Fluorescently tagged iminoalditol glycosidase inhibitors as novel biological probes and diagnostics

Article abstract of DOI:10.1016/j.bmc.2005.10.021

1,5-Dideoxy-1,5-imino-d-glucitol, the corresponding d-manno and l-ido epimers as well as the powerful β-glucosidase inhibitor isofagomine were N-alkylated with di-, tri-, as well as tetraethylene glycol derived straight chain spacer arms by a set of simple standard procedures. The terminal functional groups of the spacer arms, primary amines, were employed to introduce fluorescent dansyl moieties. Resulting derivatives showed glycosidase inhibitory activities comparable to those of the parent compounds'.

Full text of DOI:10.1016/j.bmc.2005.10.021

Bioorganic & Medicinal Chemistry 14 (2006) 1737–1742
Fluorescently tagged iminoalditol glycosidase inhibitors as
novel biological probes and diagnostics
Inge Lundt,b Andreas J. Steiner,a Arnold E. Stutz,a,* Chris A. Tarling,c Stefan Ully,a
¨
Stephen G. Withersc and Tanja M. Wrodnigga
aGlycogroup, Institut fu¨r Organische Chemie, Technische Universita¨t Graz, Stremayrgasse 16, A-8010 Graz, Austria
bDepartment of Chemistry, Technical University of Denmark (DTU), Building 201, DK-2800 Kgs. Lyngby, Denmark
cDepartment of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
Received 1 August 2005; revised 4 October 2005; accepted 13 October 2005
Available online 7 November 2005
Abstract—1,5-Dideoxy-1,5-imino-D-glucitol, the corresponding D-manno and L-ido epimers as well as the powerful b-glucosidase
inhibitor isofagomine were N-alkylated with di-, tri-, as well as tetraethylene glycol derived straight chain spacer arms by a set
of simple standard procedures. The terminal functional groups of the spacer arms, primary amines, were employed to introduce
fluorescent dansyl moieties. Resulting derivatives showed glycosidase inhibitory activities comparable to those of the parent
compoundsÕ.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
imino-D-mannitol (or DMDP) are powerful inhibitors
exceeding the parent compoundÕs activity by 2 orders
of magnitude.6
Iminosugars including iminoalditols and related bicyclic
alkaloids are well known, (usually) competitive, glycosi-
dase inhibitors.1 Representatives of this class of com-
pounds have found important roles as biological
probes, such as in the investigation of glycoprotein trim-
ming glycosidases2 or as pharmaceutical substances such
as in the treatment of diabetes type II symptoms.3 Other
biological activities associated with their glycosidase
inhibitory properties are anti-viral, anti-cancer and
anti-metastatic, anti-infective as well as insect anti-fee-
dant and plant growth regulating effects.4
2. Results and discussion
With the aim of evaluating a more general means of
tagging of iminosugars than was employed in the previ-
ously mentioned, structurally very restricted case, we
resorted to the connection of fluorophores to the iminosu-
gars via medium length spacer-arms which had been
found suitable in previous immobilization studies.5
In particular, oligoethylene glycol derived spacer arms,
which minimise the non-specific adsorption of virtually
all proteins over a wide range of conditions, have
frequently been prepared and employed as spacer arms
between ligands and their molecular tags as well as func-
tioning as constituents of self-assembling monolayers.7
It was demonstrated that immobilized N-alkylated imi-
noalditols can be employed as affinity ligands in glycosi-
dase isolation and purification protocols.5 This
derivatisation has frequently been associated with a loss
of inhibitory activity of around 1–2 orders of magnitude
but in this case the derivatives were found to be suffi-
ciently active for the purpose.5
2.1. Syntheses
Recently, we have found that some fluorescently labelled
derivatives of the glucosidase inhibitor 2,5-dideoxy-2,5-
Initially deoxynojirimycin (1a) was alkylated with three
linker arms of different lengths to yield the three
azido-terminated species 1b–d. The azides on each of
these linkers were then reduced and treated with dansyl
chloride to yield the three fluorescent derivatives 1e–g.
Compounds in the D-manno (2a–c), L-ido (3a–c) and
isofagomine (4a–c) series were each converted into a
Keywords: Iminoalditol; Glycosidase inhibitor; Fluorescent tag;
Diagnostic.
