Powerful probes for glycosidases: Novel, fluorescently tagged glycosidase inhibitors

Article abstract of DOI:10.1016/S0960-894X(01)00209-8

1-Amino-1,2,5-trideoxy-2,5-imino-D-mannitol was fluorescently tagged by reaction with dansyl chloride at N-1 or by attachment of a dansyl amide bearing spacer to this centre. Compounds obtained are highly potent inhibitors of β-glucosidase exhibiting K_i values in the single figure nanomolar range. The 1-N-dansyl substituted inhibitor was successfully exploited for binding studies with β-glucosidase from Agrobacterium sp. employing fluorescence spectrometric methods.

Full text of DOI:10.1016/S0960-894X(01)00209-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1339–1342
Powerful Probes for Glycosidases:
Novel, Fluorescently Tagged Glycosidase Inhibitors
1
a
a
b,
c
¨
Albin Hermetter, Hubert Scholze, Arnold E. Stutz, * Stephen G. Withers
b
and Tanja M. Wrodnigg
aInstitut fu¨r Biochemie der Technischen Universita¨t Graz, Petersgasse 12, A-8010 Graz, Austria
b
Glycogroup, Institut fu¨r Organische Chemie der Technischen Universita¨t Graz, Stremayrgasse 16, A-8010 Graz, Austria
c
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
Received 7 February 2001; accepted 26 March 2001
Abstract—1-Amino-1,2,5-trideoxy-2,5-imino-d-mannitol was fluorescently tagged by reaction with dansyl chloride at N-1 or by
attachment of a dansyl amide bearing spacer to this centre. Compounds obtained are highly potent inhibitors of b-glucosidase
exhibiting K values in the single figure nanomolar range. The 1-N-dansyl substituted inhibitor was successfully exploited for bind-
ing studies with b-glucosidase from Agrobacterium sp. employing fluorescence spectrometric methods. # 2001 Elsevier Science Ltd.
All rights reserved.
i
Iminosugars are potent reversible inhibitors of glycoside
hydrolases and consequently important tools in glyco-
In order to extend our understanding of the structure–
activity relationships that are operative in this range of
compounds, some examples of analogues of 3 bearing
lipophilic side chains attached to C-1 were prepared and
screened. In the course of this programme we also
became interested in derivatives of 3 featuring extended
aromatic systems, in particular fluorescent tags. Such
compounds should bear great potential for the direct
investigation and mapping of glycosidase active sites as
well as for specific labelling of particular enzymes and,
consequently, for diagnostic purposes.
2
biology and glycotechnology. 1-Deoxynojirimycin (1,5-
2
dideoxy-1,5-imino-d-glucitol, 1), the paradigmatic d-
glucosidase inhibitor in this class of inhibitors, exhibits
4
,5
2
similar properties as castanospermine (2), another
2
important glucosidase inhibiting sugar mimetic. These
and related compounds cause notable biological
effects such as anti-diabetes, anti-retroviral as well as
2
anti-tumour properties.
Exploiting the Amadori rearrangement reaction of
readily available 5-azidodeoxy-d-glucofuranose with
dibenzyl amine followed by catalytic hydrogenation
with concomitant intramolecular reductive amination of
the resulting 5-azido-1-(dibenzyl)amino-1,5-dideoxy-d-
fructopyranose and nitrogen deprotection provided
1-amino-1,2,5-trideoxy-2,5-imino-d-mannitol (4), the
The iminoalditol 2,5-dideoxy-2,5-imino-d-mannitol
3
4
(3), yet another typical representative of sugar analo-
gues with basic nitrogen instead of oxygen in the ring, is
1-aminodeoxy derivative of 3 in 75% yield. Based on
this simple synthetic approach, a range of C-1 modified
derivatives of compound 3 became available for eva-
a powerful reversible inhibitor of d-glucosidases and
invertase.
2
5
luation of their glucosidase inhibitory activities. In
particular, analogues bearing lipophilic substituents
6
showed interesting biological properties.
