Synthesis and biological evaluation of novel biotin-iminoalditol conjugates

Article abstract of DOI:10.1016/j.bmcl.2010.05.084

Biotin-iminosugar conjugates of different configuration such as d-gluco, d-galacto, l-ido as well as a furanoid representative in the d-manno configuration have been synthesised and exhibit powerful inhibition of β-glucosidase from Agrobacterium sp. with K_i values in the range of the respective parent compounds. Such molecular probes have potential for activity-based protein profiling taking advantage of the biotin-(strept)avidin interaction.

Full text of DOI:10.1016/j.bmcl.2010.05.084

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4077–4079
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel biotin–iminoalditol conjugates
Gerit Pototschnig a, Christian Morales De Csáky a, Jose R. Montenegro Burke a, Georg Schitter a,
Arnold E. Stütz a, Chris A. Tarling b, Stephen G. Withers b, Tanja M. Wrodnigg a,
*
a Glycogroup, Institut für Organische Chemie, Technische Universität Graz, Stremayrgasse 16, A-8010 Graz, Austria
b Department of Chemistry, University of British Columbia, Room W300-6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3
a r t i c l e i n f o
a b s t r a c t
Article history:
Biotin–iminosugar conjugates of different configuration such as D-gluco, D-galacto, L-ido as well as a fura-
Received 13 April 2010
Revised 18 May 2010
Accepted 19 May 2010
Available online 27 May 2010
noid representative in the D-manno configuration have been synthesised and exhibit powerful inhibition
of b-glucosidase from Agrobacterium sp. with Ki values in the range of the respective parent compounds.
Such molecular probes have potential for activity-based protein profiling taking advantage of the biotin–
(strept)avidin interaction.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Iminoalditol
Biotin
Biotin–iminoalditol conjugate
Glycosidase inhibitor
Iminosugars including iminoalditols and related bicyclic alka-
loids are the most prominent family of low molecular weight com-
petitive glycosidase inhibitors.1 Paradigmatic examples such as
compounds 1, 2, 3 and 4 (Fig. 1) and quite a few of their derivatives
have found important roles as diagnostic compounds such as in the
investigation of glycoprotein-trimming glycosidases2 or as phar-
maceutical substances for example in the treatment of diabetes
type II symptoms3 as well as hereditary enzyme deficiency dis-
eases.4 Other significant biological activities associated with their
glycosidase inhibitory properties are anti-viral, anti-cancer and
anti-metastatic, anti-infective as well as insect anti-feedant and
plant growth regulatory effects.5
Activity-based protein profiling (ABPP) of exo- as well as endo-
glycosidases has been shown to be a versatile tool for the investi-
gation of their assignment of structures and their functions.8 A typ-
ical probe for ABPP represents a mechanism-based inactivator
specific to the active site of the enzyme under investigation conju-
gated via a noncleavable or cleavable linker to biotin which allows
for affinity chromatography or fluorescence spectroscopy.9 In gen-
eral, the biotin–(strept)avidin interaction has been proven to be an
indispensable tool for biorecognition in the context of diagnostics,
biotechnology and nanotechnology.10
We have prepared three biotinylated representatives of
six-membered ring iminosugars with the D-gluco, D-galacto as well
Recently, we have found that various fluorescently labelled imi-
nosugar derivatives such as compounds 1a, 2a, 2b as well as 4a are
strikingly powerful glycosidase inhibitors exceeding the parent
compounds’ activities by up to two orders of magnitude (Fig. 2,6,7).
Such labelled inhibitors were deemed to be highly useful diag-
nostic tools for activity-based high-throughput analysis as well as
gel staining. For example, we designed and constructed an imino-
alditol chip with three typical glycosidase-inhibiting iminoalditols
attached to a polyamine surface displayed on a silicon chip.7 Expo-
sure to representative b-glucosidases revealed selective binding
events reflecting the different structural features of the inhibitors,
providing a proof-of-concept for the successful exploitation of
microarrays of typical reversible glycosidase inhibitors of the imi-
nosugar family.
as L-ido configuration 10, 11 and 15. In addition, we have prepared
the biotinylated derivative 6 of the five membered glycosidase
inhibitor 2,5-dideoxy-2,5-imino-D-mannitol (2).
Biotin–iminosugar conjugate 6 (Scheme 1) was obtained by
coupling of 1-amino-1,2,5-trideoxy-2,5-imino-D
-mannitol (5)11 to
biotin employing O-benzotriazole-N,N,N0,N0-tetramethyluronium
hexafluoro-phosphate (HBTU) as the coupling reagent in DMF
and in the presence of triethylamine in 50% yield.
Compounds 10 as well as 11 (Scheme 2) were obtained by cou-
pling of aminoethylaminobiotin 9 to N-(carboxypentyl)-1-deoxy-
nojirimycin 712 and N-(carboxypentyl)-1-deoxygalactonojirimycin
8, respectively, under the same coupling conditions and yields of
58% and 50%. Mono-biotinyl-1,2-diaminoethane (9) was prepared
by coupling of biotin to one terminus of ethylendiamine using HBTU
as the coupling reagent.
For the preparation of
L-ido iminosugar 15 (Scheme 3) we
* Corresponding author. Tel.: +43 316 873 8744; fax: +43 316 873 8740.
E-mail address: t.wrodnigg@tugraz.at (T.M. Wrodnigg).
started from 1-deoxy-
L
-idonojirimycin 12.13 Reductive amination
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.084