*
Corresponding author. Fax: +43 316 873 8740; e-mail: stuetz@
orgc.tu-graz.ac.at
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.021

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I. Lundt et al. / Bioorg. Med. Chem. 14 (2006) 1737–1742
OH
OH
H
N3
N
N
b
a
HO
HO
HO
HO
O
n
HO
HO
1a
1b: n=1
1c: n=2
1d: n=3
OH
O
H
N
S
N
HO
HO
O
n
O
N
HO
1e: n=1
1f: n=2
1g: n=3
OH
OH
OH
OH
HO
OH
R
N
HO
HO
HO
R
N
R
HO
N
OH
2a
2b
2c
3a
3b
3c
4a
4b
4c
a: R = H
b: R = (CH2)2O(CH2)2N3
c: R = (CH2)2O(CH2)2NHdansyl
Scheme 1. Reagents: (a) DMF, Na2CO3, TosOCH2(CH2OCH2)nCH2N3, b: n = 1, c: n = 2, d: n = 3; (b) i: Pd/C, MeOH, H2; ii: Dansy1Cl.
single linker arm derivative with the 5-azido-3-oxapentyl
linker obtaining 2b, 3b and 4b, and then in each case this
derivative was reduced and dansylated to yield 2c, 3c
and 4c. The choice of this specific linker arm length
was based upon the kinetic results obtained with the
deoxynojirimycin series, as described below (Scheme 1).
parent deoxynojirimycin (1e–g, Table 1). The substan-
tially worse inhibition seen for the azido-terminated link-
er arms likely has its origins in the relatively high water
solubility of the polyethylene glycol-based linker arm,
an effect which increases with length. Partitioning of
the linker arm of the inhibitor between free aqueous
solution and the (relatively) hydrophobic interior of the
enzyme active site becomes progressively worse as the
linker arm lengthens. These differences in affinity are
2.2. Inhibitory activities
Inhibition constants determined for each of the com-
pounds synthesized are presented in Tables 1–3. The
deoxynojirimycin analogues were tested as inhibitors of
the Agrobacterium sp. b-glucosidase.8 Addition of the
azido linker arms (1b–d, Table 1) substantially reduced
activities relative to deoxynojirimycin 1a, with the inhibi-
tion worsening as the linker arm lengthened. This is pre-
sumably a consequence both of conversion of the
secondary amine function to a tertiary amine, thereby
possibly sterically hindering the interaction of the nitro-
gen with the active site carboxylic acid. This interaction
has been shown to be important in structural studies with
other Clan GH-A members.9 Further, mutants of the
Agrobacterium sp. b-glucosidase in which the acid/base
side-chain carboxylate has been replaced are known to
bind deoxynojirimycin significantly more weakly. Fortu-
nately, subsequent dansylation of the reduced linker arm
largely restored the inhibitory potency to that of the
Table 1. Ki values of N-substituted compounds against Agrobacterium
sp. b-glucosidase
Compound
Ki (lM)
OH
H
N
1a
12
HO
HO
HO
OH
OH
N3
N
1b n = 1
1c n = 2
1d n = 3 1400
74
HO
HO
O
O
n
n
800
HO
O
S
H
N
N
HO
HO
1e n = 1
1f n = 2
1g n = 3
12
39
34
O
N
HO

I. Lundt et al. / Bioorg. Med. Chem. 14 (2006) 1737–1742
1739
Table 2. Ki values of N-substituted 1-deoxymannonojirimycin ana-
logues against jack bean a-mannosidase
precoated aluminium plates silica gel 60 F254 (Merck
5554), detected with UV light (254 nm), 5% vanillin/
sulfuric acid as well as ceric ammonium molybdate
(100 g ammonium molybdate/4 g ceric sulfate in 1 L
10% H2SO4) and heated on a hotplate. For column
chromatography silica gel 60 (230–400 mesh, Merck
9385) was used. Optical rotation was measured on a
Perkin Elmer 341 polarimeter at the wavelength of
589 nm and a path length of 10 cm at 20 ꢁC.
Compound
Ki (lM)
OH
OH
N
2a R = H
2b R = (CH2)2O(CH2)2N3
2c R = (CH2)2O(CH2)2Nhdansyl
18
155
78
HO
HO
R
Table 3. Ki values of N-substituted 1-deoxyidonojirimycin and isof-
agomine analogues against Agrobacterium sp. b-glucosidase
3.1.1. Kinetic studies. Agrobacterium sp. b-glucosidase
was purified and assayed as described.11 Kinetic studies
were performed at 37 ꢁC in pH 7.0 sodium phosphate
buffer (50 mM) containing 0.1% bovine serum albumin,
using 7.2 · 10ꢀ5 mg/mL enzyme. Approximate values of
Ki were determined using a fixed concentration of
substrate, 4-nitrophenyl-b-D-glucopyranoside (0.11 mM =
1.5 · Km), and inhibitor concentrations ranging from
0.2 times to 5 times the Ki value ultimately determined.
