1-Deoxynojirimycins with dansyl capped N-substituents as probes for Morbus Gaucher affected cell lines

Article abstract of DOI:10.1016/j.carres.2010.04.015

Cyclization by double reductive amination of D-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave (methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl- 1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both compounds are strong inhibitors of D-glucosidases and could also be shown to distinctly improve, at subinhibitory concentrations, the activity of β-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line through chaperoning of the enzyme to the lysosome.

Full text of DOI:10.1016/j.carres.2010.04.015

Carbohydrate Research 345 (2010) 1371–1376
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
1-Deoxynojirimycins with dansyl capped N-substituents as probes
for Morbus Gaucher affected cell lines
Richard F. G. Fröhlich a, Richard H. Furneaux a, Don J. Mahuran b, Brigitte A. Rigat b, Arnold E. Stütz c,
,
*
Michael B. Tropak b, Jacqueline Wicki d, Stephen G. Withers d, Tanja M. Wrodnigg c
a Carbohydrate Chemistry Team, Industrial Research Limited, PO Box 31-310, 5040 Lower Hutt, New Zealand
b Department of Laboratory Medicine and Pathobiology, Sick Kids Hospital, 555 University Avenue, University of Toronto, Ontario, Canada M5G 1X8
c Glycogroup, Institut für Organische Chemie, Technische Universität Graz, Stremayrgasse 16, A-8010 Graz, Austria
d Chemistry Department, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclization by double reductive amination of D-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave
Received 20 January 2010
Received in revised form 8 April 2010
Accepted 19 April 2010
(methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl-
1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with
bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both
Available online 24 April 2010
compounds are strong inhibitors of D-glucosidases and could also be shown to distinctly improve, at sub-
inhibitory concentrations, the activity of b-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line
through chaperoning of the enzyme to the lysosome.
Keywords:
Iminoalditol
N-Alkylation
Ó 2010 Elsevier Ltd. All rights reserved.
Glucosidase inhibitor
Molecular chaperone
Gaucher’s disease
1. Introduction
For Gaucher’s disease, which is characterized by deficient activity
of acid b-glucosidase (glucocerebrosidase), N-modified derivatives
Iminoalditols and structurally related alkaloids such as com-
pounds 1–3 are one of the most prominent families of low molec-
ular weight reversible glycosidase inhibitors.1 By virtue of their
powerful interactions with many glycosidases, they exert activity
against retroviruses, tumor growth and metastasis, symptoms of
diabetes type 2 as well as quite a few others.1a,1b,1e
Various N-alkylated derivatives including the N-butyl (1a), N-
nonyl (1b), and N-hydroxyethyl (1c) derivatives of compound 1
(Fig. 1) have become pharmaceutical substances in the treatment
of diabetes type 2 symptoms and other metabolic disorders includ-
ing Gaucher’s disease.2 Earlier, it had been demonstrated that
immobilized N-alkylated iminoalditols, for example, 1e, can be
employed as affinity ligands in glycosidase isolation and purifica-
tion protocols.3
of 1-deoxynojirimycin, were investigated and employed in sub-
strate reduction therapy (the N-butyl derivative 1a is marketed as
Zavesca).6 Subsequently, it was suggested that sub-inhibitory con-
centrations of active site specific molecules (pharmacological chap-
erones) could be exploited for chaperone-mediated therapy (CMT).4
Based upon this concept, these small molecules bind to and stabilize
mutant proteins such as lysosomal b-glucosidase, b-galactosidase,
or b-N-acetylhexosaminidase, once they have reached their func-
tional folded conformations enabling exit from the endoplasmatic
reticulum and subsequent transport to the lysosome. Mutant pro-
teins that cannot obtain or retain their functional conformation
are recognized as misfolded by the quality control machinery in
the endoplasmatic reticulum and are eventually targeted for
degradation.