4078
G. Pototschnig et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4077–4079
H
N
H
N
H
HO
N
N
HO
HO
HO
OH
OH
HO
OH
HO
OH
OH
HO
OH
HO
OH
OH
1
2
3
4
Figure 1. Structures of iminosugars 1–4.
with adipic acid methyl ester hemicarbaldehyde 13 in methanol
using Pd/C as the catalyst led to N-(methoxycarbonylpentyl)-1-
iminosugars. In line with previous observations with N-modified
compounds in the D-galacto series, compound 11 exhibits nicely
deoxy-
L
-ido-nojirimycin 14 in a yield of 51%. Saponification of
improved selectivity for the b-selective enzymes probed.14 Thus,
these probes are clearly recognised by the enzyme and may have
great potential for various applications including glycosidase
profiling.
the ester in 1,6-dioxane/water gave the terminal carboxylic acid
which again was coupled to biotin derivative 9 using HBTU in
DMF and triethylamine yielding the respective biotinylated L-ido
iminosugar 15 in 43% yield.
In conclusion we have synthesised the first biotin–iminosugar
conjugates and have shown that these N-modified structures act
as glycosidase inhibitors. The synthetic strategy can be applied to
a wide range of iminosugars and allows for easy modification of
Inhibition constants (Ki values, Table 1) of biotinylated com-
pounds 6, 10, 11 and 15 for the Agrobacterium sp. b-glucosidase
were found in the range of those of the corresponding parent
O
O
R
(CH2)4
(CH2)4
COOMe
(CH2)5
COOMe
(CH2)5
N
N
N
HO
HO
HO
NHdansyl
NHdansyl
OH
NHdansyl
OH
HO
HO
OH
HO
OH
OH
1a:
= 6.2 µM
2a: R = H,
= 2.4 nM
4a:
= 3.1 µM
Ki
Ki
Ki
O
COOMe
(H2C)4
2b: R =
= 0.9 µM
(CH2
)
5
Ki
NHdansyl
Figure 2. Structure and Ki values against Agrobacterium sp. b-glycosidase of paradigmatic example of fluorescently labelled iminosugars.
O
H
N
H
N
S
HO
NH2
N
H
HO
a
OH
HO
HO
OH
HN
NH
5
6
O
Scheme 1. Synthesis of compound 6. Reagents and conditions: (a) Bitoin, HBTU, Et3N, DMF, rt, 5 h, 50%.
O
OH
S
H2
N
N
N
H
HO
a
O
+
1
R
OH
HN
NH
2
R
OH
9
O
7
8
: R1 = OH, R2 = H
: R1 = H, R2 = OH
O
H
N
S
N
N
H
HO
O
1
R
HN
OH
NH
2
R
OH
10: R1 = OH, R2 = H
11: R1 = H, R2 = OH
O
Scheme 2. Synthesis of biotin–iminosugar conjugates 10 and 11. Reagents and conditions: (a) HBTU, Et3N, DMF, rt, 5 h, 58% (10) and 50% (11).