A horizontal line drawn through 1/Vmax in a Dixon plot
of these data (1/V vs [I]) intersects the experimental line
at an inhibitor concentration equal to ꢀKi. Full Ki
determinations, where required, were performed using
the same range of inhibitor concentrations while also
varying substrate (4-nitrophenyl glucoside) concentra-
tions from approximately 0.015 to 0.6 mM. Data were
analysed by direct fit to the Michaelis Menten equation
describing the reaction in the presence of inhibitors using
the program GraFit.12
Compound
Ki (lM)
OH
HO
OH
R
N
3a R = H
3b R = (CH2)2O(CH2)2N3
3c R = (CH2)2O(CH2)2NHdansyl
26
720
32
OH
OH
4a R = H
4b R = (CH2)2O(CH2)2N3
4c R = (CH2)2O(CH2)2NHdansyl
0.007
31
19
HO
R
HO
N
largely suppressed in the dansylated derivatives, possibly
because the hydrophobic dansylated terminus dominates
its properties. Similar effects had been observed with
coumarin, dapoxyl as well as acrylodan fluorophors.10
Based upon these results, only the shortest linker arm was
employed in the synthesis of the manno- and ido-deoxy-
nojirimycin and iosofagomine derivatives. Once again,
in each case, introduction of the linker reduced affinity,
but this deleterious effect was again largely reversed upon
dansylation (Tables 2 and 3). This effect of linker arm
addition was largest in the isofagomine series—most
likely because interactions of the active site nucleophile
with the ring nitrogen seem to be particularly important,
as seen in structural studies on the Clan GH-A enzyme
Cex.9 These classes of fluorescently tagged inhibitors are
now under investigation as tools for proteomic analyses.
Their uses could include activity-based staining of poly-
acrylamide gel separations of proteomes or in competitive
displacement assays of other enzyme inhibitors for
arrayed enzymes.
a-Mannosidase from jack bean was purchased from
Sigma. Kinetic studies were performed at 25 ꢁC in pH
6.8 sodium phosphate buffer (50 mM) containing 0.1%
bovine serum albumin, using 3.7 · 10ꢀ3 mg/mL enzyme.
Approximate values of Ki were determined using a
fixed concentration of substrate, 4-nitrophenyl-a-D-
mannopyranoside (1.3 mM = 1.5 · Km), and inhibitor
concentrations ranging from 0.2 times to 5 times the Ki
value ultimately determined. A horizontal line drawn
through 1/Vmax in a Dixon plot of these data (1/V vs [I])
intersects the experimental line at an inhibitor concentra-
tion equal to ꢀKi.
3.2. General procedures
3.2.1. General procedure for a-azidodeoxy-x-O-tosyloli-
goethyleneglycol formation. A 10% solution of the
respective ethylene glycol di-tosylate in dry N,N-dimeth-
ylformamide (DMF) was stirred with NaN3 (1.1 equiv)
at ambient temp for 20 h. CH2Cl2 was added to the reac-
tion mixture to precipitate the salts. After filtration, the
solution was concentrated under reduced pressure and
the remaining material was chromatographed on silica
gel to furnish the respective mono-azido compound as
colourless to slightly yellow oil.
3. Experimental
3.1. General methods
1H NMR spectra were recorded on a Varian INOVA
500 operating at 499.925 MHz. 13C NMR spectra were
recorded at 75.47 or 50.29 MHz. Residual non-deuterat-
ed solvent was used as internal standard for determina-
tion of chemical shifts. Signals of aromatic systems and
their substituents were found in the expected regions
and are not listed explicitly. Mass spectra were recorded
on an HP 1100 series MSD, Hewlett Packard. Samples
were dissolved in acetonitrile or acetonitrile/water mix-
tures. 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. Analytical TLC was performed on
3.2.2. General procedure for N-alkylation of iminoalditols.
A 2% solution of the respective iminoalditol in dry DMF
was stirred with the respective azidodeoxyethylenegylcol
tosylate at 45 ꢁC for 120 h. The solvent was removed un-
der reduced pressure and the remaining material was
chromatographed on silica gel (CHCl3/MeOH/concd
NH4OH 4:1:1). To remove counterions, 5% aqueous

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I. Lundt et al. / Bioorg. Med. Chem. 14 (2006) 1737–1742
solutions of the resulting products were treated with
strongly basic ion exchange resin Merck III. After filtra-
tion and removal of H2O, compounds were passed over
short plugs of silica gel to yield free bases.
H-10b), 2.36 (dd, 1H, H-1a), 2.26 (m, 1H, H-5). MS Calcd
for [C10H20N4O5]: m/z 276.295. Found: m/z 276.29.
3.7. N-(8-Azido-3,6-dioxaoctyl)-1-deoxynojirimycin, 1c
3.2.3. General procedure for hydrogenation of terminal
azide groups. 2% methanolic solutions of the respective
terminal azido compounds were stirred at ambient temp
under an atmosphere of H2 at ambient pressure for 20 h
employing Pd/C (10%) as catalyst. After removal of the
catalyst by filtration, MeOH was removed under re-
duced pressure and the crude, air-sensitive resulting
materials were immediately used in the next step.
