Miniaturization of Microwave‐Assisted Carbohydrate Functionalization to Create Oligosaccharide Microarrays

Glycomics focuses on studying the interactions of carbohydrates with other biopolymers, particularly proteins, to gain a better understanding of the roles of sugars in biological processes. 2] Challenges faced in glycomics have been met in the fields of genomics and proteomics. These include developing reliable tools, such as mass spectrometry and gene and protein arrays, that can be used to aid biological investigations. Developing equivalent tools for glycomics suffers from the difficulty in obtaining pure carbohydrates, since neither the isolation nor synthesis of defined carbohydrates is straightforward. Microarrays offer an attractive platform for probing the interactions of carbohydrates with other biopolymers in a high-throughput manner. The advantage of this technique is that each screening experiment requires only picomoles of material and it allows parallel screening of thousands of interactions. Several studies have been undertaken to develop a general high-throughput screening (HTS) platform that can be used in glycomics investigations. These studies evaluated different surfaces, modes of attachment and linkers, by using either isolated or synthetic oligoand polysaccharides. In two of the above examples, maleimide-functionalized glycosylamines were immobilized onto thiol-derivatized glass slides or thiol-functionalized oligosaccharides were immobilized onto maleimide-functionalized surfaces to create high-density arrays that were spotted by using an arraying robot. The small volumes that are used with a robotic arrayer offer the advantage of assay miniaturization, since only picomoles of material are required for each compound to be screened. Such slides have been applied to address biological problems including the identification of HIV vaccine candidate antigens, the evaluation of aminoglycoside antibiotics and the detection of pathogenic bacteria. The most significant challenge that remains for the construction of carbohydrate arrays is the generation of carbohydrates that can be immobilized onto glass surfaces. Given the host of applications of carbohydrate microarrays, finding solutions to this problem will allow easier access to array-based tools for performing glycomics studies. The Functional Glycomics Consortium in the USA developed a carbohydrate array with 200 structures that are 90% synthetic. Despite this, faster access to a host of complex carbohydrates is needed. Here we describe a chemical route to introduce a linker at the reducing end of carbohydrates. A microwave-assisted variation of the Kochetkov amination reaction is the key in a two-step procedure to N-glycosides containing a linker suitable for immobilization onto glass slides. Each reaction is performed without a purification step and is therefore attractive when functionalizing a large number of structures. This simple protocol allows for as little as 100 mg of carbohydrate to be immobilized on an array and displayed such that it binds to a protein. This method could streamline the major issue in the construction of carbohydrate arrays—access of arrayable material. Glycosylamines have previously been synthesized by different routes to access glycoconjugates for construction of carbohydrate microarrays. Treatment of reducing sugars with ammonium carbonate to create anomeric amines (Kochetkov reaction) is a common method for glycoconjugate synthesis. This reaction is hampered by slow reaction rates. When the Kochetkov amination was carried out in a microwave reactor, we found that product was formed in as little as 90 min, compared to the 5 days required when using “traditional” conditions. Importantly, this improvement was observed even when only a fivefold excess of ammonium carbonate over sugar was used. This is in contrast to traditional reactions, which require up to a 50-fold excess of ammonium carbonate. The tenfold reduction in the amount of ammonium carbonate required greatly simplifies the removal of uncoupled ammonium carbonate, which can interfere with subsequent conjugation steps. During the course of our studies, Bejugam and Flitsch reported similar findings for the synthesis of glycosylamines using ammonium carbonate in dimethyl sulfoxide (DMSO) with microwave irradiation. We found that methanol proved superior to DMSO as solvent since methanol is more readily removed than is DMSO and it allowed for a larger number of carbohydrates to be converted to glycosylamines. Nine carbohydrates were converted to 1-amino-1-deoxy sugars. A fivefold excess of ammonium carbonate in methanol was heated in a microwave reactor at 40 8C (10 W maximum power) for 90 min (Scheme 1, Table 1) and reaction products were characterized by H NMR and mass spectroscopy. Only a small amount of diglycosylamine was detected, an important finding since the dimer is a common by-product in conventional reactions. Removal of the diglycosylamines from the reaction product was not required because they do not interfere with the subsequent acylation. [a] M. A. Brun, Dr. M. D. Disney, Prof. Dr. P. H. Seeberger Laboratory for Organic Chemistry Swiss Federal Institute of Technology (ETH) Z)rich HCI F315 Wolfgang-Pauli-Strasse 10, 8093 Z)rich (Switzerland) Fax: (+41)1633-1235 E-mail : seeberger@org.chem.ethz.ch [b] Dr. M. D. Disney Current address: Department of Chemistry University at Buffalo The State University of New York Buffalo 657 Natural Sciences Complex, NY 14260-3000 (USA) [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.