Recently, iminosugars have been introduced as therapeutic
principles for various forms of lysosomal disorders.4–6 This group
of over 40 hereditary metabolic diseases arises from mutations in
specific genes that lead to deficiencies in enzymes involved in
the lysosomal degradation of glycolipids and glycans.
N-Nonyl-1-DNJ (1b) has become one of the current benchmark
molecules in this context. Recently, unusual structures such as
bicyclic nojirimycin derivatives7 and a lipophilic derivative of isof-
agomine8 have also been found to exert excellent activities in this
context. Other laboratories such as Wong’s9 as well as Aerts and
Overkleeft and their co-workers10 have observed that large, lipo-
philic N-alkyl substituents, in particular adamantyl-substituted
spacer arms, provide excellent interaction between the inhibitor
and the lysosomal b-glucosidase. These leading references, in line
* Corresponding author. Tel.: +0043 316 873 8244; fax: +0043 316 873 8740.
E-mail address: stuetz@tugraz.at (A.E. Stütz).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.04.015

1372
Richard F. G. F. G. Fröhlich et al. / Carbohydrate Research 345 (2010) 1371–1376
R
H
N
N
HO
N
HO
HO
OH
HO
OH
HO
OH
HO
OH
OH
OH
1
R = H
2
3
1a
1b
1c
1d
1e
R = (CH2)3CH3
R = (CH2)8CH3
R = (CH2)2OH
R = (CH2)5CO2Me
R = (CH2)5CO2H
Figure 1. Typical glucosidase inhibitors.
with our interest in fluorescently tagged glycosidase inhibitors for
diagnostic purposes,11,12 have recently led us to initiate the prepa-
O
O
S
c
ration of a range of N-modified 1,5-iminoglucitols from
hexos-5-ulose (4), amongst them compounds 10 and 11.
D-xylo-
HO
(CH2)6
NH2
RO
d
(CH2)6
NH
5
6
: R = H
: R = Ms
2. Results and discussion
N
Both compounds were synthesized from known13 N-carboxy-
pentyl-1-deoxynojirimycin (1e), which, in turn, had been accessed
H
N
e
6
R
dansyl
by reaction of D
-xylo-hexos-5-ulose11 (4) with methyl 6-aminohex-
anoate under reductive amination conditions (H2, Pd(OH)2–C, in
MeOH at ambient pressure) to give 1d, followed by saponification
of the methyl ester with NaOH in water–dioxane (Scheme 1).
Reaction of intermediate 1e with N-dansyl-1,6-diaminohexane
8 (prepared in four simple steps from 6-aminohexanol via N-dan-
syl derivative 5, the corresponding mesylate 6 and azide 7, Scheme
2) under standard peptide coupling conditions (TBTU, triethyl-
amine, DMF) gave iminoalditol 10 as a yellow-green fluorescent
syrup in 43% yield after extensive purification on silica gel.
For the synthesis of compound 11, N-dansyl-1,6-diaminohex-
ane 8 was reacted with N-dansyl-O-mesyl-6-aminohexanol (6) in
N,N-dimethylformamide in the presence of sodium carbonate
(Scheme 3).
7: R = N3
a
8: R = NH2
Scheme 2. Reagents and conditions: (c) dansylCl, NEt3, CH2Cl2; 68%; (d) MsCl, NEt3,
CH2Cl2; 92%; (e) NaN3, DMF, 90%.
NH-dansyl
NH-dansyl
(CH2)6
(CH2)6
f
+
6
8
HN
9
Scheme 3. Reagents and condition: (f) Na2CO3, DMF, 51%.
The resulting bis(6-dansylaminohexyl)amine
9
reacted
10 and 11, significantly inhibited lysosomal b-glucosidase (Cere-
smoothly with intermediate 1e (TBTU, triethylamine, DMF) to give
final product 11 in 84% yield after chromatography (Scheme 4).
In a general screen against three selected glucosidases, com-
pound 10 was found to be a powerful inhibitor of b-glucosidases
zyme). Compound 10 had a Ki value of 3.8
lM, whereas compound
11 was double as active with Ki = 1.7 M. Thus, both inhibitors
l
were tested as potential pharmacological chaperones.
