Chemoenzymatic Preparation of Novel Cyclic Imine Sugars and Rapid Biological Activity Evaluation Using Electrospray Mass Spectrometry and Kinetic Analysis

Cyclic imine sugars were prepared by a novel chemoenzymatic strategy in which azido-sugars, constructed by enzymatic aldol reactions, were hydrogenated under acidic conditions. These cyclic imine sugars were found to be potent inhibitors of glycoprocessing enzymes having Ki’s in the nanomolar and micromolar range for a variety of glycosidases. In comparison with their fully hydrogenated counterparts the cyclic imine sugars generally showed comparable or better inhibition against the glycosidases tested. Because these cyclic imines are so readily available and since imines are key intermediates in a variety of cycloadditions, condensations, and nucleophilic additions, they are valuable as versatile synthetic intermediates for the preparation of novel iminocyclitols and derivatives. An example of such synthetic utility is demonstrated by the synthesis of amino-iminocyclitol 24 via a three-center, twocomponent Strecker reaction. A novel method for rapidly screening glycosidase inhibitors using electrospray mass spectrometry is also described and shown to be capable of identifying potent fucosidase inhibitors for detailed kinetic analysis. Also, in the reductive amination of azido-sugars for the preparation of the five-membered ring iminocyclitol 8, rhodium was found to exhibit superior face selectivity when compared to palladium or platinum catalysts. The pivotal roles that carbohydrates play in biological processes have become increasingly evident.1 Efforts to understand and control the bioprocessing of carbohydrates is contributing much to glycobiology and to the development of new therapeutic strategies.2-8 Among agents capable of controlling glycoprocessing enzymes, iminocyclitols have been shown to be one of the most effective9-11 as protonated iminocyclitols mimic the oxonium ion transition state of sugar transfer reactions (Figure 1).11,12 Though several routes are available for the synthesis of these polyhydroxylated heterocycles,9,10,13,14 the use of aldolases followed by hydrogenation of the azido-sugar products is one of the most versatile and has been used for the preparation of various five-, six-, and seven-membered ring iminocyclitols (Figure 2).15-18 This paper describes a new strategy that allows facile chemoenzymatic synthesis of cyclic imine sugars which were found to be potent glycosidase inhibitors and useful building blocks for the synthesis of iminocyclitol derivatives and libraries. Also described is a method for rapidly identifying glycoprocessing enzyme inhibitors using electrospray mass spectrometry. X Abstract published in AdVance ACS Abstracts, August 15, 1997. (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Sears, P.; Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12086-12093. (3) Winchester, B.; Fleet, G. W. J. Glycobiology 1992, 2, 199-210. (4) Kim, C. U.; Lew, W.; Williams, M. A.; Liu, H.; Zhang, L.; Swaminathan, S.; Bischofberger, N.; Chen, M. S.; Mendel, D. B.; Tai, C. Y.; Laver, G.; Stevens, R. C. J. Am. Chem. Soc. 1997, 119, 681-690. (5) Lee, R. E.; Mikusová, P. J.; Brennan, P. J.; Besra, G. S. J. Am. Chem. Soc. 1995, 117, 11829-11832. (6) Nishimura, Y.; Satoh, T.; Adachi, H.; Kondo, S.; Takeuchi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka, Y. J. Am. Chem. Soc. 1996, 118, 30513052. (7) von Itzstein, M.; Wu, W.-Y.; Kok, G. B.; Pegg, M. S.; Dyason, J. C.; Jin, B.; Phan, T. V.; Smythe, M. L.; White, H. F.; Oliver, S. W.; Colman, P. M.; Varghese, J. N.; Ryan, D. M.; Woods, J. M.; Bethell, R. C.; Hotham, V. J.; Cameron, J. M.; Penn, C. R. Nature 1993, 363, 418-423. (8) Hendrix, M.; Wong, C.-H. Pure Appl. Chem. 1996, 68, 2081-2087. (9) Ganem, B. Acc. Chem. Res. 1996, 29, 340-347. (10) Hughes, A. B.; Rudge, A. J. Nat. Prod. Rep. 1994, 135-162. (11) Legler, G. AdV. Carbohydr. Chem. Biochem. 1990, 48, 319-384. (12) Sinnott, M. L. Chem. ReV. 1990, 90, 1171-1202. (13) Fleet, G. W. J. Chem. Br. 1989, 25, 287-292. (14) Paulsen, H.; Todt, K. AdV. Carbohydr. Chem. 1968, 23, 115-232. (15) Gijsen, H. J. M.; Qiao, L.; Fitz, W.; Wong, C.-H. Chem. ReV. 1996, 96, 443-473. (16) Look, G. C.; Fotsch, C. H.; Wong, C.-H. Acc. Chem. Res. 1993, 26, 182-190. (17) Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34, 412-432, 521-546. (18) Morı́s-Varas, F.; Xin-Hua, Q.; Wong, C.