Table Of ContentNew Chemical Tools for Fluorescent Detection of Hydrogen Peroxide in Living Cells
by
Duangkhae Srikun
A dissertation submitted in partial satisfaction of the
requirements for the degree of
Doctor of Philosophy
in
Chemistry
in the
Graduate Division
of the
University of California, Berkeley
Committee in charge:
Professor Christopher J. Chang, Chair
Professor Richmond Sarpong
Professor Michelle C. Chang
Professor David Schaffer
Fall 2011
New Chemical Tools for Fluorescent Detection of Hydrogen Peroxide in Living Cells
© 2011
by Duangkhae Srikun
Abstract
New Chemical Tools for Fluorescent Detection of Hydrogen Peroxide in Living Cells
by
Duangkhae Srikun
Doctor of Philosophy in Chemistry
University of California, Berkeley
Professor Christopher J. Chang, Chair
As one of the toxic by-product of aerobic metabolism, hydrogen peroxide (H2O2), at
uncontrolled levels and distributions, is a sign of oxidative stress, aging and disease. However,
H2O2 also plays an essential part in normal physiological system. H2O2 levels are regulated by
many enzymes and metabolites that generate or break-down H2O2. In macrophages, the presence
of invading pathogens activates the production of microbicidal levels of H2O2 by NADPH
oxidase (Nox). Isoforms of Nox are expressed in many non-phagocytic cells and tissues. Nox-
generated H2O2 is a secondary messenger involved in signaling for growth, proliferation,
differentiation and controlled cell death; these variations in downstream biological effects are
regulated by both the spatial and temporal production of H2O2. Small molecule fluorescent
probes bearing boronate ester moieties have been developed for chemoselective detection of
H2O2 in both oxidative stress levels and cellular signaling events. This dissertation describes the
design, synthesis, characterization and application of new boronate-based fluorescent probes
with added functionality. Peroxy-Lucifer-1 (PL1) and Peroxy-Naphthalene-1 (PN1) are
ratiometric fluorescent probes that can detect oxidative bursts in immune response events.
Ratiometric probes allow simultaneous detection of two signals from the reacted and unreacted
probes in the same sample, providing a built–in correction for variations such as uneven probe
loading, sample environment and detection efficiency. PN1 also has a high two-photon cross
section. The increased penetration depth of near-infrared excitation light allows the detection of
H2O2 in tissue specimens with PN1. SNAP-Peroxy-Green-1 (SPG1) and SNAP-Peroxy-Green-2
(SPG2) are capable of detecting local concentration of H2O2 in subcellular compartments such as
mitochondria, endoplasmic reticulum, nucleus, and plasma membrane. The precise localization
of probes to the targeted organelle is facilitated by highly specific recognition of the SNAP
ligand bound to the probe by the SNAP fusion protein. Furthermore, simultaneous detection of
H2O2 at two different locations is feasible by using a SNAP tag with an orthogonal CLIP tag;
such combined use of SNAP and CLIP tags is assisted by the expanding color palette of SNAP
and CLIP peroxy probes. Multi-modal probes using PAMAM-G5 dendrimer platform was
developed for the real-time imaging of the interplay between H2O2 and other physiological
events. Coordination of the oxidative burst and progressive acidification in phagosomes of
macrophages was elucidated with G5-SNARF2-PF1-Ac, a nanoprobe decorated with the H2O2
sensing module PF1 and pH sensor SNARF2.
1
For my parents
i
Table of Contents
Acknowledgement iii
Chapter 1. An ICT-Based Approach to Ratiometric Fluorescence Imaging of 1
Hydrogen Peroxide Produced in Living Cells
Chapter 2. A Two-Photon Fluorescent Probe for Ratiometric Imaging of 36
Hydrogen Peroxide in Live Tissue
Chapter 3. Organelle-Targetable Fluorescence Probes for Imaging Hydrogen 64
Peroxide in Living Cells via SNAP-Tag Protein Labeling
Chapter 4. Expanding the Palette of Fluorescent Probes for Multi-site 105
Detection of H2O2 with SNAP and CLIP Tag in Living Cells
Chapter 5. A Dendrimer-Based Platform for Simultaneous Dual Fluorescence 139
Imaging of Hydrogen Peroxide and pH Gradients Produced in
Living Cells
Appendix 1. Fluorescent Reporters for Hydrogen Peroxide using Styryl Dyes 174
Appendix 2. Attempts Toward Ratiometric Fluorescent Detection of Hydrogen 195
Peroxide on BODIPY Platform.
