Transcript Slide 1

Design and Synthesis of 5-Membered
Azasugars for Glycosidase Inhibition
Margaret L. Wong
Kiessling Group
15 November 2007
Carbohydrates and Cellular
Recognition Events
Image adapted from cover of Nature 1995, 373.
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Processing of Oligosaccharides and
Glycosidase Function
Catalyze polysaccharide hydrolysis
Family of hydrolase enzymes: glucosidases, galactosidases,
mannosidases, etc.
Glycosidases are imperative to biosynthesis of cellular oligosaccharides.
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Impact of Glycosidase Inhibition
Glycosidase inhibition can give rise to antiviral, anticancer,
antibacterial activity.
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Azasugars as Natural Products and
Glycosidase Inhibitors
Nojirimycin (1966)
Antibiotic product of Streptomyces
1-Deoxynojirimycin (DNJ) (1968)
Natural product of Streptomyces, Bacillus and Morus
mulberry trees
2,5-dideoxy-2,5-imino-D-mannitol (DMDP) (1976)
Isolated from the leaves of legume Derris elliptica.
Asano, N. Curr. Top. Med. Chem. 2003, 3, 471-484.
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Azasugars
Polyhydroxylated 5- and 6- membered N-heterocycles
Common names: “iminosugars” and “iminocyclitols”
Inhibitors of cellular glycosidases
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5-Membered Azasugars as Therapeutic
Targets in Glycosidase Inhibition
I.
Transition state analogue of natural sugars
II.
Synthetic strategies for iminosugar core
III. Combinatorial libraries to probe efficacy of inhibition
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5-Membered Azasugars as Therapeutic
Targets in Glycosidase Inhibition
I.
Transition state analogue of natural sugars
II.
Synthetic strategies for iminosugar core
III. Combinatorial libraries to probe efficacy of inhibition
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Glycoside Hydrolysis
Glycosidases hydrolyze polysaccharides with inversion or retention.
McCarter, J. Withers, S.G. Curr. Opin. Struct. Biol. 1994, 4, 885-892.
Davies, G.; Henrissat, B. Structure 1995, 3, 853-859
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Saccharide-Bound Glucosidase
Trapped intermediate during hydrolysis
C-2 OH is an important stabilizing
interaction.
≡
Caines, M.E. et al. Angew. Chem. Int. Ed. 2007, 26, 4474-4476.
Caines, M.E. et al. J. Biol. Chem. 2007, 282, 14300-14308
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Isofagomine-Bound Glucosidase
Azasugar mimics transition state
Isofagomine-bound glucosidase uses
N as an anomeric carbon.
Loss of hydrogen bonding with “C-2”
hydroxyl group.
≡
Caines, M.E. et al. J. Biol. Chem. 2007, 282, 14300-14308
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DMDP Derivative-Bound Glucosidase
5-membered azasugar mimics transition state
Enzyme binds N like the anomeric
carbon of O-sugar substrate.
“C-2” hydroxyl group, hydrophobic
portions are stabilizing
≡
Caines, M.E. et al. J. Biol. Chem. 2007, 282, 14300-14308
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Side-Chain Interactions with Azasugar
Charged endocyclic nitrogen
C-6 Hydroxyl group & aglycon, hydrophobic substitution
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5-Membered Azasugars as Therapeutic
Targets in Glycosidase Inhibition
I.
Transition state analogue
II.
Synthetic strategies of iminosugar core
III. Combinatorial libraries to probe efficacy of inhibition
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Azasugar via Amadori Rearrangement
Amadori rearrangement & cyclization:
Azido nitrogen becomes endocyclic nitrogen in azasugar.
Dibenzylamino nitrogen is a second site of reactivity.
Wrodnigg, T.M.; Stutz, A.E.; Withers, S.G. Tet. Lett. 1997, 38, 5463.
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Amadori Rearrangement of 5-Azido-DeoxyD-Glucofuranose
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Diastereoselectivity of Imine Reduction
Conditions
2R:2S
10% Pd/C, 50 psi, H2
90:10
10% Pd/C, 15 psi H2
85:15
5% Rh/Al2O3, 15 psi H2
98:02
Kajimoto, T. et al J. Am. Chem. Soc. 1991, 113, 6678-6680.
Takayama, S. et al. J. Am. Chem. Soc. 1997, 119, 8146.
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Epoxides as Chiral Precursors
Via Sharpless asymmetric epoxidation
Takebayashi, M. et al J. Org. Chem. 1999, 64, 5280-5291.
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Reduction of Pyrrole
Diastereoselectivity:
Donohoe, T.J. et al. Org. Lett. 2003, 5, 999-1002.