-H. J. Am. Chem. Soc. 1996, 118, 7647-7652. Figure 1. Postulated transition-state structures for glycosyl transferases and glycosidases and comparison with cyclic imine sugars. Figure 2. Preparation of azido-sugars and iminocyclitols using aldolases. 8146 J. Am. Chem. Soc. 1997, 119, 8146-8151 S0002-7863(97)01695-8 CCC: $14.00 © 1997 American Chemical Society Azido-sugars obtained from aldolase reactions are usually converted to iminocyclitols by catalytic hydrogenation under hydrogen pressures of 50 psi or more. This one-pot hydrogenation procedure consists of two or three reduction steps: (1) reduction of azide to amine; (2) reductive deoxygenation in the case of phosphates and butyrates and; (3) intramolecular reductive amination (Figure 3).19,20 The utility of azido-sugars would be greatly expanded if the hydrogenation process could be stopped after the first reduction step. Trapping this intermediate would give access to cyclic imine sugars whose shape and charge should mimic the transition-state stabilized by glycoprocessing enzymes (Figure 1). Additionally, since imines are versatile intermediates capable of various reactions such as cycloadditions, condensations, and nucleophilic additions, the cyclic imine sugars should be useful for the synthesis of iminocyclitol derivatives and libraries. We performed our studies on azido-sugars 2-5 and 7 which were prepared according to Scheme 1. The known enzymatic aldol condensation of 2-azido-3-hydroxy propanal (1) and dihydroxyacetone phosphate (DHAP) gave a diastereomeric mixture of 2 and 3 which were separated by silica gel chromatography after enzymatic butyration of the primary hydroxyl group. Azido-sugar 7 was prepared chemoenzymatically according to published procedures.21 Since hydrogenation of azido-sugar 2 to iminocylitol 8 had been performed using hydrogen pressures of 50 psi or more in previous reports,23-26 we initially anticipated that the reductive amination step might be avoided by using a lower pressure of hydrogen. However, we found that an atmospheric pressure (15 psi) of hydrogen or even formic acid was sufficient to convert the azido-sugar 2 to iminocyclitol 8 within 10 min (Figure 4). In the case of the butyrated compound 4, hydrogenation gave the deoxygenated iminocyclitol, as in the case of phosphate sugars25 (Figure 3). Different catalysts were also investigated but each gave either complete hydrogenation or no reaction. Because the rearrangement of the amino-carbonyl sugar to its cyclic imine form is a fast intramolecular process and the subsequent reduction also very facile, it was not possible to prevent over-reduction under these conditions. We also observed that, among various catalysts, rhodium-alumina gave a higher face selectivity in the reductive amination of azidosugar 2 to iminocyclitol 8 as seen in Figure 4. Unlike the completely face-selective reductive amination frequently observed for the preparation of six-membered ring iminocyclitols, palladiumor platinum-catalyzed reductive amination for the preparation of many five-membered ring iminocyclitols such as 824-26 results in only 85-95% face selectivity at best. Rhodium is a superior catalyst for achieving high face-selectivity in the preparation of 8 and perhaps is useful for the highly faceselective reductive amination of other azido-sugars. To prevent the azido-sugars from full hydrogenation to iminocyclitols, an intermediate had to be trapped in some form. We decided to try trapping the amino-carbonyl sugar intermediate as its hydrochloride salt. Protonation of the amine would greatly disfavor rearrangement to the cyclic imine, a step required for the subsequent reductive amination, and should (19) Kajimoto, T.; Liu, K. K.-C.; Pederson, R. L.; Zhong, Z.; Ichikawa, Y.; Porco, J. A. J.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113, 61876196. (20) Liu, K. K.-C.; Kajimoto, T.; Chen, L.; Zhong, Z.; Ichikawa, Y.; Wong, C.-H. J. Org. Chem. 1991, 56, 6280-6289. (21) Qiao, L.; Murray, B. W.; Shimazaki, M.; Schultz, J.; Wong, C.-H. J. Am. Chem. Soc. 1996, 118, 7653-7662. (22) Pathak, V. P. Syn. Commun. 1993, 23, 83-85. (23) Pederson, R. L.; Kim, M.-J.; Wong, C.-H. Tetrahedron Lett. 1988, 29, 4645-4648. (24) Ziegler, T.; Straub, A.; Effenberger, F. Angew. Chem., Int. Ed. Engl. 1988, 27, 76-717. (25) Kajimoto, T.; Chen, L.; Liu, K. K.-C.; Wong, C.-H. J. Am. Chem. Soc. 1991, 113, 6678-6680. (26) Hung, R. R.; Straub, J. A.; Whitesides, G. M. J. Org. Chem. 1991, 56, 3849-3855. Figure 3. The steps involved in the hydrogenation of an azido-sugar to an iminocyclitol.