Appendix 3. Green and Yellow Fluorescent Probes for Hydrogen Peroxide 211
using Rhodamine Fluorophores
Appendix 4. A Naphthalimide-Based Fluorescent Probe for Hydrogen Peroxide 225
with Pyridinone-Methide Self-Immolative Linker
Appendix 5. Attempts Toward Red Fluorescent Probes for Hydrogen Peroxide 235
Based on Acridinone Fluorophores
Appendix 6. Red Fluorescent Probes for Detection of Hydrogen Peroxide 249
Based on Seminaphthorhodafluors (SNARFs) and
Naphthofluorescein
Appendix 7. Development of A Genetically-Encoded Red Fluorescent Protein 266
Sensor for Hydrogen Peroxide Imaging
ii
Acknowledgement
I’m grateful to Professor Christopher Chang, for his support and mentorship, and the
colorful projects that he allowed me to work on. I’m thankful for all members of the Chang lab
that have help created a joyful and supporting research lab. Special thanks to the peroxide group:
Evan Miller helped me start with fluorescence imaging, Christine Nam taught me all the
molecular biology techniques, Aaron Albers and Bryan Dickinson helped with synthesis. The
original members (Hill Harman, Han Sen Soo, Emily Que, Dylan Domaille) were also very
helpful in everything: from getting to know Berkeley to preparing for the qualifying exam.
I’m also thankful for Ministry of Science, Thailand for the scholarship that supported my
education here in the United States for the past 10 years. I have had the best education and the
best time of my life.
iii
Chapter 1
An ICT-Based Approach to Ratiometric Fluorescence Imaging of
Hydrogen Peroxide Produced in Living Cells
Portions of this work were published in the following scientific journal:
Srikun, D.; Miller, E. W.; Domaille, D. W. and Chang C. J. “An ICT-Based Approach to
Ratiometric Fluorescence Imaging of Hydrogen Peroxide Produced in Living Cells.” Journal of
the American Chemical Society. 2008, 130, 4596 - 4597.
Portions of this work were done in collaboration with the following people:
Evan Miller and Dylan Domaille assisted in confocal fluorescence imaging.
1
Abstract
We present the synthesis, properties, and biological applications of Peroxy Lucifer (PL),
a new series of fluorescent probe for imaging H2O2 produced in living cells by a ratiometric
response. PL probes utilize a chemoselective boronate-based switch to detect H2O2 by
modulation of internal charge transfer (ICT) within a 1,8-naphthalimide dye. PL features high
selectivity for H2O2 over similar reactive oxygen species, including superoxide and nitric oxide,
and a 65 nm shift in emission from blue-colored fluorescence to green-colored fluorescence upon
reaction with H2O2. Confocal microscopy experiments in live macrophages show that PL probes
can ratiometrically visualize localized H2O2 bursts generated in living cells at immune response
levels.
Introduction
The chemistry of hydrogen peroxide (H2O2) in living systems is receiving increasing
attention owing to its diverse contributions as a marker for oxidative stress and damage events
1 2-4
associated with aging and disease, as a second messenger for cellular signal transduction, or
5
as a killing agent generated by immune cells to combat microbial invasion. A key step towards
elucidating the complex biological roles of this reactive oxygen metabolite is the development of
new methods to monitor dynamic changes in peroxide concentrations generated within localized
regions of the cell, as spatial and temporal variations in cellular H2O2 flows can lead to
dramatically different physiological or pathological consequences. Small-molecule reporters
6
offer one approach to meet this need and indicators that give a turn-on emission increase in
7-15
response to H2O2 have been reported and applied for the study of peroxide biology.
Despite advances in the development of new chemical probes, a limitation of intensity-
based probes is that variability in excitation and emission efficiency, sample environment, and
probe distribution pose potential problems for use in quantitative measurements. In contrast,
ratiometric probes provide the practical advantage of built-in corrections for such variants by
allowing simultaneous detection of two signals resulting from reacted and unreacted forms of the
16
probe in the same sample. Our approach to ratiometric fluorescence detection of cellular H2O2
relies on controlling internal charge transfer (ICT) within a dye platform to promote a change in
its emission color upon reaction with H2O2. Specifically, modulation of the electron-donating
amine at the 4-position on a 1,8-naphthalimide (e.g., Lucifer Yellow) affects both ICT and
emission color, as making this substituent more electron deficient results in ICT-induced blue
shifts in emission maxima. We reasoned that modifying the 4-amino donor into a more electron-
withdrawing carbamate group that could be specifically decaged by H2O2 back to the amine
would provide a switch for ratiometric detection of H2O2, a strategy that is inspired by indicators
17 18 19,20 21
for pH, anions, metal ions, and sugars.
