Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars

Department of Chemistry, Stanford University, Stanford, California, USA.
Nature Protocol (Impact Factor: 9.67). 02/2009; 4(7):1044-63. DOI: 10.1038/nprot.2009.85
Source: PubMed


Transient interactions among glycoconjugates underlie developmental, immunological and metastatic recognition. Glycan-mediated interactions have low binding affinities and rapid dissociation rates. As a result, these complexes dissociate when removed from their cellular context, complicating characterization. Photocrosslinkers introduce a covalent bond between glycoconjugates and their binding partners, allowing physiologically relevant complexes to be isolated. This protocol describes metabolic incorporation of a diazirine photocrosslinker into sialic acids in cellular glycoconjugates. Subsequent irradiation results in photocrosslinking of sialic acid to neighboring macromolecules, providing a photochemical 'snapshot' of binding events. As photocrosslinking sugars are light activated, these reagents have the potential to be used for temporally and/or spatially restricted crosslinking. We provide instructions for the synthesis of photocrosslinking sugar precursors, cell culture for metabolic incorporation, flow cytometry to evaluate metabolic incorporation, photoirradiation and analysis of the crosslinked complexes. Synthesis of photocrosslinking sugars requires 4-6 d, and photocrosslinking experiments can be completed in an additional 6 d.

Download full-text


Available from: Michelle R Bond, Mar 12, 2014
  • Source
    • "A covalent linkage generated in vivo or in vitro between a small molecule and a macromolecule can be used to characterize and map multiple structural features (Brunner 1996) or to probe for specific receptors (Bond et al. 2009; Hatanaka and Sadakane 2002). In this way, the development of bioconjugate reactions using affinity labels has permitted many elegant and structurally informative studies (Hino et al. 2005; Suchanek et al. 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability to incorporate non-canonical amino acids into proteins by genetic or chemical methods allows one to introduce novel chemical properties into a protein at a defined residue. Such a residue may then be modified using common organic transformations. In this way, the structure or function of the peptide may be altered without perturbing any of the other neighbouring amino acids in the peptide chain. Here, we describe the syntheses and potential applications of multiple para-substituted phenylalanine derivatives comprising an isothiocyanate, α-diazoketone, or nitrone functionality. In all, three novel amino acids were synthesized in good overall yields. These non-canonical amino acids permit the further development of in vitro and in vivo chemoselective and regioselective bioconjugate reactions not possible with other reagents.
    Amino Acids 11/2010; 39(5):1381-4. DOI:10.1007/s00726-010-0594-3 · 3.29 Impact Factor
  • Source

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This report provides a perspective on metabolic glycoengineering methodology developed over the past two decades that allows natural sialic acids to be replaced with chemical variants in living cells and animals. Examples are given demonstrating how this technology provides the glycoscientist with chemical tools that are beginning to reproduce Mother Nature's control over complex biological systems – such as the human brain – through subtle modifications in sialic acid chemistry. Several metabolic substrates (e.g., ManNAc, Neu5Ac, and CMP-Neu5Ac analogs) can be used to feed flux into the sialic acid biosynthetic pathway resulting in numerous – and sometime quite unexpected – biological repercussions upon nonnatural sialoside display in cellular glycans. Once on the cell surface, ketone-, azide-, thiol-, or alkyne-modified glycans can be transformed with numerous ligands via bioorthogonal chemoselective ligation reactions, greatly increasing the versatility and potential application of this technology. Recently, sialic acid glycoengineering methodology has been extended to other pathways with analog incorporation now possible in surface-displayed GalNAc and fucose residues as well as nucleocytoplasmic O-GlcNAc-modified proteins. Finally, recent efforts to increase the “druggability” of sugar analogs used in metabolic glycoengineering, which have resulted in unanticipated “scaffold-dependent” activities, are summarized.
    Glycobiology 09/2009; 19(12):1382-401. DOI:10.1093/glycob/cwp115 · 3.15 Impact Factor
Show more