Derivatization of Free Natural Glycans for Incorporation onto Glycan Arrays: Derivatizing Glycans on the Microscale for Microarray and Other Applications

Xuezheng Song1, Jamie Heimburg‐Molinaro1, David F. Smith1, Richard D. Cummings1

1 Emory University, Atlanta, Georgia
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100194
Online Posting Date:  April, 2011
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Abstract

Nature possesses an unlimited number and source of biologically relevant natural glycans, many of which are too complicated to synthesize in the laboratory. To capitalize on the naturally occurring plethora of glycans, a method is presented here to fluorescently tag isolated free glycans while maintaining the closed‐ring structure. After purification of the labeled glycans, they can be printed on a glass surface to create a natural glycan microarray, suitable for interrogation with potential glycan‐binding proteins. The derivatization of these natural glycans has vastly expanded the number of glycans available for functional studies. Curr. Protoc. Chem. Biol. 3:53‐63 © 2011 by John Wiley & Sons, Inc.

Keywords: fluorescence; reductive amination; glycan microarray; conjugation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Closed‐Ring Glycan‐AEAB from Free Reducing Glycans
  • Alternate Protocol 1: Preparation of AEAB Conjugates Using Commercial Chemicals
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Closed‐Ring Glycan‐AEAB from Free Reducing Glycans

  Materials
  • 0.05 to 1 mg free reducing glycan, lyophilized (e.g., Sigma‐Aldrich, V‐labs, Carbosynth)
  • Milli‐Q purified water (Millipore), or equivalent
  • Ammonium bicarbonate
  • 50% (v/v) acetonitrile, HPLC grade (Fisher Scientific)/10 mM ammonium bicarbonate
  • 10 mM ammonium bicarbonate
  • Sodium bicarbonate
  • Saturated sodium bicarbonate solution, ice‐cold
  • Acryloyl chloride (e.g., Sigma‐Aldrich)
  • Sodium borohydride (e.g., Sigma‐Aldrich), optional
  • Acetic acid, ACS grade (Fisher Scientific), optional
  • 50% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid (TFA), HPLC grade (Fisher Scientific)
  • Methanol
  • Ethanol
  • Ozone
  • Nitrogen gas
  • Methyl sulfide
  • 7:3 (v/v) dimethyl sulfoxide (DMSO), ACS grade (Fisher Scientific)/acetic acid
  • Sodium cyanoborohydride
  • 2‐(N‐aminoethyl)‐amino benzamide (AEAB) hydrochloride (Song et al., )
  • Acetonitrile
  • 1% (v/v) trifluoroacetic acid (TFA), HPLC grade (Fisher Scientific)
  • 1.5‐ml screw‐cap polypropylene centrifuge tubes
  • 55°C and 65°C heating block or water bath
  • 150 mg, 300 mg, and 1 g carbograph solid phase extraction (SPE) columns (Alltech)
  • Rotary evaporator (e.g., SpeedVac, Thermo Scientific)
  • Lyophilizer
  • 15‐ml conical polypropylene centrifuge tubes
  • Porous graphitized carbon (PGC) analytical HPLC column (Thermo Scientific)
  • High‐performance liquid chromatography (HPLC) system

Alternate Protocol 1: Preparation of AEAB Conjugates Using Commercial Chemicals

  Materials
  • 7:3 (v/v) dimethyl sulfoxide (DMSO)/acetic acid solution
  • p‐nitrophenyl anthranilate, 98% (PNPA, Fisher Scientific)
  • Sodium cyanoborohydride, 95% (Sigma‐Aldrich)
  • 0.05 to 1 mg free reducing glycan, lyophilized (e.g., Sigma‐Aldrich, V‐labs, Carbosynth)
  • Acetonitrile
  • 1% (v/v) trifluoroacetic acid (TFA)
  • Milli‐Q purified water, or equivalent
  • Ethylenediamine solution: dissolve 100 µl ethylenediamine in 1 ml DMSO
  • 10% (v/v) acetic acid
  • 65°C heating block or water bath
  • C18 analytical column
  • High‐performance liquid chromatography (HPLC) system
  • Rotary evaporator (e.g., SpeedVac, Thermo Scientific)
  • Lyophilizer
  • Hypercarb (PGC) HPLC column
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Figures