Following the general procedure for N-alkylation of imi-
noalditols, 1-deoxynojirimycin (1a, 67 mg, 0.41 mmol)
gave 1c (48 mg, 36.5%) as a colourless wax. 13C NMR
(MeOH-d4): d 79.0 (C-3), 70.7, 70.4, 70.3, 70.0, 69.5,
68.4, 66.5, 58.2, 57.3, 51.5 (C-1, C-2, C-4, C-5, C-6,
1
C-10, C-20, C-40, C-50, C-70) 50.6 (C-80). H NMR: d
3.92 (dd, 1H, J5,6a 2.4 Hz, J6a,6b 11.7 Hz, H-6a), 3.83
(dd, 1H, J5,6b 2.4 Hz, H-6b), 3.70–3.61 (m, 8H), 3.46
(ddd, 1H, J1e,2 4.9 Hz, J2,3 9.3 Hz, H-2), 3.41–3.36 (m,
3H), 3.15 (dd, 1 H, J3,4 9.3 Hz, H-3), 3.06 (dd, 1H,
J1a,1e 11.2 Hz, H-1e), 3.04 (m, 1H, H-10a), 2.70 (m, 1H,
H-10b), 2.29 (dd, 1H, J1a,2 10.8 Hz, H-1a), 2.20 (m, 1H,
J4,5 9.7 Hz, H-5). MS: Calcd for [C12H24N4O6]: m/z
320.448. Found: m/z 320.44.
3.2.4. General procedure for N-dansylation. To a 2%
DMF solution of the respective terminal amine, dansyl
chloride (1.7 equiv) and Et3N (2 equiv) were added
and the mixture was stirred at ambient temp for 3 h.
Excess MeOH was added to the mixture and the
solvents were removed under reduced pressure. Chro-
matography of the green-fluorescent residue gave the
respective dansylamino-tagged iminoalditol.
3.8. N-(11-Azido-3,6,9-trioxaundecanyl)-1-deoxynojiri-
mycin, 1d
3.3. a-Azidodeoxy-x-O-tosyldiethyleneglycol (5-azido-3-
oxapentyl-1-toluenesulfonate), n = 1
Following the general procedure for N-alkylation
of iminoalditols, 1-deoxynojirimycin (1a, 50 mg,
0.31 mmol) gave 1d (48 mg, 42.5%) as a colourless
wax. 13C NMR (MeOH-d4): d 79.2 (C-3), 70.6, 70.4,
70.3 (2 C), 70.2, 70.0, 69.4, 68.3, 66.5, 58.1, 57.2, 51.5
(C-1, C-2, C-4, C-5, C-6, C-10, C-20, C-40, C-50, C-70,
Following the general procedure for a-azidodeoxy-x-O-
tosyloligothyleneglycol formation, diethyleneglycol ditosy-
late (7.55 g, 18.2 mmol) gave the monoazide (2.50 g,
48%) as a yellowish oil. 13C NMR (CDCl3): d 70.4,
69.4, 68.9, 50.8.
1
C-80, C-100), 50.6 (C-110). H NMR: d 3.93 (dd, 1H,
J5,6a 2.9 Hz, J6a,6b 12.2 Hz, H-6a), 3.84 (dd, 1H, J5,6b
2.9 Hz, H-6b), 3.70–3.61 (m, 12H), 3.47 (m, 1H), 3.41
(m, 2H), 3.16 (t, 2H), 3.07 (dd, 1H, J1e,2 4.9 Hz, J1a,1e
11.3 Hz, H-1e), 3.04 (m, 1H), 2.67 (m, 1H), 2.27
(dd, 1H, J1a,2 10.7 Hz, H-1a), 2.21 (ddd, J4,5 9.3 Hz,
H-5). MS: Calcd for [C10H20N4O5]: m/z 364.502. Found:
m/z 364.50.
3.4. a-Azidodeoxy-x-O-tosyltriethyleneglycol (8-azido-
3,6-dioxaoctyl-1-toluenesulfonate), n = 2
Following the general procedure for a-azidodeoxy-x-O-
tosyloligothyleneglycol formation, triethyleneglycolditosy-
late (2.00 g, 4.36 mmol) gave the monoazide (615 mg,
42.8%) as a colourless syrup. 13C NMR (CDCl3): d 71.0,
70.3, 69.5, 69.0, 50.9.
3.9. N-(5-Dansylamino-3-oxapentyl)-1-deoxynojirimycin,
1e
3.5. a-Azidodeoxy-x-O-tosyltetraethyleneglycol (11-azi-
do-3,6,9-trioxaundecanyl-1-toluenesulfonate), n = 3
Following the general procedures for azide hydrogena-
tion and N-dansylation, 1b (60 mg, 0.22 mmol) gave
dansyl-tagged compound 1e as a green-fluorescent glass
20
(25 mg, 23.8%). ½aꢁ ꢀ1.4 (c 1.3, MeOH). 13C NMR
Following the general procedure for a-azidodeoxy-x-O-
tosyloligothyleneglycol formation, tetraethyleneglycol
ditosylate (2.05 g, 4.08 mmol) gave the monoazide
(691 mg, 45.4%) as a slightly yellow syrup. 13C NMR
(CDCl3): d 71.0, 70.9, 70.8, 70.3, 69.5, 68.9, 50.8.