We now present the synthesis and application of Peroxy Lucifer (PL), a new series of
ratiometric fluorescent reporters for imaging H2O2 produced in living systems. Owing to its dual
emission readout, PL probes are capable of visualizing highly localized changes in H2O2
concentrations generated by live cells in response to phagocytic stimulation while retaining the
ability to spatially monitor relative [H2O2] fluxes throughout the rest of the sample.
Results and Discussion
Design and Synthesis of Peroxy Lucifer (PL). Previously, our lab has developed Ratio-
Peroxyfluor-1 (RPF1), a ratiometric fluorescent sensor for H2O2 based on a fluorescence
2
resonance energy transfer (FRET) mechanism between a coumarin donor and a boronate-
protected fluorescein receptor. RPF1 possess a large dynamic range; the green to blue emission
ratio changes from 0.45 in the absence of H2O2 to 3.7 after treatment with 200 µM H2O2 for 1 h.
However, RPF1 has limitations in live-cell imaging due to its poor membrane permeability
resulting from the rigidity of the compound structure. In the PL series of probes, we utilized the
manipulation of the ICT state as the mechanism for ratiometric fluorescence response (Scheme
1). The ICT probes require only one fluorophore which allows for a smaller and less rigid probe.
In the uncaged naphthalimide probe, Lucifer Yellow (LY), the fluorophore contains both an
electron donating and an electron withdrawing group, which results in greater stabilization and
lower energy of the internal charge transfer excited state. However, when the amino group is
caged by the carbamate moiety, the push-pull mechanism of the ICT state is disrupted, giving a
blue-shifted fluorescence emission from the higher energy excited state.
We synthesized Peroxy-Lucifer-0 (PL0) starting from the commercially available 4-
amino-1,8-naphthalic anhydride (Scheme 2). Lucifer-Yellow-0 (LY0) was obtained from
condensation of naphthalic anhydride and alkylamine under microwave irradiation. The H2O2
22
responsive 4-(dihydroxyboryl)benzyloxycarbonyl (Dobz) was installed by the reaction of
isocyanate generated in situ by refluxing LY0 with triphosgene in dry toluene, with compound 4.
Observing that LY0 and PL0 display a tendency for aggregation in aqueous buffer, we proceeded
to synthesize LY1 and PL1, replacing butylamine with more hydrophilic 2-(2-
aminoethoxy)ethanol.
In diversifying the alkylamine substitution (PL2 - PL5), we have revised the synthetic
route (Scheme 3-5). The overall yield is greatly improved by masking the 4-amino group as 4-
nitro until the very last step before carbamate formation. The 4-nitro substitution eliminates the
hydrogen bonding found in the 4-amino substitution, resulting in better solubility and ease of
purification in organic solvent.
Spectroscopic Properties and Optical Responses to H2O2. We assessed the
spectroscopic properties of PL dyes and their uncaged LY dyes under physiological-like
conditions (20 mM HEPES, pH 7.4) (Table 1). In the absence of H2O2, PL displays one major
absorption band centered at 375 nm with a corresponding blue- colored fluorescence maximum
at 475 nm. The relative blue-shift of these absorption and emission features compared with the
uncaged LY dyes (λabs = 475 nm, λem = 545 nm) is consistent with ICT involving the relatively
electron-poor carbamate donor. Alkyl chloride and carboxylic acid ester functional group did not
affect the spectroscopic properties of PL and LY dyes. However, maleimide functionality in PL5
and LY5 results in a marked fluorescence quenching (Φ < 0.1). Addition of glutathione (1 mM)
results in an instantaneous increase in fluorescence emission in both PL5-GSH (Φ = 0.47), and
23,24
LY5-GSH (Φ = 0.33). The thiol-ene reaction of PL5 with GSH can be used for trappability
of probes, in addition to the improved signal-to-noise due to the low quantum yield of PL5 with
unreacted maleimide.
