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UC Berkeley Electronic Theses and Dissertations
Title
Chemical Tools for Imaging Glycans in Living Systems
Permalink
https://escholarship.org/uc/item/7027t6kr
Author
Chang, Pamela
Publication Date
2010
Peer reviewed|Thesis/dissertation
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University of California
Chemical Tools for Imaging Glycans in Living Systems
by
Pamela Vivian Chang
A dissertation submitted in partial satisfaction of the
requirements for the degree
Doctor of Philosophy
in
Chemistry
in the
Graduate Division
of the
University of California, Berkeley
Committee in charge:
Professor Carolyn R. Bertozzi, Chair
Professor Christopher J. Chang
Professor Matthew D. Welch
Spring 2010
Chemical Tools for Imaging Glycans in Living Systems
© 2010
By Pamela Vivian Chang
Abstract
Chemical Tools for Imaging Glycans in Living Systems
by
Pamela Vivian Chang
Doctor of Philosophy in Chemistry
University of California, Berkeley
Professor Carolyn R. Bertozzi, Chair
Glycans are important mediators of many biological processes, including cell-cell
adhesion and communication as well as development, inflammation, and cancer cell metastasis.
The composition and expression of these biomolecules are known to change during these
physiological processes, and, accordingly, there has been much interest in profiling and imaging
glycans in vivo. While proteins can be visualized using fluorescent proteins such as GFP, glycans
are challenging targets for imaging because they are not genetically encoded. We have developed
a two-step method to equip glycans with reporter tags for isolation and visualization from living
systems known as the chemical reporter strategy. In our approach, an unnatural sugar, which
bears a small reactive functional group known as a chemical reporter, is metabolically
incorporated into cell-surface glycans. These reporters can then be detected via a covalent,
bioorthogonal reaction such as the Staudinger ligation or Cu-free click chemistry by delivery of
exogenous phosphine or cyclooctyne probes, respectively. If an affinity tag or imaging agent is
appended to the probe, this method allows for the enrichment and visualization of the labeled
glycoconjugates.
This thesis describes the development of new chemical tools for profiling and imaging
glycans and their extension to living systems. Chapter 1 provides an overview of recent advances
in imaging technologies, including the chemical reporter strategy, for visualizing non-
proteinaceous biomolecules such as glycans, lipids, nucleic acids, and small molecule
metabolites. Chapter 2 describes the application of Cu-free click chemistry using a panel of
cyclooctyne probes to glycan labeling in mice. Chapter 3 describes the synthesis of phosphine
and cyclooctyne imaging probes and the evaluation of their ability to image glycans in vivo.
Chapter 4 outlines the development of a new chemical reporter for detecting the monosaccharide
sialic acid and its application to the labeling of murine glycoconjugates. Finally, Chapter 5
presents a novel strategy for cell-selective labeling of glycans using a caged metabolic precursor.
1
This dissertation is dedicated to my family,
Mou-Hsiung, Yuen-man, Dennis and Jeff
i
Chemical Tools for Imaging Glycans in Living Systems
Table of Contents
List of Figures v
List of Tables vii
List of Schemes vii
Acknowledgements vi
Chapter 1. Imaging beyond the proteome
Introduction 1
Fluorescent proteins 1
GFP-tagged binding domains for imaging phosphoinositides 1
Förster resonance energy transfer (FRET)-based sensors of signaling 1
molecules
FRET-based sensors of protein post-translational modifications 3
Small molecule sensors 4
2+
Fluorescent Ca indicators 5
2+
Small molecule Zn indicators 5
+ 2+ 3+
Small molecule Cu and Fe /Fe indicators 6
Small molecule ROS and RNS indicators 7
Chemical reporter strategy 9
Lectins and antibodies for imaging glycans 9
Bioorthogonal chemical reporter strategy for imaging glycans 9
Bioorthogonal chemical reporter strategy for imaging lipids 13
Bioorthogonal chemical reporter strategy for imaging nucleic acids 14
Label-free imaging methods 15
Imaging mass spectrometry 15
Raman and coherent anti-Stokes Raman scattering (CARS) 16
microscopy
Non-optical imaging methods 17
Positron emission tomography (PET) imaging 18
ii
Single photon emission computed tomography (SPECT) imaging 18
MRI and magnetic resonance spectroscopic imaging (MRSI) 18
Conclusions 20
References 21
Chapter 2. Copper-free click chemistry in living animals
Introduction 41
Results and discussion 43
Evaluation of cyclooctyne reagents on cultured cells 43
Comparison of cycloctyne and phosphine probes in vivo 4
DIFO-FLAG binds mouse serum albumin (MSA) with high affinity 49
Conclusions 50
Materials and methods 52
References 58
Chapter 3. Imaging glycans using the chemical reporter strategy
Introduction 61
Results and discussion 63
Synthesis and photophysical characterization of fluorescent 63