D
(MeOH-d4): d 79.2 (C-3), 70.7, 69.6, 69.4, 67.8, 66.7,
58.2, 57.1, 51.4 (C-1, C-2, C-4, C-5, C-6, C-10,C-20, C-
1
40), 42.7 (C-50). H NMR: d 3.82 (dd, 1H, J5,6a 2.9 Hz,
J6a,6b 12.2 Hz, H-6a), 3.77 (dd, 1H, J5,6b 2.9 Hz, H-
6b), 3.44 (ddd, J1a,2 10.3 Hz, J1e,2 4.9 Hz, J2,3 9.3 Hz,
H-2), 3.35–3.21 (m, 4H), 3.13 (dd, 1H, H-3), 3.04 (t,
2H), 2.97 (dd, 1H, J1a,1e 11.2 Hz, H-1e), 2.87–2.81 (m,
1H, H-10a), 2.44 (m, 1H, H-10b), 2.15 (dd, 1H, H-1a),
2.10 (ddd, 1H, J4,5 9.3 Hz, H-5). MS: Calcd for
[C22H33N3O7S]: m/z 483.588. Found: m/z 483.59.
3.6. N-(5-Azido-3-oxapentyl)-1-deoxynojirimycin, 1b
Following the general procedure for N-alkylation of imi-
noalditols, 1-deoxynojirimycin (1a, 100 mg, 0.613 mmol)
20
D
gave 1b (60 mg, 50%) as a colourless wax. ½aꢁ +8.9
(c 1.25, MeOH). 13C NMR (MeOH-d4): d 79.2 (C-3),
70.5, 69.8, 69.4, 68.1, 66.4, 58.0, 57.3, 51.5 (C-1, C-2,
3.10. N-(8-Dansylamino-3,6-dioxaoctyl)-1-deoxynojiri-
mycin, 1f
1
C-4, C-5, C-6, C-10, C-20, C-40) 50.6 (C-50). H NMR: d
3.94 (dd, 2H, J5,6a = J5,6b 2.4 Hz, J6a,6b 11.7 Hz, H-6a,
H-6b), 3.68 (m, 4H, 2H-20, 2H-40), 3.48 (ddd, 1H, J1a,2
9.9 Hz, J1e,2 4.9 Hz, J2,3 9.3 Hz, H-2), 3.41–3.36 (m,
3H), 3.17 (dd, 1H, J3,4 9.3 Hz, H-3), 3.11 (dd, 1H, J1a,1e
11.2 Hz, H-1e), 3.08 (m, 1H, H-10a), 2.80 (m, 1H,
Following the general procedures for azide hydrogena-
tion and N-dansylation, 1c (48 mg, 0.15 mmol) gave
dansyl-tagged compound 1f as a green-fluorescent glass
(31 mg, 39%). 13C NMR (MeOH-d4): d 79.3 (C-3), 70.5,

I. Lundt et al. / Bioorg. Med. Chem. 14 (2006) 1737–1742
1741
70.0, 69.8, 69.5, 69.4, 68.6, 66.6, 58.0, 57.5, 51.3 (C-1, C-
2, C-4, C-5, C-6, C-10, C-20, C-40, C-50, C-70), 42.7 (C-
80). 1H NMR: d 3.91 (dd, 1H, J5,6a 2.9 Hz, J6a,6b
12.2 Hz, H-6a), 3.85 (dd, J5,6b 2.4 Hz, H-6b), 3.63–3.45
(m, 3H), 3.40–3.37 (m, 3H), 3.32–3.24 (m, 4H), 3.17
(dd, 1H, J2,3 8.8 Hz, J3,4 9.3 Hz, H-3), 3.04–2.98 (m,
4H, incl H-1e and H-10a), 2.58 (m, 1H, H-10b), 2.26
(dd, 1H, J1a,1e = J1e,2 10.7 Hz, H-1a), 1.98 (ddd, 1H,
J4,5 9.3 Hz, H-5). MS: Calcd for [C24H37N3O8S]: m/z
527.642. Found: m/z 527.64.
1.5 Hz, H-1a), 2.09 (ddd, J4,5 8.8 Hz, H-5). MS: Calcd
for [C22H33N3O7S]: m/z 483.588. Found: m/z 483.58.
3.14. N-(5-Azido-3-oxapentyl)-1-deoxy-L-idonojirimycin,
3b
Following the general procedure for N-alkylation of
iminoalditols, 1-deoxy-L-idonojirimycin, 3a (140 mg,
0.86 mmol) gave 3b (60 mg, 25%) as a colourless wax.