Donohoe, T. J.; Sintim, H. O.; Hollinshead, J. J. Org. Chem. 2005, 70-7297-7304.
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Enzyme-Catalyzed
Asymmetric Aldol Reaction
DHAP-dependent Aldolase catalyzed reaction
Donor: dihydroxyacetone phosphate (DHAP)
Acceptor: aliphatic aldehydes
α-heteroatom substituted aldehydes
monosaccharides
Selectivity: dependent on enzyme
Machajewski, T.D.; Wong, C.-H. Angew. Chem. Int. Ed. 2000, 39, 1352-1374.
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Selectivity of Aldolases
Machajewski, T.D.; Wong, C.-H. Angew. Chem. Int. Ed. 2000, 39, 1352-1374.
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Chemoenzymatic Synthesis of
N-Acetylglucosamine
Other diastereomers accessible through acceptors with different
α-substitution.
Takaoka, Y.; Kajimoto, T.; Wong, C.-H. J. Org. Chem. 1993, 58, 4809.
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D-Fructose-6-Phosphate Aldolase (FSA)
FSA: a new aldolase enzyme for iminocyclitol synthesis
One-pot synthesis of iminocyclitol
Wider substrate tolerance
reduces number of steps to
iminocyclitol products
without loss of selectivity.
Sugiyama, M.et al. J. Am. Chem. Soc. 2007, ASAP.
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Synthetic Approaches Toward
2,5-Dideoxy-2,5-Imino-Glucofuranoses
Amadori rearrangement
Tandem Staudinger reduction/cyclization via chiral epoxides
Reduction of pyrrole
Chemoenzymatic synthesis with aldolases
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5-Membered Azasugars as Therapeutic
Targets in Glycosidase Inhibition
I.
Transition state analogue
II.
Synthetic strategies of iminosugar core
III. Combinatorial libraries to probe efficacy of inhibition
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Aglycon Derivatization
Structure-activity relationship: C-1’ modification
Loss of inhibition with short, polar C1-substituent
Increased inhibitory activity with extended C1 alkylation
Combination of alkylation and aromaticity afforded the best inhibitors
Wrodnigg, T.M., et al. Bioorg. Med. Chem. 2004, 12, 3485-3495.
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Aglycon Derivatization of Amadori Products
Synthesis of library
Inhibition assay readout: p-nitrophenol glucopyranoside
Wrodnigg, T.M.; Withers, S.G.; Stütz, A.E. Bioorg. Med. Chem. Lett. 2001, 11, 1063-1064
Wrodnigg, T.M., et al. Bioorg. Med. Chem. 2004, 12, 3485-3495.
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C-1’ Substitution Effects on βGlucosidase Inhibition
Wrodnigg, T.M.; Withers, S.G.; Stütz, A.E. Bioorg. Med. Chem. Lett. 2001, 11, 1063-1064
Wrodnigg, T.M., et al. Bioorg. Med. Chem. 2004, 12, 3485-3495.
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Second Aglycon Library Screen of DMDP
Amide Derivatives
Modified Amadori conditions
Liang, P.-H. et al. ChemBioChem 2006, 7, 165-173.
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2nd Aglycon Library
Baker’s yeast α-glucosidase: 93%; 94% inhibition
IC50 = 0.15; 0.28 μM
Ki = 0.053; 0.077
Almond β-glucosidase: 67% inhibition
IC50 = 2.4 μM
Ki = 1.2
Liang, P.-H. et al. ChemBioChem 2006, 7, 165-173.
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Library of α- and β-Glucosidase Inhibitors
Azasugars diversified at C-1’ position to access a hydrophobic
binding pocket.
Rapid, in situ screening of azasugar-coupled acids facilitates
identification of glycosidase inhibitors.
Bicyclic, aromatic substituents increased potency of inhibition.
Aglycon libraries each inhibited different glucosidases.
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Conclusions
Azasugars core can be synthesized from chiral and achiral starting
materials.
Evolution of azasugar libraries utilize synthetic methods.
Inhibitor assays identified lipophilic C-1’ modifications increase
inhibition potentcy against α- and β-glucosidases.
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Evaluation of Current Approaches
X-ray crystallography of enzymes with bound 5-membered azasugar
inhibitors
Rigorous studies to identify specific inhibition modes per glycosidase.
Shorter synthetic routes to polyhydroxylated azasugars for more rapid
inhibitor identification.
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Acknowledgements
Professor Laura L. Kiessling
Kiessling Group
Charles P. Allen
Chris Shaffer
Rick McDonald
Tamas Benkovics
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