phosphine and cyclooctyne probes
Chemical tagging of azide-containing protein with fluorescent 66
phosphines
Imaging cell-surface glycans using the chemical reporter strategy 66
Visualization of glycans in healthy mice using fluorescence imaging 70
Design and synthesis of “smart” fluorogenic phosphines for in vivo 72
imaging
Screen of cancer cell lines for conversion of Ac4ManNAz to cell- 74
surface SiaNAz
Evaluation of tumor xenografts for azidosugar metabolism 75
Conclusions 76
Materials and methods 77
References 84
iii
Chapter 4. Development of new chemical reporters of sialic acid
biosynthesis
Introduction 87
Results and discussion 88
Alkynyl analog of ManNAc (Ac4ManNAl) is converted to SiaNAl 88
and incorporated into cell-surface glycans
Ac4ManNAl is more efficiently metabolized than Ac4ManNAz in 89
mammalian cell lines
Ac4ManNAl is metabolized to SiaNAl in laboratory mice 90
Ac4ManNAl is more efficiently metabolized than Ac4ManNAz in 91
laboratory mice
Conclusions 91
Materials and methods 93
References 96
Chapter 5. A strategy for the cell-selective delivery of azidosugars
Introduction 9
Results and discussion 99
Design and synthesis of caged metabolic precursors to sialic acid 100
Stability studies of caged azidosugars 101
In vitro enzymatic uncaging of caged azidosugar 5.1 using the 101
prostate-specific antigen protease (PSA)
Selective labeling of cell-surface glycans using caged azidosugar 5.1 102
and PSA
Fluorescence microscopy analysis of cells labeled with caged 105
azidosugar 5.1 in the presence of PSA
Conclusions 105
Materials and methods 106
References 12
Appendix 114
iv
List of Figures
Figure 1–1 Genetically-encoded fluorescent protein-based FRET 2
reporters of small molecules and post-translational
modifications
2+
Figure 1–2 Small molecule fluorescent Ca sensors 4
2+
Figure 1–3 Small molecule fluorescent Zn sensors 5
+ 2+ 3+
Figure 1–4 Small molecule fluorescent Cu and Fe /Fe sensors 7
Figure 1–5 Small molecule fluorescent ROS/RNS sensors 8
Figure 1–6 Bioorthogonal chemical reactions 10
Figure 1–7 Bioorthogonal chemical reporter strategy 11
Figure 1–8 Cyclooctyne reagents for Cu-free click chemistry 12
Figure 1–9 Chemical reporters for profiling and visualizing lipids in 14
living systems
Figure 1–10 Metabolic labels for nucleic acids 15
Figure 2–1 Cu-free click chemistry in mice 42
Figure 2–2 Chemical tagging of azido glycans in vitro and in vivo with 45
cyclooctyne probes
Figure 2–3 Comparison of in vivo and subsequent ex vivo reactions on 46
isolated splenocytes
Figure 2–4 Chemical tagging of serum glycoproteins in vivo using Cu- 47
free click chemistry
Figure 2–5 Comparison of Cu-free click chemistry and the Staudinger 48
ligation for labeling splenocyte cell-surface azides in vivo
Figure 2–6 Cu-free click chemistry and Staudinger ligation products are 49
observed in a variety of tissues in vivo
Figure 2–7 DIFO-FLAG binds mouse serum albumin (MSA) 50
Figure 3–1 Altered glycosylation patterns are a hallmark of cancer 61
Figure 3–2 A strategy for noninvasive imaging of glycans 62
Figure 3–3 Phosphine and cyclooctyne probes for glycan-specific 64
imaging
Figure 3–4 Specific labeling of azido-DHFR with 3.1-3.3 6
Figure 3–5 Flow cytometry analysis of Jurkat cels labeled with 3.2 6
Figure 3–6 Flow cytometry analysis of Jurkat cels labeled with 3.3 67
v
Figure 3–7 Flow cytometry analysis of Jurkat cells labeled with 3.1 or 67
phosphine oxide of 3.1 (3.1-ox)
Figure 3–8 Fluorescence microscopy of CHO cels labeled with 3.1 68
Figure 3–9 Colocalization studies of CHO cels labeled with 3.1 68
Figure 3–10 Fluorescence microscopy of CHO cells labeled with 3.4 or 69
3.5
Figure 3–11 Labeling of live cells bearing two bioorthogonal chemical 70
reporters
Figure 3–12 Fluorescence imaging of mice with 3.1 71
Figure 3–13 Fluorescence imaging of mice with 3.5 71
Figure 3–14 Fluorescence imaging of mice with 3.4 72
Figure 3–15 Activatable probes for Staudinger ligation 73
Figure 3–16 Metabolism of Ac4ManNAz in various mammalian cancer 75
cell lines
Figure 4–1 Metabolic labeling of cellular glycans with Ac4ManNAl and 88
detection by CuAAC
Figure 4–2 Western blot analysis of lysates from Jurkat cells treated 88
with Ac4ManNAl or no sugar
Figure 4–3 Fluorescence micrographs of CHO cells treated with 50 μM 89
Ac4ManNAl or no sugar for 3 d
Figure 4–4 Experimental overview for probing Ac4ManNAl metabolism 90
in vivo
Figure 4–5 Western blot analysis of tissue lysates from B6D2F1/J mice 90
administered Ac4ManNAl or vehicle
Figure 4–6 Ac4ManNAl is converted to the corresponding sialic acid 91
more efficiently than Ac4ManNAz in mouse organs
Figure 5–1 A strategy for tissue-specific release of Ac3ManNAz via 100
enzymatic activation
Figure 5–2 Hydrolytic stability test of 5.1 and 5.2 103
Figure 5–3 Compound 5.1 serves as a substrate for PSA in vitro 103
Figure 5–4 Cell-selective metabolic labeling of glycans using 5.1 and 104
PSA
Figure 5–5 Flow cytometry analysis of CHO cells treated with 5.1 and 104
PSA
Figure 5–6 Cell viability assay 104
vi