G. Pototschnig et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4077–4079
4079
(CH2)5COOMe
H
N
N
O
HO
HO
a
O
+
HO
O
OH
HO
OH
13
OH
OH
12
14
O
H
N
S
N
NH
HO
b
O
HN
HO
OH
NH
OH
15
O
Scheme 3. Synthesis of biotinylated iminosugar 15. Reagents and conditions: (a) Pd/C, H2, MeOH, rt, 10 h, 51%; (b) (i) NaOMe, water/dioxane, rt, 12 h; (ii) compound 9, HBTU,
Et3N, DMF, rt, 12 h, 43%.
Table 1
compounds) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2010.05.084.
Inhibitory activities (Ki values in lM) of biotinylated iminosugars 6, 10, 11 as well as
15 with b-glycosidase from Agrobacterium sp. (b-glu/gal Abg), E. coli (b-gal E. coli) as
well as with the
a-galactosidase from green coffee beans (
a-gal GCB)
References and notes
Compound
Ki ( M) Ki ( M)
l
l
Ki (
lM)
b-glu/gal Abg
b-gal E. coli
a
-gal GCB
1. (a)For example: Iminosugars: From Synthesis to Therapeutic Applications;
Compain, P., Martin, O. R., Eds.; WILEY-VCH: Weinheim, 2008; (b) Martin, O.
R.; Compain, P. Curr. Top. Med. Chem. 2003, 3, 541; (c) Asano, N.; Nash, R. J.;
Molyneux, R. J.; Fleet, G. W. J. Tetrahedron Asymmetry 2000, 11, 1645; (d)
Wrodnigg, T. M. In Timely Research Perspectives in Carbohydrate Chemistry;
Schmid, W., Stütz, A. E., Eds.; Springer: Vienna, 1999; pp 393–426; (e) Vasella,
A. T. Angew. Chem., Int. Ed. 1999, 38, 750; (f)Iminosugars as Glycosidase
Inhibitors; Stütz, A. E., Ed.; Wiley-VCH: Weinheim, 1999; (g) Bols, M. Acc.
Chem. Res. 1998, 31, 1; (h) Legler, G. Adv. Carbohydr. Chem. Biochem. 1990, 48,
319.
6 (2)
0.5 (0.2)
60 (12)
11 (100)
30 (26)
nd
nd
2.3 (13)
nd
nd
nd
10 (1)
11 (4)
15 (12)
1.1 (0.013)
nd
nd—not determined.
the linker length. Such biotinylated glycosidase inhibitor conju-
gates are envisaged as helpful tools for activity-based protein pro-
filing of glycoside hydrolases. Additionally, the biotin covalently
bound to the enzyme inhibitor allows for detection or purification
of proteins by affinity binding to (strept)avidin-carrying molecules
or surfaces.
2. (a) Spiro, R. G. In Carbohydrates in Chemistry and Biology; Ernst, B., Hart, G. W.,
Sinay, P., Eds.; Wiley-VCH: Weinheim, 2000; Vol. 3, pp 65–79; (b) Elbein, A. D.;
Molyneux, R. J. In Iminosugars as Glycosidase Inhibitors; Stütz, A. E., Ed.;
Weinheim: Wiley-VCH, 1999; pp 216–251.
3. MiglitolÒ by Bayer.
4. Butters, T. D. Expert Opin. Pharmacother. 2007, 8, 427.
5. Simmonds, M. S. J.; Kite, G. C.; Porter, E. A. In Iminosugars as Glycosidase
Inhibitors; Stütz, A. E., Ed.; Wiley-VCH: Weinheim, 1999; pp 8–30.
6. Hermetter, A.; Scholze, H.; Stütz, A. E.; Withers, S. G.; Wrodnigg, T. M. Bioorg.
Med. Chem. Lett. 2001, 11, 1339.
Acknowledgements
7. Steiner, A. J.; Stütz, A. E.; Wrodnigg, T. M.; Tarling, C. A.; Withers, S. G.;
Hermetter, A.; Schmidinger, H. Bioorg. Med. Chem. Lett. 2008, 18, 1922.
8. (a) Hekmat, O.; He, S.; Warren, R. A.; Withers, S. G. J. Proteom. Res. 2008, 7,
3282; (b) Williams, S. J.; Hekmat, O.; Withers, S. G. ChemBioChem 2006, 7, 116.
9. Uttamchandani, M.; Li, J.; Sun, H.; Yao, S. Q. ChemBioChem 2008, 9, 667.
G.S. thanks the Institute of Organic Chemistry for financial sup-
port (January–June 2009). Financial support by the Canadian Insti-
tutes of Health Research (CIHR Team Grant CTP-82944) is
gratefully acknowledged.
´
10. Wilchek, M.; Miron, T.; Bayer, E. A. Khimiya be Yisrael. 2005, 20, 23.
11. Wrodnigg, T. M.; Stütz, A. E.; Withers, S. G. Tetrahedron Lett. 1997, 38, 5463.
12. Hettkamp, H.; Legler, G.; Bause, E. Eur. J. Biochem. 1984, 142, 85.
13. Fowler, P. A.; Haines, A. H.; Taylor, R. J. K.; Chrystal, E. J. T.; Gravestock, M. B.
Carbohydr. Res. 1993, 246, 377.
Supplementary data
14. Schitter, G.; Scheucher, E.; Steiner, A. J.; Stuetz, A. E.; Thonhofer, M.; Tarling, C.
A.; Withers, S. G.; Wicki, J.; Fantur, K.; Paschke, E.; Mahuran, D. J.; Rigat, B. A.;
Tropak, M.; Wrodnigg, T. M. Beilstein JOC 2010, 6, 21.
Supplementary data (general methods, biological data as well
as experimental and analytical details for the synthesised