Functionalized C-Glycoside Ketohydrazones: Carbohydrate Derivatives that Retain the Ring Integrity of the Terminal Reducing Sugar Neil P. J. Price,* ,† Michael J. Bowman, † Sophie Le Gall, ‡ Mark A. Berhow, † David F. Kendra, † and Patrice Lerouge ‡ National Center for Agricultural Utilization Research (NCAUR), USDA-ARS, 1815 North University Street, Peoria, Illinois 61604, and Laboratoire Glycobiologie et Matrice Extracellulaire Ve ´ge ´ tale, EA 4358, IFRMP 23, Universite ´ de Rouen, 76821 Mont Saint-Aignan, France Glycosylation often mediates important biological pro- cesses through the interaction of carbohydrates with complementary proteins. Most chemical tools for the functional analysis of glycans are highly dependent upon various linkage chemistries that involve the reducing terminus of carbohydrates. However, because of ring opening, the structural integrity of the reducing sugar ring (pyranose or furanose) is lost during these techniques, resulting in derivatized carboydrates that markedly differ from the parent molecule. This paper describes a new aqueous-based, one-pot strategy that involves first con- verting the sugar to a C-glycoside ketone, followed by conversion to ketohydrazones or oximes. Hence, the C-glycoside ketones are tagged with fluorescence, colored, cationic or biotin-labeled groups or immobilized onto hydrazine-functionalized beads. No activating or protect- ing groups are required, and the chemistry is mild enough for a wide range of carbohydrates. We demonstrate the versatility of the approach to diverse glycans, including bead immobilization and lectin analysis of acarbose, an antidiabetic drug, to dabsyl-tagged enzyme substrates to screen cellulases, and for the analysis of plant cell wall hemicellulosics. Genomic and proteomic techniques are widely used to eluci- date the role of genes and proteins in biological systems. Until now, the corresponding techniques to explore the role of carbo- hydrates in biology, the growing discipline of glycomics, has tended to lag behind. Carbohydrates have long been known to be important in a variety of cellular process, including inflamma- tion, signal transduction, fertilization, development, and cell-cell, bacterium-cell, and virus-cell recognition. 1-5 A number of glycomics tools are becoming available, including fluorescent and colorimetric glycoconjugates, sugar microarrays, quantum-dot conjugates, affinity-tagged carbohydrates, derivatized micro- spheres, and affinity resins. 6-10 However, rapid advances have been hindered by the structural complexity and diversity of monosaccharides, glycoconjugates, oligosaccharides, and poly- saccharides, and the relative difficulty of isolating, characterizing, and synthesizing such complex heterologous structures. Most glycomics tools are highly dependent upon various linkage chemistries that involve the reducing-terminus of carbo- hydrates. 11-17 Numerous techniques for the derivatization of sugars have been described, including reductive amination and the formation of reducing sugar hydrazones, thiazolidines, and oximes (Figure 1). However, because of ring-opening, the struc- tural integrity of the reducing sugar ring (pyranose or furanose) is not retained during these techniques, which thus results in derivatized oligosaccharides that markedly differ from the parent molecule. Reductive amination methods are based on imine formation between an amine reagent and the anomeric carbonyl group of a reducing carbohydrate, followed by reduction to an open-chain glycamine derivative. The imines themselves are generally unstable, with the equilibrium favoring free carbonyl, except when stabilized by alpha-nitrogens such as in hydrazones and oximes. 18 Hydrazones and oximes are excellent derivatives for the analysis of simple aldehydes and ketones, but sugars do not usually form simple hydrazones. Rather, they react with three equivalents of aryl- or acyl-hydrazines at both the C-1 and C-2 carbons to give sugar osazones. An imine initially forms at the anomeric C-1 carbon. The adjacent hydroxyl is then oxidized to a carbonyl group, which reacts with a third equivalent of the * Corresponding author. E-mail: neil.price@ars.usda.gov. Tel: 1-309-681-6246. Fax. 1-309-681-6040. † USDA-ARS. ‡ Universite ´ de Rouen. (1) Dwek, R. A. Chem. Rev. 1996, 96, 683–720. (2) Varki, A. Glycobiology 1993, 3, 97–130. (3) Raman, R.; Raguram, S.; Venkataraman, G.; Paulson, J. C.; Sasisekharan, R. Nat. Methods 2005, 2, 817–824. (4) Paulson, J. C.; Blixt, O.; Collins, B. E. Chem. Biol. 2006, 2, 238–248. (5) Prescher, J. A.; Bertozzi, C. R. Cell 2006, 126, 851–854. (6) Ohtsubo, K.; Marth, J. D. Cell 2006, 126, 855–867. (7) Turnbull, J. E.; Field, R. A. Nat. Chem. Biol. 2007, 3, 74–77. (8) Ratner, D. M.; Adams, E. W.; Disney, M. D.; Seeberger, P. H. ChemBioChem 2004, 5, 1375–1383. (9) Houseman, B. T.; Mrksich, M. Chem. Biol. 2002, 9, 443–454. (10) Feizi, T.; Mulloy, B. Curr. Opin. Struct. Biol. 2003, 13, 602–604. (11) O’Shannessy, D. J.; Wilchek, M. Anal. Biochem. 1990, 191, 1–8. (12) Xia, B.; Kawar, Z. S.; Ju, T.; Alvarez, R. A.; Sachdev, G. P.; Cummings, R. D. Nat. Methods 2005, 2, 845–850. (13) Larsen, K.; Thygesen, M. B.; Guillaumie, F.; Willats, W. G. T.; Jensen, K. J. Carbohydr. Res. 2006, 341, 1209–1234. (14) Yarema, K. J.; Bertozzi, C. R. Curr. Opin. Chem. Biol. 1998, 2, 49–61. (15) Fukui, S.; Feizi, T.; Galustian, C.; Lawson, A. M.; Chai, W. Nat. Biotechnol. 2002, 20, 1011–1017. (16) Hase, S. J. Chromatogr., A 1996, 720, 173–182. (17) Lamari, F. N.; Kuhn, R.; Karamanos, N. K. J. Chromatogr., B 2003, 793, 15–36. (18) Sander, E. G.; Jencks, W. P. J. Am. Chem. Soc. 1968, 90, 6154–6162. Anal. Chem. 2010, 82, 2893–2899 10.1021/ac902894u  2010 American Chemical Society 2893 Analytical Chemistry, Vol. 82, No. 7, April 1, 2010 Published on Web 03/01/2010