Design and Synthesis of 5-Membered Azasugars for Glycosidase Inhibition

1. Design and Synthesis of 5-Membered Azasugars for Glycosidase Inhibition Margaret L. Wong Kiessling Group 15 November 2007

2. Carbohydrates and Cellular Recognition Events Image adapted from cover of Nature1995, 373.

3. 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.

4. Impact of Glycosidase Inhibition Glycosidase inhibition can give rise to antiviral, anticancer, antibacterial activity.

5. 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.

6. Azasugars • Polyhydroxylated 5- and 6- membered N-heterocycles • Common names: “iminosugars” and “iminocyclitols” • Inhibitors of cellular glycosidases

7. 5-Membered Azasugars as Therapeutic Targets in Glycosidase Inhibition • Transition state analogue of natural sugars • Synthetic strategies for iminosugar core • Combinatorial libraries to probe efficacy of inhibition

8. 5-Membered Azasugars as Therapeutic Targets in Glycosidase Inhibition • Transition state analogue of natural sugars • Synthetic strategies for iminosugar core • Combinatorial libraries to probe efficacy of inhibition

9. 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. Structure1995, 3, 853-859

10. 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

11. ≡ 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

12. 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

13. Side-Chain Interactions with Azasugar • Charged endocyclic nitrogen C-6 Hydroxyl group & aglycon, hydrophobic substitution

14. 5-Membered Azasugars as Therapeutic Targets in Glycosidase Inhibition • Transition state analogue • Synthetic strategies of iminosugar core • Combinatorial libraries to probe efficacy of inhibition

15. 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.

16. Amadori Rearrangement of 5-Azido-Deoxy-D-Glucofuranose

17. Diastereoselectivity of Imine Reduction Kajimoto, T. et alJ. Am. Chem. Soc.1991, 113, 6678-6680. Takayama, S. et al. J. Am. Chem. Soc.1997, 119, 8146.

18. Epoxides as Chiral Precursors Via Sharpless asymmetric epoxidation Takebayashi, M. et alJ. Org. Chem. 1999, 64, 5280-5291.

19. 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.

20. 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.

21. Selectivity of Aldolases Machajewski, T.D.; Wong, C.-H. Angew. Chem. Int. Ed.2000, 39, 1352-1374.

22. 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.

23. 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.

24. 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

25. 5-Membered Azasugars as Therapeutic Targets in Glycosidase Inhibition • Transition state analogue • Synthetic strategies of iminosugar core • Combinatorial libraries to probe efficacy of inhibition

26. 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.

27. 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.

28. C-1’ Substitution Effects on β-Glucosidase 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.

29. Second Aglycon Library Screen of DMDP Amide Derivatives Modified Amadori conditions Liang, P.-H. et al. ChemBioChem2006, 7, 165-173.

30. 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. ChemBioChem2006, 7, 165-173.

31. 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.

32. 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.

33. 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.

34. Acknowledgements • Professor Laura L. Kiessling Kiessling Group Charles P. Allen Chris Shaffer Rick McDonald Tamas Benkovics