20
D
½aꢁ ꢀ13.5 (c 0.8, MeOH). 13C NMR (MeOH-d4): d
74.8 (C-3), 71.3, 69.9, 69.8, 69.6, 63.9, 56.8, 54.1, 51.7
(C-1, C-2, C-4, C-5, C-6, C-10, C-20, C-40), 50.6 (C-50).
1H NMR: d 3.84 (dd, 1H, J5,6a 4.4 Hz, J6a,6b 11.7 Hz,
H-6a), 3.80 (dd, 1H, J5,6b 7.3 Hz, H-6b), 3.71 (dd, 1H,
J3,4 9.3 Hz, J4,5 5.4 Hz, H-4), 3.66–3.60 (m, 4H), 3.54
(ddd, 1H, J1a,2 9.8 Hz, J1e,2 5.4 Hz, J2,3 8 Hz, H-2),
3.07–2.96 (m, 2H), 2.91 (m, 1H, H-5), 2.87 (dd, 1H,
J1a,1e 12.7 Hz, H-1e), 2.67 (dd, 1H, H-1a). MS: Calcd
for [C10H20N4O5]: m/z 276.295. Found: m/z 276.28.
3.11. N-(11-Dansylamino-3,6,9-trioxaundecanyl)-1-
deoxynojirimycin, 1g
Following the general procedures for azide hydrogena-
tion and N-dansylation, 1d (51 mg, 0.14 mmol) gave
dansyl-tagged compound 1g as a green-fluorescent glass
(45 mg, 56%). 13C NMR (MeOH-d4): d 79.0 (C-3), 70.6,
70.1 (3 C), 69.9, 69.4 (2 C), 68.2, 66.5, 58.1, 58.0, 51.4
(C-1, C-2, C-4, C-5, C-6, C-10, C-20, C-40, C-50, C-70,
1
C-80, C-100), 42.7 (C-110). H NMR: d 3.90 (dd, 1H,
3.15. N-(5-Dansylamino-3-oxapentyl)-1-deoxy-L-idonoj-
irimycin, 3c
J5,6a 2.9 Hz, J6a,6b 12.2 Hz, H-6a), 3.83 (dd, J5,6b
2.4 Hz, H-6b), 3.62–3.51 (m, 6H), 3.47 (ddd, J1a,2
9.8 Hz, J1e,2 4.4 Hz, J2,3 3 Hz, H-2), 3.41–3.29 (m, 7H),
3.18 (dd, 1H, J3,4 8.8 Hz, H-3), 3.08–2.98 (m, 4H, incl
H-1e, H-10a), 2.65 (m, 1H, H-10b), 2.27 (dd, 1H,
J1a,1e = J1a,2 10.7 Hz, H-1a), 2.21 (ddd, 1H, J4,5 9.3 Hz,
H-5). MS: Calcd for [C26H41N3O9S]: m/z 571.695.
Found: m/z 571.68.
Following the general procedures for azide hydrogena-
tion and N-dansylation, 4b (40 mg, 0.15 mmol) gave
dansyl-tagged compound 3c as a green-fluorescent glass
20
D
(5.3 mg, 10%). ½aꢁ ꢀ8 (c 0.3, MeOH). 13C NMR
(MeOH-d4): d 74.7 (C-3), 71.3, 69.9, 69.5, 69.1, 63.8,
56.9, 54.0, 51.8 (C-1, C-2, C-4, C-5, C-6, C-10, C-20, C-
1
40), 34.8 (C-50). H NMR: d 3.82 (dd, 1H, J5,6a 5 Hz,
3.12. N-(5-Azido-3-oxapentyl)-1-deoxymannojirimycin,
2b
J6a,6b 11.3 Hz, H-6a), 3.78 (dd, 1H, J5,6b 7.3 Hz, H-
6b), 3.68 (dd, 1H, J3,4 8.8 Hz, J4,5 5.4 Hz, H-4), 3.57
(t, 2H), 3.52 (m, 1H, H-2), 3.50–3.45 (m, 2H), 3.42 (t,
2H), 3.01 (m, 1H), 2.88–2.74 (m, 2H, H-1e, H-5), 2.62
(dd, 1H, J1a,1e12.7 Hz, J1a,2 9.8 Hz, H-1a). MS: Calcd
for [C22H33N3O7S]: m/z 483.588. Found: m/z 483.58.
Following the general procedure for N-alkylation of
iminoalditols, 1-deoxymannojirimycin (2a, 75 mg,
0.46 mmol) gave 2b (62 mg, 49%) as a colourless wax.
20
D
½aꢁ ꢀ38.4 (c 0.7, MeOH). 13C NMR (MeOH-d4): d
75.3 (C-3), 69.7, 68.5, 68.4, 68.3, 66.0, 58.1, 56.1, 51.6
(C-1, C-2, C-4, C-5, C-6, C-20, C-40, C-50), 50.7 (C-10).