Their influence on residual glucocerebrosidase in fibroblasts
homozygous for the common N370S mutation found in type
Gaucher patients was compared with Ambroxol (ABX) (Fig. 2).
Although ABX has been used to treat humans for airway mucus
hyper-secretion diseases such as chronic bronchitis, it was recently
found to have a secondary side activity as a glucocerebrosidase
from Agrobacterium sp. (Ki = 0.99
as well as from almonds (Ki = 0.73
against yeast -glucosidase. Compound 11 was more active than
inhibitor 10 against the Agrobacterium sp. enzyme (Ki = 0.22 M)
but was much less potent with the almond glucosidase (Ki ca.
80 M) and, akin to 1 and 10, did not inhibit the -selective gluco-
l
M; compound 1: Ki = 12
lM)
l
M) but devoid of activity
a
l
l
a
inhibitor (Ki = 23
3
l
M at pH 5.6) and pharmacological chaper-
sidase screened (1e was not an inhibitor of these enzymes). Both,
OH
OR
O
OH
OH
N
HO
HO
H2N-(CH2)5-CO2Me
a
O
HO
HO
OH
1d
: R = Me
b
1e
: R = H
OH
OH
4
1d: R = Me
1e: R = H
Scheme 1. Reagents and conditions: (a) H2, Pd–C, MeOH, 69%; (b) NaOH, dioxane–water, 95%.

Richard F. G. F. G. Fröhlich et al. / Carbohydrate Research 345 (2010) 1371–1376
1373
NH
NH-dansyl
N
(CH2)6
10
8
HO
HO
O
O
HO
OH
g
OH
N
1e
NH-dansyl
NH-dansyl
(CH2)6
(CH2)6
N
9
HO
OH
11
OH
Scheme 4. Reagents and conditions: (g) DBTU, NEt3, DMF (10: 43%; 11: 84%).
range of concentrations probed and showed a statistically signifi-
cant maximal activity increase of 60% at 30 M. This effect is spe-
cific for glucocerebrosidase, as the relative activity of another
lysosomal enzyme, b-N-acetylhexosaminidase, did not signifi-
cantly differ from DMSO treated patient cells at this concentration
or other concentrations of 11.
Based on the encouraging, albeit preliminary, results of this
exploratory study, we are now engaged in several structural re-
iterative modifications of the N-substituent in compound 11
looking for an optimal balance between undesired inhibition of
lysosomal and other glucosidases and beneficial sub-inhibitory
chaperoning effects of the particular enzyme under consideration
here.
Br
Br
l
NH
OH
NH2
Figure 2. Structure of Ambroxol (ABX).
one.14 Relative to DMSO treated patient cells, ABX is effective at
enhancing mutant lysosomal glucocerebrosidase activity more
than 50% at 10–30
l
M concentrations. Compound 10 was found
somewhat less effective than ABX with best effects at a 20-
centration causing a 25% relative increase of enzymatic activity
l
M con-
3. Experimental
(Fig. 3). Compound 11 was found to be more effective across the
3.1. General methods
Optical rotations were measured on a Perkin Elmer 341 polar-
imeter at the wavelength of 589 nm and a path length of 10 cm
at 20 °C. NMR spectra were recorded at 300.13 MHz (1H) and at
75.4 MHz (13C). CDCl3 was employed for protected compounds
and methanol-d4 for unprotected inhibitors. Chemical shifts are
listed in delta employing residual, non-deuterated solvent as the
internal standard. The signals of the protecting groups were found
in the expected regions and are not listed explicitly. Electrospray
mass spectra were recorded on an HP 1100 series MSD, Hewlett
Packard. Samples were dissolved in acetonitrile or acetonitrile/
water mixtures. The scan mode for positive ions (mass range
100–1000 D) was employed varying the fragmentation voltage
from 50 to 250 V with best molecular peaks observed at 150 V.