These encouraging observations have now led to the
design and synthesis of novel, fluorescently labelled
analogues such as 5, which were easily obtained by
*
8
Corresponding author. Tel.: +43-316-873-8241; fax: +43-316-873-
740; e-mail: stuetz@orgc.tu-graz.ac.at
7
0
960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00209-8

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A. Hermetter et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1339–1342
coupling dansyl chloride directly to N-1 of the inhibitor
(Scheme 1). Alternatively, in the case of spacer-armed
N-Dansyl ethanolamine (9) exhibited a K value of
i
4
500 mM documenting the importance of the sugar
mimicking iminoalditol moiety for the inhibitory activity
found with compounds 5 and 6.
compound 6, a convergent synthesis was performed
by coupling iminoalditol intermediate 4 with the
fluorescently labelled spacer arm 8 employing HBTU
0
0
[
hexafluorophosphate] as the coupling reagent.
(O-benzotriazol-1-yl)-N,N,N ,N -tetramethyluronium
8
Characterisation of fluorescence properties of com-
pound 5 as well as the corresponding complex with
Agrobacterium sp. b-glucosidase was conducted as fol-
1
0
lows. Different amounts of enzyme were incubated
with compound 5 and electrophoresis was performed on
native polyacrylamide gel without detergent (Fig. 1A).
Other proteins such as b-galactosidase from Aspergillus
oryzae, glucose isomerase, as well as albumin served as
probes for the selectivity of 5 for b-glucosidase activity.
Scheme 1.
Compound 8 was prepared from dansyl chloride and
methyl 6-aminohexanoate to give methyl ester 7 which
was subsequently saponified (Schemes 2 and 3).
Scheme 2.
In both cases, the primary amine in compound 4 reacted
highly selectively in the presence of the unprotected ring
nitrogen furnishing the desired 1-N-acylated products 5
and 6.
Figure 1. Visualization of the fluorescent complex of 5 with Agro-
bacterium sp. b-glucosidase (Abg) on native PAGE. (A) Different
amounts of the enzyme and other related enzymes were preincubated
with 2 nmol of 5 at room temperature for 15 min. Lanes: 1, 1 mg Abg;
Scheme 3.
2
, 2 mg Abg; 3, 3 mg Abg; 4, 4 mg Abg; 5, 5 mg Abg; 6, 7.5 mg Abg; 7,
0 mg Abg; 8, 40 mg galactosidase; 9, 40 mg glucose isomerase; 10, 40 mg
In order to probe any undesired inhibitory effect of the
dansylamino moiety itself, N-dansyl ethanolamine (9)
was prepared.
1
albumin. (B) Plot of fluorescence intensity versus enzyme mass.
Only the desired glucosidase–inhibitor complex could be
detected as a single strongly fluorescing band nicely
demonstrating the purity of the enzyme with respect to
glucosidase activity as well as the selectivity of the
inhibitor.
A plot of fluorescence intensity versus concentration of
enzyme showed a linear concentration-dependent effect
(Fig. 1B).
Strong inhibition was observed with both new deriva-
tives of compound 4 employing b-glucosidase from
Agrobacterium sp. (Abg) at pH 6. Their inhibitory
power was found to be two orders of magnitude better
than parent compound 3 (3: K 200 nM; 5: K 2.4 nM; 6:
The quenching of the intrinsic tryptophan fluorescence
of a defined amount of Agrobacterium sp. b-glucosidase
by titration with compound 5 was also examined. The
dansyl fluorophor is an energy acceptor for the first
i
i
K 2.1 nM) and thus amongst the most potent of this
i
3
,9
type of reversible b-glucosidase inhibitors.

A. Hermetter et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1339–1342
1341
excited singlet state of tryptophan. Consequently, bind-
ing of dansyl labelled inhibitor 5 to the enzyme
suppresses the protein fluorescence highly efficiently.
7. Experimental procedures: Compound 5. To a 3% solution
of compound 4 in CH CN/MeOH 1:4 (v/v), 1.0 equiv of dan-
syl chloride (Aldrich) was added and the mixture was kept at
ambient temperature until all starting material was consumed
3
(
TLC: MeOH/CHCl /NH OH concd 25:75:2). Concentration
3 4
of the solution under reduced pressure and chromatographic
purification of the residue (silica gel, Merck 60; MeOH/
CHCl /NH OH concd 10:80:1) furnished sulfonamide 7 in
3 4
2
D
0
13
4
0% yield. ½ꢀꢀ 22.1 (c 1.0 in MeOH). C NMR(50.9 MHz,
ꢁ
3
CD
OD, 25 C): d 79.5, 78.3, 63.3, 62.2, 61.5, 45.1, 44.6 (2C).