1H NMR: d 3.94 (dd, 1H, J5,6a 2.9 Hz, J6a,6b 11.7 Hz,
H-6a), 3.89 (dd, 1H, J5,6b 2.4 Hz, H-6b), 3.81 (m, 1H,
H-2), 3.68–3.62 (m, 5H), 3.38 (t, 2H), 3.33 (m, 1H),
3.04 (dd, 1H, J1a,1e 12.7 Hz, J1e,2 3.9 Hz, H-1e), 3.02
(m, 1H, H-10a), 2.85 (m, 1H, H-10b), 2.65 (dd, 1H,
J1a,2 1.5 Hz, H-1a), 2.27 (ddd, 1H, J4,5 9.3 Hz, H-5).
MS: Calcd for [C10H20N4O5]: m/z 276.295. Found: m/z
276.30.
3.16. N-(5-Azido-3-oxapentyl)-isofagomine, N-(5-azido-
3-oxapentyl)-(3R,4R,5R)-3,4-dihydroxy-5-hydroxymeth-
yl-piperidine, 4b
Following the general procedure for N-alkylation of
iminoalditols, isofagomine, 4a (88.3 mg, 0.48 mmol)
20
D
gave 4b (101 mg, 81%) as a colourless wax. ½aꢁ +13.5
(c 1.5, MeOH). 13C NMR (MeOH-d4): d 74.8 (C-4),
71.9 (C-3), 69.8, 68.6 (C-20, C-30), 61.5 (C-50), 59.0 (C-
1
2), 57.2 (C-10), 55.7 (C-6), 50.5 (C-40), 43.8 (C-4) H
0
0
0
NMR: d 3.80 (dd, 1H, J5,5 a 3.9 Hz, J5 a,5 b 11.0 Hz, H-
50a), 3.64 (dd, 2H, H-20, H-30), 3.52 (m, 2H, J2ax,3
6.8 Hz, J2eq,3 3.4 Hz, J3,4 7.3 Hz, H-3, H-50b), 3.37 (t,
2H, H-40), 3.08 (m, 3H, H-2ax, H-4, H-6ax), 2.67 (m,
2H, H-10), 1,98 (m, 2H, J2ax,2eq = 6ax,6eq 11.7 Hz, J2eq,3
2.9 Hz, J6eq,5 2.4 Hz, H-2eq, H-6eq), 1.76 (m, 1H, H-
5). MS: Calcd for [C10H20N4O4]: m/z 260.295. Found:
m/z 260.29.
3.13. N-(5-Dansylamino-3-oxapentyl)-1-deoxymannojiri-
mycin, 2c
Following the general procedures for azide hydrogena-
tion and N-dansylation, 2b (44 mg, 0.16 mmol) gave dan-
syl-tagged compound 2c as a green-fluorescent glass
20
(41 mg, 68%). ½aꢁ ꢀ21 (c 2.0, MeOH). 13C NMR
D
(MeOH-d4): d 75.2 (C-3), 69.5, 68.3, 68.2, 67.7, 66.3,
57.8, 55.7, 51.4 (C-1, C-2, C-4, C-5, C-6, C-10, C-20, C-
3.17. N-(5-Dansylamino-3-oxapentyl)-isofagomine,N-(5-
dansylamino-3-oxapentyl)-(3R,4R,5R)-3,4-dihydroxy-5-
hydroxymethyl-piperidine, 4c
1
40), 42.8 (C-50). H NMR: d 3.85 (dd, 1H, J5,6a 2.5 Hz,
J6a,6b 11.7 Hz, H-6a), 3.81 (dd, 1H, J5,6b 2.5 Hz, H-6b),
3.79 (m, 1H, H-2), 3.65 (dd, 1H, J2,3 9.3 Hz, J3,4 8.8 Hz,
H-3), 3.36–3.26 (m, 6H), 3.04 (t, 2H), 2.92 (dd, 1H, J1e,2
3.9 Hz, J1a,1e 12.2 Hz, H-1e), 2.88–2.82 (m, 7H, aryl-
NMe2, H-10a), 2.44 (m, 1H, H-10b), 3.99 (dd, 1H, J1a,2
Following the general procedures for azide hydrogena-
tion and N-dansylation, 4b (70 mg, 0.27 mmol) gave
dansyl-tagged compound 4c as a green-fluorescent glass

1742
I. Lundt et al. / Bioorg. Med. Chem. 14 (2006) 1737–1742
20
D
(79 mg, 63%). ½aꢁ +7.0 (c 1.5, MeOH). 13C NMR
1999, 38, 750; Stutz, A. E., Ed.; Iminosugars as Glyco-
¨
(MeOH-d4): d 74.8 (C-4), 71.7 (C-3), 69.2, 67.6 (C-20,
C-30), 61.5 (C-50), 58.8 (C-2), 57.1 (C-10), 55.6 (C-6),
44.7 (2C, 2· NCH3), 43.6 (C-5), 42.7 (C-40). 1H
sidase Inhibitors; Wiley-VCH: Weinheim, 1999; Bols, M.