MALDI mass spectra were recorded on a MALDI Micro MX (Waters)
time-of-flight instrument used in reflectron mode with 2.3 m effec-
tive flight path. Analytical TLC was performed on precoated alumi-
num plates Silica Gel 60 F254 (E. Merck 5554), detected with UV
light (254 nm), 10% vanillin–sulfuric acid as well as ceric ammo-
nium molybdate (100 g ammonium molybdate/8 g ceric sulfate
in 1 L 10% H2SO4) and heated on a hotplate. For column chromatog-
raphy Silica Gel 60 (230–400 mesh, E. Merck 9385) was used.
3.2. Kinetic studies
Almond b-glucosidase and Saccharomyces cerevisiae (yeast)
a
-glucosidase were obtained from Sigma. Agrobacterium sp. b-glu-
Figure 3. Relative lysosomal b-glucosidase and b-N-acetylhexosaminidase activi-
ties in Gaucher fibroblast (N370S/N370S) cell-line treated with ABX, 10 and 11.
Lysosomal enzyme activities of b-glucosidase (A) and b-N-acetylhexosaminidase (B)
for each dose of ABX, 10 or 11 were normalized to activities found in lysates from
vehicle (0.1% DMSO) treated patient cells. All treatments were performed in
triplicate. Treatment groups showing a statistically significant increase relative to
DMSO treated cells are marked with an asterisk (p <0.05). Dashed line demarcates
relative activity levels greater or less than 1.
cosidase was produced and purified as described previously.13,15
Purified human recombinant lysosomal acid b-glucocerebrosidase
was purchased from Genzyme (Cambridge, MA, USA). All buffer
chemicals and substrates were obtained from Sigma (USA).
Kinetic studies using non-lysosomal enzymes were performed
at 37 °C in 50 mM sodium phosphate buffer (pH 7.0 or pH 6.5).

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Richard F. G. F. G. Fröhlich et al. / Carbohydrate Research 345 (2010) 1371–1376
Approximate values of Ki were determined using a fixed concentra-
tion of substrate, either 4-nitrophenyl b- -glucopyranoside or 4-
nitrophenyl -glucopyranoside at a concentration equal to 1–2
mixture was stirred for 16 h in the dark. Solvents were removed
under reduced pressure and the remaining residue was dissolved
in dichloromethane (2.5 mL) and purified on silica gel employing
cyclohexane–EtOAc 1:3 v/v as the eluant to give compound 5
(633 mg, 68.5%) as a yellow oil. 13C NMR (125 MHz, CDCl3):
d = 152.0, 135.1, 130.4, 130.1, 129.8, 129.6, 128.5, 123.4, 119.2,
115.3, 62.6, 45.6 (2C), 43.2, 32.4, 29.5, 26.2, 25.2. MS calcd for
[C18H26N2O3S]: m/z 350.48352; found 351.490 [M+H]+, 373.470
[M+Na]+.
D
a
-D
times the Km value, and inhibitor concentrations ranging from
0.2 to 5 times the Ki value ultimately determined. Initial rates were
determined by continuous measurement of the release of nitro-
phenolate using a Unicam UV4 spectrophotometer equipped with
a temperature controlled circulating water bath. Reactions were
initiated by the addition of enzyme, and the release of nitrophen-
olate was monitored at 400 nm. The concentration of enzyme
added and the length of time that the reaction was monitored in
each case were selected such that less than 10% of the total sub-
strate was converted to product. A horizontal line drawn through
1/Vmax in a Dixon plot of these data (1/V vs [I]) intersects the exper-
imental line at an inhibitor concentration equal to ꢀKi.