Videos

Literature Cited

Literature Cited
   Alvarez, R.A. and Blixt, O. 2006. Identification of ligand specificities for glycan‐binding proteins using glycan arrays. Methods Enzymol. 415:292‐310.
   Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., and Parekh, R.B. 1995. Nonselective and efficient fluorescent labeling of glycans using 2‐amino benzamide and anthranilic acid. Anal. Biochem. 230:229‐238.
   Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J., van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.H., and Paulson, J.C. 2004. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U.S.A. 101:17033‐17038.
   Cummings, R.D. 2009. The repertoire of glycan determinants in the human glycome. Mol. Biosyst. 5:1087‐1104.
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   Feizi, T. and Chai, W. 2004. Oligosaccharide microarrays to decipher the glyco code. Nat. Rev. Mol. Cell Biol. 5:582‐588.
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   Luyai, A., Lasanajak, Y., Smith, D.F., Cummings, R.D., and Song, X. 2009. Facile preparation of fluorescent neoglycoproteins using p‐nitrophenyl anthranilate as a heterobifunctional linker. Bioconjug. Chem. 20:1618‐1624.
   Manger, I.D., Rademacher, T.W., and Dwek, R.A. 1992. 1‐N‐glycyl beta‐oligosaccharide derivatives as stable intermediates for the formation of glycoconjugate probes. Biochemistry 31:10724‐10732.
   Paulson, J.C., Blixt, O., and Collins, B.E. 2006. Sweet spots in functional glycomics. Nat. Chem. Biol. 2:238‐248.
   Smith, D.F., Song, X., and Cummings, R.D. 2010. Use of glycan microarrays to explore specificity of glycan‐binding proteins. Methods Enzymol. 480:417‐444.
   Song, X., Lasanajak, Y., Rivera‐Marrero, C., Luyai, A., Willard, M., Smith, D.F., and Cummings, R.D. 2009a. Generation of a natural glycan microarray using 9‐fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag. Anal. Biochem. 395:151‐160.
   Song, X., Lasanajak, Y., Xia, B., Smith, D.F., and Cummings, R.D. 2009b. Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem. Biol. 4:741‐750.
   Song, X., Xia, B., Stowell, S.R., Lasanajak, Y., Smith, D.F., and Cummings, R.D. 2009c. Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem. Biol. 16:36‐47.
   Song, X., Lasanajak, Y., Xia, B., Heimburg‐Molinaro, J., Rhea, J.M., Ju, H., Zhao, C., Molinaro, R.J., Cummings, R.D., and Smith, D.F. 2011. Shotgun glycomics: A microarray strategy for functional glycomics. Nat. Methods 8:85‐90.
   Stevens, J., Blixt, O., Paulson, J.C., and Wilson, I.A. 2006. Glycan microarray technologies: Tools to survey host specificity of influenza viruses. Nat. Rev. Microbiol. 4:857‐864.
   Stowell, S.R., Arthur, C.M., Dias‐Baruffi, M., Rodrigues, L.C., Gourdine, J.P., Heimburg‐Molinaro, J., Ju, T., Molinaro, R.J., Rivera‐Marrero, C., Xia, B., Smith, D.F., and Cummings, R.D. 2010. Innate immune lectins kill bacteria expressing blood group antigen. Nat. Med. 16:295‐301.
   Xia, B., Kawar, Z.S., Ju, T., Alvarez, R.A., Sachdev, G.P., and Cummings, R.D. 2005. Versatile fluorescent derivatization of glycans for glycomic analysis. Nat. Methods 2:845‐850.
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