Acc. Chem. Res. 1998, 31, 1; Legler, G. Adv. Carbohydr.
Chem. Biochem. 1990, 48, 319.
2. Spiro, R. G. In Carbohydrates in Chemistry and Biology.
In Part II: Biology of Saccharides; Ernst, B., Hart, G. W.,
Sinay, P., Eds.; Wiley-VCH: Weinheim, 2000; Vol. 3, pp
65–79; Elbein, A. D.; Molyneux, R. J. In Iminosugars as
0
0
0
NMR: d 3.80 (dd, 1H, J5,5 a 3.4 Hz, J5 a,5 b 11.0 Hz,
H-50a), 3.52 (m, 2H, H-3, H-50b), 3.25 (m, 4H, H-20,
H-30), 3.06 (m, 3H, H-2ax, H-4, H-6ax), 2.97 (m, 2H,
H-40), 2.35 (m, 2H, H-10), 1.83 (m, 2H, J2ax,2eq = 6ax,6eq
11.2 Hz, J2eq,3 2.9 Hz, J6eq,5 3.4 Hz, H-1eq, H-5eq),
1.75 (m, 1H, H-5). MS: Calcd for [C22H33N3O6S]: m/z
467.589. Found: m/z 467.59.
Glycosidase Inhibitors; Stutz, A. E., Ed.; Wiley-VCH:
¨
Weinheim, 1999; pp 216–251.
3. Miglitolꢂ by BAYER.
4. Simmonds, M. S. J.; Kite, G. C.; Porter, E. A. In
Iminosugars as Glycosidase Inhibitors; Stutz, A. E., Ed.;
¨
Wiley-VCH: Weinheim, 1999; pp 8–30.
5. De Raadt, A.; Ekhart, C.; Legler, G.; Stutz, A. E. In
¨
Wiley-VCH: Weinheim, 1999; pp 207–215.
6. Hermetter, A.; Scholze, H.; Stutz, A. E.; Withers, S. G.;
¨
Wrodnigg, T. M. Bioorg. Med. Chem. Lett. 2001, 11, 1339.
7. Bertozzi, C. R.; Bednarski, M. J. Org. Chem. 1991, 56,
4326; Schwabacher, A. W.; Lane, J. W.; Schiesher, M. W.;
Leigh, K. M.; Johnson, C. W. J. Org. Chem. 1998, 63,
1727; Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.;
Whitsides, G. M. Exp. Cell Res. 1997, 235, 305; Sigal, G.
B.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc.
1998, 120, 3464.
¨
Iminosugars as Glycosidase Inhibitors; Stutz, A. E., Ed.;
Acknowledgments
Financial support by the Austrian Fonds zur Fo¨rderung
der Wissenschaftlichen Forschung (FWF), Vienna (Pro-
ject 15726-N03), as well as by the Protein Engineering
Network of Centres of Excellence of Canada (PENCE)
is gratefully acknowledged. A. E. S. thanks the Danish
Technical University for a guest professorship in March
2005.
8. Kempton, J. B.; Withers, S. G. Biochemistry 1992, 31,
9961.
References and notes
9. Notenboom, V.; Williams, S. J.; Hoos, R.; Withers, S. G.;
Rose, D. R. Biochemistry 2000, 39, 11553.
1. For example: Martin, O. R.; Compain, P., Eds., Imi-
nosugars: Recent Insights into their Bioactivity and
Potential as Therapeutic Agents; Curr. Top. Med. Chem.
2003, Vol. 3, pp 471–591; From lianas to glycobiology
tools: Twenty-five years of 2,5-dideoxy-2,5-imino-D-man-
nitol, Wrodnigg, T. M. In Timely Research Perspectives
10. Wrodnigg, T. M.; Withers, S. G.; Stutz, A. E. Bioorg.
Med. Chem. Lett. 2001, 11, 1063.
¨
11. (a) Prade, H.; Mackenzie, L. F.; Withers, S. G. Carbohydr.
Res. 1998, 305, 371; (b) Kempton, J. B.; Withers, S. G.
Biochemistry 1992, 31, 9961.
12. Leatherbarrow, R. J. GraFit Version 4.0, Erithacus
Software, Staines, U.K., 1992.
in Carbohydrate Chemistry; Schmid, W., Stutz, A. E.,
¨
Eds.; Springer: Vienna, New York, 2002; pp 43–76;
Heightman, T. D.; Vasella, A. T. Angew. Chem., Int. Ed.