3.5. N-Dansyl-O-mesyl-6-aminohexanol (6)
To a 3% solution of compound 5 (570 mg, 1.63 mmol) in dichlo-
romethane containing triethylamine (0.7 mL), methanesulfonyl
chloride (0.15 mL, 2.0 mmol) was added at 0 °C and the mixture
was stirred for 1 h. The reaction mixture was diluted with dichlo-
romethane and the solution was washed with water, then dried
(Na2SO4), filtered and the solvent was removed under reduced
pressure to give 640 mg (92%) of the product which was suffi-
ciently pure for the next step. 1H NMR (500 MHz, CDCl3): d = 8.53
(d, 1H, J = 8.5 Hz), 8.29 (d, 1H, J = 8.5 Hz), 8.25–8.22 (m, 1H),
759–7.49 (m, 2H), 7.18 (d, 1H, J = 7.5 Hz), 4.91 (br s, 1H), 4.10 (m,
2H), 2.97 (s, 3H), 2.90–2.85 (m, 8H), 1.60–1.52 (m, 2H), 1.42–1.33
(m, 2H), 1.30–1.14 (m, 4H). 13C NMR (125 MHz, CDCl3): d = 152.3,
135.0, 134.9, 130.7, 130.1, 129.8, 128.7, 123.5, 119.1, 115.5, 70.1,
45.7 (2C), 43.2, 37.6, 29.5, 29.0, 25.9, 25.0. MS calcd for
[C19H28N2O5S2]: m/z 428.57341; found 428.580 [M+H]+.
Kinetic studies using b-glucocerebrosidase (7.5 lg/mL) were
performed in 100 mM citrate phosphate buffer pH 5.5 containing
0.2% taurodeoxycholate at 37 °C for 30 min. Experiments were car-
ried out using 4-nitrophenyl b-D-glucopyranoside substrate at con-
centrations ranging from approximately 0.2 to 5 times Km in the
presence of inhibitors at final concentrations ranging from 0.5 to
3 times the Ki value that was ultimately ascertained. Ki values were
determined by fitting the data to the competitive model described
by KmObserved = Km ꢁ (1 + [I]/Ki) and the Michaelis–Menten equation
using non-linear regression function within Prism 5.0 (Graphpad)
software (where KmObserved is the Km observed in the presence of
inhibitor).
3.3. Treatment of Gaucher patient cells and intracellular
lysosomal enzyme activity
3.6. N-Dansyl-1-azido-6-aminohexane (7)
Primary skin fibroblasts derived from a patient with Gaucher
disease homozygous for the N370S mutation (c.1226A>G/
c.1226A>G) were provided by the Hospital for Sick Children Tissue
Culture Facility. Fibroblast cells were grown in complete minimal
essential medium from Wisent Inc. (Canada) in the presence of
5% antibiotics (penicillin and streptomycin, Invitrogen), supple-
mented with 10% fetal bovine serum (FBS, Wisent Inc., Canada),
and incubated at 37 °C in a humidified atmosphere with 5% CO2.
To evaluate the intracellular enzyme activity enhancement of the
compounds, cells were grown to 70–80% confluence in 96-well tis-
sue culture plates. Threefold serial dilutions of the compounds in
DMSO were prepared and added to growth media at a 100-fold
dilution. Patient cells were grown in the presence of the com-
pounds for five days at 37 °C as described above. Prior to enzyme
activity analysis, media were removed, cells were washed with
two changes of phosphate buffered saline and lysed in the presence
of 200 mM citrate phosphate buffer pH 5.5 containing 0.4% Triton
X-100 and 0.2% taurodeoxycholate at 4 °C for 1 h. Aliquots of the
lysate were added to an equal volume of 10 mM 4-methylumbel-
To a 4% solution of mesylate 6 (257 mg, 0.60 mmol) in N,N-
dimethylformamide, sodium azide (119 mg, 3 equiv) was added
and the mixture was stirred at 90 °C for 2 h when TLC indicated com-
pleted conversion of the starting material. The reaction mixture was
concentrated under reduced pressure and the residue was parti-
tioned between water and dichloromethane. The aqueous layer
was extracted with dichloromethane and the combined organic lay-
ers were dried (Na2SO4). After filtration and removal of the solvent
under reduced pressure the residue was purified by chromatogra-
phy to give 205 mg (90%) of product 7. 1H NMR (500 MHz, CDCl3):
d = 8.53 (d, 1H, J = 8.5 Hz), 8.32 (d, 1H, J = 8.5 Hz), 8.24 (d, 1H,
J = 7.3 Hz), 7.58–7.49 (m, 2H), 7.18 (d, 1H, J = 7.5 Hz), 5.01 (m, 1H),
3.10 (m, 2H), 2.93–2.84 (m, 8H), 1.42–1.30 (m, 4H), 1.18–1.07 (m,
4H); 13C NMR (125 MHz, CDCl3): d = 152.2, 135.0, 130.6, 130.1,
129.8, 129.75, 128.6, 123.4, 119.0, 115.4, 51.4, 45.7 (2C), 43.4, 29.5,
28.7, 26.2, 26.1. MS calcd for [C18H25N5O2S]: m/z 375.49625; found
376.500 [M+H]+.