OD): d 3.80–3.58 (m, 2H, H-3, H-4),
=11.0 Hz, J5,6=4.0 Hz, J5,6 =6.1 Hz,
1
H NMR(200 MHz, CD
3
3
.65–3.42 (m, 2H, J6,6
0
0
0
H-6, H-6 ), 3.37–3.27 (m, 1H, NH), 3.08–2.79 (m, 10H, H-1,
0
H-1 , H-2, H-5, NMe
dansyl chloride in CH
2
). Compound 6. To a 3% solution of
Cl , triethylamine (2.1 equiv) and
Figure 2. Quenching of the intrinsic tryptophan fluorescence of Agro-
bacterium sp. b-glucosidase (Abg) by titration with compound 5.
2
2
methyl 6-aminohexanoate hydrochloride (1.0 equiv, Aldrich)
were added and the mixture was kept at ambient temperature
2 2
for 2 h. The mixture was partitioned between CH Cl and 6%
Gratifyingly, the plot of the tryptophan fluorescence
intensity versus the inhibitor/enzyme ratio exhibited
maximal quenching at a 1:1 ratio of enzyme to inhibitor
(Fig. 2). Larger amounts of inhibitor 5 did not further
reduce the protein fluorescence.
aqueous HCl and the organic layer was washed with aqueous
sodium bicarbonate until neutral, dried (Na SO ) and con-
2
4
centrated under reduced pressure. Conventional chromato-
graphy of the residue gave 7 (quantitative). To a 10% solution
of this compound in dioxane/water 1:1 (v/v), aqueous NaOH
(10%) was added dropwise until pH 10 was reached and the
mixture was kept at ambient temperature for 4 h. The solution
Thus, it may be concluded that one inhibitor molecule
binds to the active site of a single polypeptide without
any additional adsorbtion of label at the surface of the
protein.
was neutralised with ion exchange resin Amberlite IR120
+
[
H ], filtered and concentrated under reduced pressure to give
the free acid 8 (85%). To a 10% methanolic solution of this
compound, compound 4, triethylamine, and HBTU (1 equiv
each) were added and the mixture was kept at ambient tem-
perature for 30 min (TLC in CHCl /MeOH/NH OH 75:25:2)
Analogue 6 exhibited analogous behaviour.11
3
4
and concentrated under reduced pressure. Chromatographic
purification (CHCl /MeOH/NH OH 70:10:1) gave compound
In conclusion, compounds 5 and 6 represent a novel
type of reversible iminoalditol-based glycosidase inhi-
bitor featuring not only powerful inhibitory activities
but also excellent chemo-optical properties which, by
their unprecedented combination, are clearly eminently
suitable for exploitation in a wide range of new appli-
cations in glycobiology.
3
4
20
13
6 (70% from 4). ½ꢀꢀ 12.2 (c 1.6 in MeOH). C NMR
D
ꢁ
(50.9 MHz, CD
OD, 25 C): d 175.4, 79.9, 78.1, 63.6, 62.0,
3
6
1.7, 44.6 (2C), 42.5, 41.9, 35.7, 29.1, 26.0, 25.1. Compound 9:
Reaction of dansyl chloride with 2-aminoethanol (1.5 equiv) in
CH Cl in the presence of triethylamine (2 equiv) gave com-
2
2
pound 9, which, after concentration of the reaction mixture
under reduced pressure, was isolated in 98% yield by chro-
1
3
matography (CHCl
ꢁ
3
/MeOH 60:1). C NMR(50.9 MHz,
CD OD, 25 C): d 60.8, 44.9, 44.6 (2C). Synthetic procedures
3
Acknowledgements
are not optimised. For all compounds reported, the signals of
the aromatic system were found in the expected region.
8. Dourtoglou, V.; Ziegler, J.-C.; Gross, B. Tetrahedron Lett.
1978, 15, 1269; Li, P.; Xu, J.-C. Tetrahedron 2000, 56, 4437.