Reaction of PL probes with H2O2 triggers chemoselective cleavage of the boronate-based
carbamate protecting group to deliver the green-fluorescent LY dyes as characterized by its
absorption (λabs = 435 nm) and emission (λem = 540 nm) spectra, respectively (Figure 2). All PL
probes showed similar responses to H2O2 except PL3 that displayed slower response to H2O2 in
both absorption and fluorescence emission profile. In this case, it is likely that the hydrophobic
butylchloride moiety in PL3 induces microscopic aggregation, decreasing the interface area
between PL3 and H2O2. Under pseudo-first-order conditions (1 μM PL1 and 1 mM H2O2), the
3
-4 -1
observed rate constant for H2O2 deprotection is kobs = 8.8 × 10 s (Figure 3). The ratiometric
emission response of PL1 is highly selective for H2O2 over other reactive oxygen species (Figure
4). Compared to reactions with H2O2, the ratio of carbamate- to amine-substituted
naphthalimides does not change appreciably with tert-butylhydroperoxide, hypochlorite,
superoxide, singlet oxygen, nitric oxide, hydroxyl radical, or tert-butoxy radical. Moreover, we
demonstrated that the fluorescence emission profile of PL1 and LY1 was stable across a
biologically relevant pH range (Figure 5).
Fluorescence Detection of H2O2 in Living Cells using Confocal- and Two-Photon
Microscopy. PL and LY dyes, like other ICT based fluorescent dyes display solvatochromism
(Figure 6), therefore the fluorescence emission profile can be affected by subcellular localization
of chromophore. This phenomena is demonstrated by the uptake of PL3 into 3T3-L1 adipocytes
(Figure 7). The fluorescence emission from PL3 in lipid droplets showed higher Fgreen/Fblue ratio
correlated with the blue-shifted emission maxima of PL3 in the more hydrophobic environment.
Treatment of PL3-labeled adipocytes with 200 µM H2O2 for 30 min resulted in the overall
increase Fgreen/Fblue emission ratio (Figure 8). The ability of PL probes to detect rising H2O2
concentration in cellular environments was demonstrated in the ratiometric change in
fluorescence emission when HEK293T loaded with PL probes were treated with 200 µM H2O2
for 30 min (Figure 9). Fgreen/Fblue varied among the PL probes, the result of the different
subcellular localization induced by the functional side chain. Fgreen/Fblue is governed by the
hydrophobicity of the side chain. PL3 and LY3, with their butylchloride group, displayed the
lowest Fgreen/Fblue, and mostly localized to membrane-bound structures such as golgi apparatus
and endoplasmic reticulum (Figure 10). On the other hand, PL5 and LY5 displayed the highest
Fgreen/Fblue because PL5 and LY5 can react with glutathione rapidly to generate tripeptide
conjugates which help retaining the probes within cytoplasm. The difference in Fgreen/Fblue of PL3
and PL5 were elaborated by images obtained from lambda mode scanning. The normalized
emission spectra showed PL3 and PL5 sharing the same emission maximum at 478 nm, however
with less intensity in green emission wavelength from PL3 (Figure 11). The difference between
LY3 and LY5 is more dramatic with LY3 showing emission maximum at 517 nm and LY5
showing red-shifted emission maximum at 536 nm (Figure 12).
Having established the ratiometric response of PL probes to bath application of H2O2 in
cell cultures, we then tested the ability of PL probes to detect endogenous bursts of H2O2
produced within living cells. Treatment of RAW24.7 macrophage cells with phorbol myristate
acetate (PMA) rapidly induces phagocytosis; the event is associated with a dramatic change in
cell morphology from round to amoeboid shape packed with phagosomes, which are also visible
25,26
in brightfield images. To detect the oxidative burst in phagasome, the probe must be
hydrophilic enough to remain in solution phase, otherwise the rapid insertion of hydrophobic
probe into plasma membrane will give a population of phagosomes without fluorescence
emission signal. RAW264.7 cells were incubated with PMA (1 µg/mL) and either PL1 or PL4 (5
µM, in DMEM) for 15 min; then washed twice with fresh buffer. Confocal fluorescence images
show a heterogenous population of dormant round cells and stimulated amoeboid-liked cells.
Both PL1 (Figure 13) and PL4 (Figure 14) were able to detect the oxidative burst within
phagosome, reflecting the higher green emission from phagosome compared to the rest of the
cell body as shown in images obtained from channel mode and lambda mode.
The ratiometric fluorescence imaging with PL1 is also demonstrated using two-photon
excitation at 820 nm. The ratio image constructed from 535 - 600 nm (green) and 430 - 495 nm
(blue) fluorescence collection windows using ImageJ software gave Fgreen/Fblue of 0.6 in PL1-
4