3.7. N-Dansyl-1,6-diaminohexane (8)
liferyl b-
D-glucopyranoside or 3.2 mM 4-methylumbelliferyl b-N-
acetyl- -glucosaminide and were incubated at 37 °C for 2 h or
D
To a 1% solution of azide 7 (387 mg, 1.03 mmol) in MeOH, Pd/C
(10%, 86 mg) was added and the mixture was stirred under an
atmosphere of hydrogen at ambient pressure for 1 h. After filtra-
tion, the solvent was removed under reduced pressure and the
remaining residue was purified by chromatography on silica gel
employing a mixture of CHCl3–MeOH–concd NH4OH (30:10:1 v/
v/v) as the solvent system to give pure product 8 (280 mg, 78%)
as a fluorescent syrup. 1H NMR (500 MHz, CD3OD): d = 8.53 (m,
1H), 8.36 (m, 1H), 8.17 (m, 1H), 7.59–7.51 (m, 2H), 7.23 (m, 1H),
2.87–2.79 (m, 8H), 2.52–2.45 (m, 2H), 1.34–1.20 (m, 4H), 1.15–
1.00 (m, 4H); 13C NMR (125 MHz, CD3OD): d = 153.1, 137.2,
131.1, 131.0, 130.9, 130.1, 129.0, 124.3, 120.6, 116.4, 45.8 (2C),
43.7, 42.1, 32.9, 30.4, 27.2. MS calcd for [C18H27N3O2S]: m/z
349.49879; found 350.510 [M+H]+.
15 min to measure GCC or Hex activity, respectively. Reactions
were terminated by the addition of four volumes of 0.1 M 2-ami-
no-2-methyl-1-propanol (pH 10.5). Fluorescence was quantified
using a Molecular Devices M2 Spectrofluorometer plate reader
using excitation and emission wavelengths of 365 and 450 nm,
respectively. Activity values for each dose of ABX, 10 or 11 were
normalized to activities found in lysates from vehicle (0.1% DMSO)
treated patient cells.
3.4. N-Dansyl-6-aminohexanol (5)
To a 2% solution of 6-aminohexanol (309 mg, 2.64 mmol) in
dichloromethane containing triethylamine (2 mL), dansyl chloride
(690 mg, 2.56 mmol) was added at ambient temperature and the

Richard F. G. F. G. Fröhlich et al. / Carbohydrate Research 345 (2010) 1371–1376
1375
3.8. Bis(6-dansylaminohexyl)amine (9)
(m, 1H), 2.26 (dd, 1H, J = 10.9 Hz, J = 11.2 Hz), 2.18–2.11 (m, 2H),
1.66–1.45 (m, 4H), 1.34–1.21 (m, 6H), 1.15–1.00 (m, 4H); 13C
NMR (125 MHz, MeOH-d4): d = 175.9 (C-60), 153.1, 137.2, 131.1,
131.0, 130.9, 130.1, 129.0, 124.3, 120.6, 116.4 (dansyl), 80.2, 71.6,
70.4, 67.3, 58.9, 57.3 (C-1, C-2, C-3, C-4, C-5, C-6), 53.6, 45.8,
43.7, 40.1 (10, 100, 600, NMe2), 36.9, 30.4, 30.1, 28.0, 27.2, 27.0, 26.8,
24.7. MS: calcd for [C30H48N4O7S]: m/z 608.80366; ESIMS found:
[M+H]+ 609.8506, [M+Na]+ 631.7701.