Synthetic studies were kindly supported by the Austrian
¨
Fonds zur Forderung der wissenschaftlichen Forschung
FWF), Project P 13593 CHE. Enzymatic studies were
9. For other, related types of highly potent b-glucosidase
inhibitors see: Jespersen, T. M.; Dong, W.; Sierks, M. R.;
(
supported by funds from the Protein Engineering Net-
work of Centres of Excellence of Canada. T.M.W.
thanks the FWF for a Hertha-Firnberg-Fellowship
Skrydstrup, T.; Lundt, I.; Bols, M. Angew. Chem. 1994, 106,
1
858; Angew. Chem., Int. Ed. Engl. 1994, 33, 1778; Legler, G.;
Finken, M.-T.; Felsch, S. Carbohydr. Res. 1996, 292, 91; Pan-
day, N.; Canac, Y.; Vasella, A. Helv. Chim. Acta 2000, 83, 58.
(
T-18 CHE).
1
0. Characterisation of binding of compound 7 to Agro-
bacterium sp. b-glucosidase (Abg) and separation on native
polyacrylamide gel: gels: 3.6% stacking gel, 6% resolving gel.
For the labelling experiment, the enzyme (4 mg/mL) was dilu-
ted with 0.1 M potassium phosphate buffer pH 7.0 to a final
concentration of 0.5 mg/mL. Aliquots from 2 to 20 mL were
used and brought to a final volume of 20 mL with 0.1 M
potassium phosphate buffer pH 7.0. Compound 5 was also
dissolved in 0.1 M potassium phosphate buffer pH 7.0 to a
concentration of 1 mM. Two millilitres of the stock solution
were pipetted to the enzyme samples and incubated for 15 min
at room temperature (final concentration 0.09 nmol/mL).
Seven millilitres dissociation buffer (10 mM Tris/HCl pH 7.4,
50% glycerol, 1% Triton X-100, 0.1% bromophenol blue)
were added and samples were applied onto the gel after
10 min. Images were taken using a Herolab EASY gel doc-
umentation system with UV excitation at 254 nm. Fluores-
References and Notes
. Dedicated to Herbert Stutz (1944–2000), a dedicated che-
tz, A. E., Ed.;
1
¨
mist and good friend.
2
. Iminosugars as Glycosidase Inhibitors; Stu
VCH-Wiley: Weinheim, 1999, and references cited therein.
. Welter, A.; Jadot, J.; Dardenne, G.; Marlier, M.; Casimir,
J. Phytochemistry 1976, 15, 747.
. Wrodnigg, T. M.; Stutz, A. E.; Withers, S. G. Tetrahedron
Lett. 1997, 38, 5463.
. Wrodnigg, T. M.; Gaderbauer, W.; Greimel, P.; Ha
H.; Sprenger, F. K.; Stutz, A. E.; Virgona, C.; Withers, S. G.
J. Carbohydr. Chem. 2000, 19, 975.
. Wrodnigg, T; Withers, S. G.; Stu
Chem. Lett. 2001, 11, 1063.
¨
3
4
¨
5
¨
usler,
¨
6
¨
tz, A. E. Bioorg. Med.

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A. Hermetter et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1339–1342
cence was detected for 0.32 s corresponding to eight iteration
steps. Quenching of the intrinsic tryptophan fluorescence of
Agrobacterium sp. b-glucosidase (Abg) by titration with com-
pound 5. The enzyme was diluted with 0.1 M potassium
phosphate buffer pH 7.0 to a final concentration of 1 nmol/mL
3 nm. The inhibitor was dissolved in 0.1 M potassium phos-
phate buffer pH 7.0 (31 mM, detected by the absorption spec-
trum at 335 nm, e=4600 in methanol) and added stepwise to
the cuvette under stirring. The fluorescence intensity was
detected 2 min after the addition of the inhibitor.
(
Instrument (Shimadzu RF-5301 PC, spectrofluorophotometer)
settings were excitation 280 nm, emission 335 nm, slits 3 nm/
50 mg/mL). Two millilitres were used for the experiment.
11. Influences of the nature as well as of the length of the
spacer-arm on enzyme–inhibitor interactions are currently
being investigated.