, pH 4.5), b-mannosidase
from rat epididymis, pH 4.5), -fucosidase (from bovine epidid-
a
-glucosidase (from rice, assayed at pH 5.0; from
The enzyme a-Gal A was incubated at 48 °C in 100 mM sodium
citrate buffer (pH 4.6) containing 1 mg/mL of BSA at various incu-
bation time. After incubation, an aliquot was diluted with fourfold
volume of 100 mM sodium citrate buffer (pH 4.6), and then the
a
(
6
(
a
remaining -Gal A activity was assayed immediately using p-nitro-
a
a
-L
phenyl-a-D-galactoside as substrate.
ymis, pH 5.5), trehalase (from porcine kidney, pH 6.5), amylogluco-
sidase (from Aspergillus niger, pH 4.5), p-nitrophenyl glycosides,
and various disaccharides were purchased from Sigma–Aldrich
Co. Brush border membranes were prepared from the rat small
intestine according to the method of Kessler et al.,18 and were
assayed at pH 5.8 for rat intestinal maltase using maltose. For rice
References and notes
1.
Brady, O. R.; Gal, A. E.; Bradley, R. M.; Marlensson, E.; Warshaw, A. L.; Laster, L.
N. Eng. J. Med. 1967, 276, 1163.
2.
Garman, S. C.; Garboczi, D. N. J. Mol. Biol. 2004, 337, 319.
3. Lieberman, R. L.; D’aquino, J. A.; Ringe, D.; Petsko, G. A. Biochemistry 2009, 48,
816.
4
a-glucosidase and rat intestinal maltase activities, the reaction
4
5
.
.
Fan, J.-Q.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112.
Hamanaka, R.; Shinohara, T.; Yano, S.; Nakamura, M.; Yasuda, A.; Yokoyama, S.;
Fan, J.-Q.; Kawasaki, M.; Ishii, S. Biochem. Biophys. Acta. 2008, 1782, 408.
Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O. R.; Fan, J.-
Q. Eur. J. Biochem. 2000, 267, 4179.
Benjamin, E. R.; Flanagan, J. J.; Schilling, A.; Chang, H. H.; Agarwal, L.; Katz, E.;
Wu, X.; Pine, C.; Wustman, B.; Desnick, R. J.; Lockhart, D. J.; Valenzano, K. J. J.
Inherit. Metab. Dis. 2009, 32, 424.
mixture (0.2 mL) contained 25 mM maltose and the appropriate
amount of enzyme, and the incubations were performed for
6
.
.
1
1
0–30 min at 37 °C. The reaction was stopped by heating at
00 °C for 3 min. After centrifugation (600 g; 10 min), 0.05 mL of
7
the resulting reaction mixture were added to 3 mL of the Glucose
CII-test Wako (Wako Pure Chemical Ind., Osaka, Japan). The absor-
bance at 505 nm was measured to determine the amount of the
8
9
.
.
Fan, J.-Q.; Ishii, S. FEBS J. 2007, 274, 4962.
Shin, S. H.; Murray, G. J.; Kluepfel-Stahl, S.; Cooney, A. M.; Quirk, J. M.;
Schiffmann, R.; Brady, R. O.; Kaneski, C. R. Biochem. Biophys. Res. Commun. 2007,
359, 168.
released D-glucose. Other glycosidase activities were determined
using an appropriate p-nitrophenyl glycoside as substrate at the
optimum pH of each enzyme. The reaction mixture (1 mL)
contained 2 mM of the substrate and the appropriate amount of
enzyme. The reaction was stopped by adding 2 mL of 400 mM
Na CO . The released p-nitrophenol was measured spectrometri-
2 3
cally at 400 nm.
1
0. Asano, N.; Nishida, M.; Miyauchi, M.; Ikeda, K.; Yamamoto, M.; Kizu, H.; Kameda,
Y.; Watson, A. A.; Nash, R. J.; Fleet, G. W. J. Phytochemistry 2000, 53, 379.
1. Ikeda, K.; Takahashi, M.; Nishida, M.; Miyauchi, M.; Kizu, H.; Kameda, Y.;
Arisawa, M.; Watson, A. A.; Nash, R. J.; Fleet, G. W. J. Carbohydr. Res. 2000, 323,
1
73.
1
1
2. Saotome, C.; Kanie, Y.; Kanie, O.; Wong, C.-H. Bioorg. Med. Chem. 2000, 8, 2249.
3. Yu, L.; Ikeda, K.; Kato, A.; Adachi, I.; Godin, G.; Compain, P.; Martin, O.; Asano,
N. Bioorg. Med. Chem. 2006, 14, 7736.
The preparation and in vitro enzyme assay of a-Gal A were per-
formed according to the methods described in our previous paper
1
1
1
1
4. Kato, A.; Kato, N.; Adachi, I.; Hollinshead, J.; Fleet, G. W. J.; Kuriyama, C.; Ikeda,
K.; Asano, N.; Nash, R. J. J. Nat. Prod. 2007, 70, 993.
5. Kato, A.; Kato, N.; Miyauchi, S.; Minoshima, Y.; Adachi, I.; Ikeda, K.; Asano, N.;
Watson, A. A.; Nash, R. J. Phytochemistry 2008, 69, 1261.
6. Remeo, G.; Urso, M.; Piszcane, A.; Blum, E.; de Falco, A.; Ruffilli, A. Biochem.
Genet. 1975, 13, 615.
7. Takahata, H.; Banba, Y.; Ouchi, H.; Nemoto, H. Org. Lett. 2003, 5, 2527.
(
Asano, N., 2000). The Fabry R301Q lymphoblasts were cultured in
DMDM (Dulbecco’s modified Eagle’s medium; Sigma–Aldrich Co.)
supplemented with 10% FCS. Cells were cultured in a water-jacket
incubator at 37 °C under 5% CO
2
in the presence or absence of DIA
for 3 days. The intracellular
scribed previously.
a-Gal A assay was performed as de-
18. Kessler, M.; Acuto, O.; Strelli, C.; Murer, H.; Semenza, G. A. Biochem. Biophys.
Acta 1978, 506, 136.
6