To a solution of mesylate 6 (28 mg, 70 lmol) and amine 8
(23 mg, 66 lmol) in N,N-dimethylformamide (1 mL) Na2CO3
(42 mg, 0.40 mmol) was added and the mixture was stirred at
55 °C for 48 h. The solvent was removed under reduced pressure
and the remaining material was purified by chromatography on
silica gel employing CHCl3–MeOH–concd NH4OH 70:10:1 v/v/v as
the solvent system to give product 9 (23 mg, 51%) as a slightly yel-
low syrup. 1H NMR (500 MHz, CDCl3): d = 8.55–8.50 (m, 2H), 8.31–
8.26 (m, 2H), 8.25–8.20 (m, 2H), 7.57–7.48 (m, 4H), 7.19–7.15 (m,
2H), 2.90–2.84 (m, 16H), 2.46 (t, 4H, J = 7.3 Hz), 1.40–1.28 (m, 8H),
1.20–1.08 (m, 8H); 13C NMR (125 MHz, CDCl3): d = 152.2, 135.0,
130.6, 130.1, 130.0 (2C), 128.6, 123.5, 119.0, 115.4, 49.9, 45.7
(2C), 43.4, 29.8, 29.6, 26.8, 26.4. MS calcd for [C36H51N5O4S2]: m/z
681.99697; found 683.010 [M+H]+.
3.11. N-Bis(dansylaminohexyl)aminocarbonylpentyl-1,5-
dideoxy-1,5-imino-D-glucitol (11)
To an aqueous solution (1.5 mL) of N-methoxycarbonylpentyl-
1-deoxynojirimycin (1d, 32 mg, 0.11 mmol), 0.5 M aqueous NaOH
(0.30 mL, 0.15 mmol) was added and the mixture was kept at
ambient temperature for 60 min. The mixture was brought to pH
5 by the addition of aqueous HCl (5%) and the solvent was removed
under reduced pressure. Bis(6-dansylamino)hexylamine (9,
100 mg, 0.15 mmol) in dichloromethane (3 mL) was added to the
residue and the solvent was again removed under reduced pres-
sure. The residue was dissolved in DMF (1.5 mL), triethylamine
3.9. N-Methoxycarbonylpentyl-1,5-dideoxy-1,5-imino-D-
glucitol (1d)
To a 5% aqueous solution of D
-xylo-hexos-5-ulose11 (4, 250 mg,
1.27 mmol), triethylamine (0.25 mL, 1.78 mmol), methanol
(10 mL), methyl 6-aminocaproate hydrochloride (279 mg,
1.53 mmol, 1.2 equiv), and Pd(OH)2 (20%, 100 mg) were added
and the mixture was stirred under an atmosphere of hydrogen
for 72 h. The catalyst was removed by filtration and washed with
MeOH. Filtrate and washings were combined and concentrated un-
der reduced pressure. For easier removal of excess aminocaproate,
MeOH (10 mL) and triethylamine (0.25 mL, 1.78 mmol) followed
by di-tert-butyldicarbonate (336 mg, 1.53 mmol) were added and
the mixture was stirred at ambient temp for 30 min. The reaction
mixture was concentrated under reduced pressure and the
remaining residue was purified by chromatography on silica gel
(CHCl3–MeOH–concd NH4OH, 500:100:6 v/v/v) to give known11,16
(30 lL, 0.22 mmol) and TBTU (96%, 39 mg, 0.12 mmol) were added.
After completed conversion (TLC, ca. 10 min), the mixture was con-
centrated under reduced pressure and the residue was purified by
chromatography (CHCl3–MeOH–concd NH4OH, 50:10:1 v/v/v) pro-
viding compound 11 (87 mg, 84%) as a fluorescent yellow syrup.
