bio sphere
A versatile glycomics tool ith a vast array of monosaccharide fluorescently labeled glycans bearing primary amines. In the new method, an building blocks that can be asexcess of DAP is used so that only one sembled in almost limitless combinasugar reacts with each DAP molecule, tions, polysaccharides (glycans) have a leaving a free amine for further derivatigreater potential for structural diversity zation. Unreacted DAP is removed by than nucleic acids or proteins. Until recently, this diversity posed a key problem for glycobiologists: Individual glycans could not be purified in sufficient quantities from biological tissues for direct analysis. BeNH NH NH cause most glycans are too N N N complex to be readily syntheNH NH NH sized, researchers needed a straightforward method for converting glycans into derivatives that could be purified GDAP-microarray slides from tissues, quantified, and reacted with analytical GDAPs can be attached to microarray slides. reagents. Richard Cummings passing the reaction mixture over a carand colleagues at the University of Okbohydrate-binding cellulose filter. After lahoma Health Sciences Center recently elution, glycan–DAP conjugates (GDAPs) provided a crucial breakthrough with a are separated by HPLC on the basis of method to generate fluorescently lasize, charge, and structure. beled glycan derivatives that have reacCummings and colleagues showed tive primary amines (Nat. Methods that purified GDAPs react with a variety 2005, 2, 845–850). of analytically useful molecules. GDAPs According to James Paulson of the can be biotinylated for interaction with Scripps Research Institute, “A major streptavidin beads or covalently conjuproblem in the glycobiology field over the past 20–30 years [has been] to deriva- gated to carboxylate microspheres or protein carriers. These GDAPs are valutize sugars released from glycoproteins so able for many applications, including they could be coupled to other reagents. isolation and identification of new carThere’s an aldehyde group at the reducbohydrate-binding proteins (lectins). ing end of the sugar, but it’s a very poor For many glycobiologists, the most exreactive group, and most products are citing application of the new technique unstable unless further modified.” Inspired by the robust chemical reac- is the ability to conjugate GDAPs to activated glass slides to produce glycan tivity of protein amine groups and by microarrays. Probing glycan microarrays the known ability of glycans to react allows exploration of lectin specificity with primary aryl amines, Cummings and function. Says Cummings, “In the reasoned that reacting reducing glycans past, glycan microarrays were limited by with the fluorescent molecule 2,6-diavailability of the glycans, which was aminopyridine (DAP) would produce 644
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controlled by the organic chemists who had to synthesize them.” However, the new technique allows glycans to be directly released from glycoproteins obtained from tissues and purified in submicrogram amounts, expanding the repertoire of glycan microarrays. In addition to contributing a reactive primary amine, the DAP molecule also confers fluorescence to glycans. Michael Pierce of the University of Georgia comments, “With a fluorescently labeled carbohydrate, you can easily quantify the amount of derivatization, to make the methods highly reproducible.” Cummings hopes that the new technique will propel the glycobiology field forward by offering a simple, inexpensive derivatization approach that is accessible to the average biologist. Paulson says, “The chemistries are quite straightforward—you don’t have to be a synthetic chemist.” Pierce comments, “A large part of my lab relates to purifying glycans and determining their structure and function. People working in these areas will find this method very useful.” DAP derivatization of glycans is a major boon to the National Institutes of Health-funded Consortium for Functional Glycomics, of which Cummings is a member. “This opens up a whole new approach the consortium will take to create glycan microarrays,” he says. “Now we can prepare glycans from tissues, parasites, and pathogens. The opportunities for microarrays exploded.” a —Laura Tomky Cassiday RICHARD CUMMINGS, UNIV. OF OKLAHOMA HEALTH SCIENCES CTR.
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© 2006 AMERICAN CHEMICAL SOCIETY
