Cyclic Guanidino-Sugars with Low pK a as Transition-State Analog Inhibitors of Glycosidases: Neutral Instead of Charged Species Are the Active Forms Jin-Hyun Jeong, Brion W. Murray, Shuichi Takayama, and Chi-Huey Wong* Contribution from the Department of Chemistry, The Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, California 92037 ReceiVed May 17, 1995 X Abstract: Cyclic guanidino-sugars with different pK a values are designed and synthesized as transition-state analog inhibitors of galactosidases. Characterization of these structures (7, 10, 12) reveals that 7 and 10 are in a pH- dependent equilibrium between a furanose form and a mixture of neutral and protonated tetrahydropyrimidine forms. In contrast, the O-linked guanidino-sugar 12 exists as the tetrahydropyrimidine forms above pH 5. The furanose- tetrahydropyrimidine equilibrium can thus be modulated with the appropriate N-substituent which affects the guanidino- sugar pK a value. Enzymatic inhibition by 7, 10, and 12 is also pH-dependent, indicating that the enzymes recognize the tetrahydropyrimidine form. Evidence is presented to support a dominant role for the uncharged form of the six-membered cyclic guanidino-sugar in the inhibition of galactosidases. Though the inhibition potency is moderate (K i range 4-50 µM), the use of cyclic guanidino-sugars in the study provides new insights into the mechanism of inhibition of glycosidases. Oligosaccharide biosynthesis and degradation pathways are crucial to such diverse processes as HIV-1 replication, inflam- mation, blood clotting, cell differentiation, tumor development, cell routing, diabetes, and other metabolic disorders. Mecha- nistic understanding 1 and inhibition 2-5 of the enzymes that direct these reactions (glycosidases, glycosyltransferases) are areas of considerable interest. Both classes of enzymes are thought to proceed through a flattened half-chair (or twist-boat) conforma- X Abstract published in AdVance ACS Abstracts, April 15, 1996. (1) (a) Schramm, V. L.; Horenstein, B. A.; Kline, P. C. J. Biol. Chem. 1994, 269, 18259. (b) Sinnott, M. L. Chem. ReV. 1990, 90, 1171-1202. (c) Sharper, J. H.; Sharper, N. L. Curr. Opin. Struct. Biol. 1992, 2, 701. (d) Withers, S. G.; Warren, R. A.; Street, I. P.; Rupitz, K.; Kempton, J. B.; Abersold, R. J. J. Am. Chem. Soc. 1990, 112, 5887. (e) Singh, A. N.; Hester, L. S.; Raushel, F. M. J. Biol. Chem. 1987, 262, 2554. (f) Bell, J. E.; Beyer, T. A.; Hill, R. L. J. Biol. Chem. 1976, 251, 3003-3013. (g) Dale, M. P.; Ensley, H. E.; Kern, K.; Sastry, K. A. R.; Byers, L. D. Biochemistry 1985, 24, 3530-3539. (h) Franck, R. W. Bioorg. Chem. 1992, 20, 77. (i) Taylor, N. R.; von Itzstein, M. J. Med. Chem. 1994, 37, 616. (2) (a) Hodgson, J. Bio/Technology 1991, 9, 609. (b) Asano, N.; Kizu, H.; Oseki, K.; Tomioka, E.; Matusi, K.; Okamoto, M.; Baba, M. J. Med. Chem. 1995, 38, 2349-2356. (c) Winchester, B.; Fleet, G. W. J. Glyco- biology 1992, 2, 199-210. (d) Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res. 1993, 26, 182-190. (e) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34, 521- 546. (f) Elbein, A. D. Annu. ReV. Biochem. 1987, 56, 497-534. (g) Carchon, H.; Bruyne, C. K. D. Carbohydr. Res. 1975, 41, 175-189. (3) (a) Bernotas, R. C.; Pezzone, M. A.; Ganem, B. Carbohydr. Res. 1987, 167, 305-311. (b) Papandreou, G.; Tong, M. K.; Ganem, B. J. Am. Chem. Soc. 1993, 115, 11682-11690. (c) Schedler, D. J. A.; Bowen, B. R.; Ganem, B. Tetrahedron Lett. 1994, 35, 3845. (d) Ichikawa, M.; Igarashi, Y.; Ichikawa, Y. Tetrahedron Lett. 1995, 36, 1767-1770. (e) Oszczapowicz, J.; Orlinski, R.; Walczynska, H. Pol. J. Chem. 1979, 53, 2531. (f) Ichikawa, Y.; Igarashi, Y. Tetrahedron Lett. 1995, 36, 4585. (g) Ermert, P.; Vasella, A. HelV. Chim. Acta 1991, 74, 2043. (4) (a) Kajimoto, T.; Liu, K.-C.; Pederson, R. L.; Zhong, Z.; Ichikawa, Y.; Porco, J. A.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113, 6187. (b) Wang, Y.-F.; Dumas, D. P.; Wong, C.-H. Tetrahedron Lett. 1993, 34, 403. (c) Dumas, D. P.; Kajimoto, T.; Liu, K. K.-C.; Wong, C.-H.; Berkowitz, D. B.; Danishefsky, S. J. Bioorg. Med. Chem. Lett. 1992, 2, 33-36. (d) Truscheit, E.; Frommer, W.; Junge, B.; Mu ¨ller, L.; Schmidt, D. D.; Wingender, W. Angew. Chem., Int. Ed. Engl. 1981, 20, 744. (e) Schwarz, P. M.; Elbein, A. D. J. Biol. Chem. 1985, 26, 14452. (f) Gruters, R. A.; Neefjes, J. J.; Termette, M.; Goede, R. E. Y. D.; Tulp, A. Nature 1987, VOLUME 118, NUMBER 18 MAY 8, 1996 © Copyright 1996 by the American Chemical Society S0002-7863(95)01602-7 CCC: $12.00 © 1996 American Chemical Society