½
a 2D0
ꢂ
ꢀ 3:9 (c 1.5, MeOH); 1H NMR (500 MHz, MeOH-d4): d = 8.53–
8.46 (m, 2H), 8.36 (d, 2H, J = 8.6 Hz), 8.20–8.14 (m, 2H), 7.58–
7.49 (m, 4H), 7.24–7.17 (m, 2H), 3.89–3.78 (m, 2H), 3.69–3.53
(m, 2H), 3.37 (dd, 1H, J = 9.3 Hz, J = 9.9 Hz), 3.15 (dd, 1H), 3.11–
2.93 (m, 5H), 2.86–2.75 (m, 17H), 2.63–2.51 (m, 1H), 2.27–2.09
(m, 4H), 1.63–1.42 (m, 4H), 1.34–1.19 (m, 11H), 1.14–1.03 (m,
4H), 1.03–0.93 (m, 4H); 13C NMR (125 MHz, MeOH-d4): d = 174.8
(C-60), 153.1 (2C), 137.0 (2C), 131.1 (2C), 131.0 (2C), 130.9 (2C),
130.1 (2C), 129.1 (2C), 124.3 (2C), 120.6 (2C), 116.4 (2C), 80.5,
73.5, 71.9, 70.7, 67.3, 62.2, 59.3, 57.6, 53.6, 46.8, 45.9, 43.7, 43.6,
33.8, 30.4, 30.3, 29.7, 28.3, 28.2, 27.2, 27.1, 27.0 (2C), 26.5, 25.0.
MS: calcd for [C48H72N6O9S2]: m/z 941.27184; ESIMS found:
[M+H]+ 942.2888, [M+Na]+ 964.2890.
compound 1d (59 mg, 16% as a colorless syrup). ½a D20
ꢀ 12:7 (c 2.0,
ꢂ
MeOH); 13C NMR (125 MHz, MeOH-d4): d = 175.9 (C-60), 80.5, 72.0,
70.7, 67.3, 59.4, 57.7 (C-1, C-2, C-3, C-4, C-5, C-6), 53.5 (OMe), 52.0
(C-10), 34.7, 28.0, 25.6, 24.8 (C-20, C-30, C-40, C-50). MS: calcd for
[C13H25NO6]: m/z 291.34730; ESIMS found: [M+H]+ 292.510,
[M+Na]+ 314.250.
3.10. N-(Dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-
Acknowledgments
1,5-imino-D-glucitol (10)
Financial support by the Austrian Fonds zur Förderung der Wis-
senschaftlichen Forschung (FWF), Vienna (Project P18998-N17) as
well as Canadian Institutes of Health Research Team Grant CTP-
82944 (to D. M., S.G.W and M.B.T) is gratefully acknowledged.
We are grateful for the excellent technical assistance of Sayuri
Yonekawa.
To an aqueous solution (3 mL) of N-methoxycarbonylpentyl-1-
deoxynojirimycin (1d, 132 mg, 0.45 mmol), 0.5 M aqueous NaOH
(1.0 mL, 0.5 mmol) was added and the mixture was kept at ambi-
ent temperature for 90 min. The mixture was brought to pH 5 by
the addition of aqueous HCl (5%) and the solvent was removed un-
der reduced pressure. Mono-N-dansyl-1,6-diaminohexane (6,
200 mg, 0.57 mmol) in dichloromethane (5 mL) was added to this
crude 1e and the solvent was again removed under reduced pres-
sure. The residue was dissolved in DMF (4.5 mL), triethylamine
(125 lL, 0.9 mmol) and TBTU (96%, 167 mg, 0.5 mmol) were added.
After completed conversion (TLC, ca. 10 min), the mixture was con-
centrated under reduced pressure. Dichloromethane (8 mL), trieth-
ylamine (60 mL, 0.43 lmol), and di-t-butyldicarbonate (35 mg,
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