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Fluorogenic and fluorescent bioorthogonal labelling strategies for examining glycoproteins and phospholipids Open Access


Other title
Type of item
Degree grantor
University of Alberta
Author or creator
Key, Jessie Adam
Supervisor and department
Cairo, Christopher (Chemistry)
Examining committee member and department
Cosa, Gonzalo (Chemistry, Mcgill University)
Brindley, David (Biochemistry)
Lowary, Todd (Chemistry)
Gibbs-Davis, Julianne (Chemistry)
Brown, Alexander (Chemistry)
Department of Chemistry

Date accepted
Graduation date
Doctor of Philosophy
Degree level
Traditional genetically-encoded imaging strategies are problematic for use in analysing cell surface interactions due to the heterogeneous nature of the cell surface, and the high level of post-translational modifications. Small molecule fluorophores can provide dynamic information on cell surface interactions due to their small size, low detection limits and environmental sensitivity. Recent advances in bioconjugate strategies such as the Staudinger ligation, Sharpless-Meldal reaction and periodate oxidation with aniline-catalyzed ligation (PAL) and hydrazone/oxime forming reactions have imparted improved specificity. However, there remains a need for chromophores with improved specificity. This thesis describes the synthesis and application of novel chromophores for cell surface imaging. We employed selective labelling strategies to combat background signal, and enable detection of specific post-translational modifications. We generated a small library of alkynyl coumarin profluorophores and characterized the corresponding alkyne and triazole derivatives. We observed increased fluorescence emission quantum yields of up to 9-fold and bathochromic shift of up to 23 nm due to the formation of the triazole linkage. Although these dyes are useful for fluorogenic labelling strategies, their relatively low brightness and short excitation wavelength inspired us to examine a new series of alkynyl and azido benzoxadiazole fluorophores for labelling cell surface glycoconjugates. We were able to identify reactive chromophores which act as fluorogenic, quenched, and static fluorescent labels. The fluorogenic dye showed an almost 50-fold increase of intensity, and the quenched dyes showed up to 500-fold loss of intensity. To quantify sialic acid content, using PAL methodology, we developed a novel 4-aminooxy-7-nitro- benzoxadiazole (NBDAO) fluorophore, and compared its performance with commercially available dyes. NBDAO was excited by a 488 nm laser line and found to be suitable for in vitro and in vivo labelling studies using SDS-PAGE, microplate assays, fluorescence spectroscopy, and flow cytometry. As well, a series of bioorthogonal cyanine-5 fluorophores and choline derivatives were synthesized and examined for suitability in phospholipid metabolic labelling experiments. Propargyl- and azido- choline readily incorporated as metabolic labels of phospholipids. However, a carbonyl-choline analog did not show any incorporation. The Cy 5 dyes were found to have exceptionally high absorption coefficients and long-wavelength absorption and emission.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Key, J.A., Koh, S., Timerghazin, Q.K., Brown, A. and Cairo, C.W. (2009) Dyes and Pigments, 82, 196-203.Key, J.A. and Cairo, C.W. (2011) Dyes and Pigments, 88, 95-102.Sandbhor, M.S., Key, J.A., Strelkov, I.S. and Cairo, C.W. (2009) The Journal of Organic Chemistry, 74, 8669-8674Key, J.A., Li, C. and Cairo, C.W. (Submitted) Bioconjugate Chemistry.Key, J.A., Cairo, C.W. and Ferguson, M.J. (2008) Acta Crystallographica Section E-Structure Reports Online, 64, O1910-U3631Key, J.A., Li, M.D. and Cairo, C.W. (2011) Journal of Chemical Education, 88, 98-100.

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