ChemBioChem 2004, 5, 379 ± 383 DOI: 10.1002/cbic.200300804 ¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 379 Probing Protein±Carbohydrate Interactions with Microarrays of Synthetic Oligosaccharides Daniel M. Ratner, [a] Eddie W. Adams, [a] Jing Su, [b] Barry R. O'Keefe, [c] Milan Mrksich, [b] and Peter H. Seeberger* [a, d] Formerly a ™neglected dimension∫ of biochemistry, recent years have seen growing interest in studying the biological function of carbohydrates and glycoconjugates. An emerging understand- ing of the physiological role of these biomolecules has uncovered their vital participation in a host of fundamental cellular processes. In the form of glycopeptides, glycolipids, glycosaminoglycans, and proteoglyans, glycoconjugates are known to be involved in inflammation, [1] cell ± cell interactions, [2] signal transduction, [3] fertility, and development. [4, 5] Unfortu- nately, current methods for elucidating the biochemical roles of glycoconjugates are often cumbersome. This demonstrates the need to develop techniques that will satisfy this growing field of study by enabling rapid and facile exploration of biochemical events involving carbohydrates. Inspired by the success of DNA and protein microarrays, [6, 7] the chip-based approach has been put forward as a useful tool in the emerging field of glycomics. [8±10] Nitrocellulose-coated slides have been employed for the noncovalent immobilization of microbial polysaccharides and neoglycolipid-modified oligosac- charides. [11, 12] Hydrophobic interactions have been utilized to anchor lipid-bearing carbohydrates on polystyrene microtiter plates. [13] Self-assembled monolayers presenting benzoquinone groups enabled the Diels ± Alder-mediated immobilization of cyclopentadiene-derivatized monosaccharides on a gold sur- face. [14] Another covalent immobilization chemistry involved treating maleimide-functionalized mono- and disaccharide gly- cosylamines with a thiol-derivatized glass slide, [15] or, alterna- tively, thiol-functionalized carbohydrates with a self-assembled monolayer presenting maleimide groups. [16] Our motivation for developing a system for arraying carbohy- drates is based on the need to have microarrays that are fully phosphate buffer (0.1 M) and Perfect Hyb hybridization buffer (Sigma, 1:1 v/v) for 5 h to give double-stranded DNA assembly on the surface. The resulting surfaces were rinsed with the hybridization buffer and immersed in a solution of hemin (1.2 mM) in buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO; pH 7.4) for 12 h at room temperature. The resulting system was further treated with doxorubicin (5,5 mM) in phosphate buffer (0.1 M, pH 7.4) for 1 h at room temperature. Light-emission measurements : Light emission was performed by using a photon-counting spectrometer (Edinburgh Instruments, FLS 920) equipped with a cooled photomultiplier detection system, connected to a computer (F900 v. 6.3 software). Before the samples analyses, the background light was recorded and integrated. This background was subtracted from the recorded integrated spectra of the respective samples. Sample analyses were performed by adding the DNAzyme solution (15 mL) or the modified surface to luminol (0.5 mM) and H 2 O 2 (30 mM) in buffer solution (3.3 mL; 25 mM HEPES, 20 mM KCl, and 200 mM NaCl; pH 9.0) in a cuvette. Acknowledgements This research is supported by the German ± Israeli Foundation (GIF). Keywords: biocatalysis ¥ DNA ¥ hemin ¥ luminescence ¥ monolayers ¥ self-assembly [1] a) T. R. Cach, A. J. Zang, P. J. Grabowski, Cell, 1981, 27 , 487 ± 496; b) C. Guerrier-Takeda, K. Gardiner, T. Marsh, N. Pace, S. Altman, Cell, 1983, 35, 849 ± 857. [2] a) D. N. Frank, N. R. 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Willner, J. Am. Chem. Soc. 2003, 125, 3452±3454; b) F. Patolsky, Y. Weizmann, E. Katz, I. Willner, Angew. Chem. 2003, 115, 2474 ± 2478; Angew. Chem. Int. Ed. 2003, 42, 2372 ± 2376. [9] F. Patolsky, E. Katz, I. Willner, Angew. Chem. 2002, 114, 3548 ± 3552 ; Angew. Chem. Int. Ed. 2002, 41, 3398 ± 3402. [10] V. Pavlov, Y. Xiao, I. Willner, unpublished results. [11] D. K. Lavallee, The Chemistry and Biochemistry of N-Substituted Porphyrins, VCH, Weinheim, 1987 . Received: October 17, 2003 [Z794] [a] D. M. Ratner, E. W. Adams, Prof. Dr. P. H. Seeberger Department of Chemistry, Massachusetts Institute of Technology Cambridge, MA 02139 (USA) E-mail: seeberger@org.chem.ethz.ch [b] J. Su, Prof. Dr. M. Mrksich Department of Chemistry and Institute for Biophysical Dynamics The University of Chicago Chicago, IL 60637 (USA) [c] Dr. B. R. O'Keefe Molecular Targets Discovery Program NCI Center for Cancer Research, National Cancer Institute Frederick, MD 21702 (USA) [d] Prof. Dr. P. H. Seeberger Current address: Laboratory for Organic Chemistry, ETH-Hˆnggerberg HCI F315 Wolfgang-Pauli-Stra˚e 10, 8093 Z¸rich (Switzerland) Fax: ( 41) 1633-1235