A microtubule-localizing activity-based sensing fluorescent probe for imaging hydrogen peroxide in living cells

Article abstract of DOI:10.1016/j.bmcl.2021.128252

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species (ROS) in living systems with broad roles spanning both oxidative stress and redox signaling. Indeed, owing to its potent redox activity, regulating local sites of H₂O₂ generation and trafficking is critical to determining downstream physiological and/or pathological consequences. We now report the design, synthesis, and biological evaluation of Microtubule Peroxy Yellow 1 (MT-PY1), an activity-based sensing fluorescent probe bearing a microtubule-targeting moiety for detection of H₂O₂ in living cells. MT-PY1 utilizes a boronate trigger to show a selective and robust turn-on response to H₂O₂ in aqueous solution and in living cells. Live-cell microscopy experiments establish that the probe co-localizes with microtubules and retains its localization after responding to changes in levels of H₂O₂, including detection of endogenous H₂O₂ fluxes produced upon growth factor stimulation. This work adds to the arsenal of activity-based sensing probes for biological analytes that enable selective molecular imaging with subcellular resolution.

Full text of DOI:10.1016/j.bmcl.2021.128252

Bioorg. Med. Chem. Lett. 48 (2021) 128252
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A microtubule-localizing activity-based sensing fluorescent probe for
imaging hydrogen peroxide in living cells
,b,*
Shang Jiaa, Christopher J. Changa
a Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA
b Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hydrogen peroxide (H2O2) is a major reactive oxygen species (ROS) in living systems with broad roles spanning
both oxidative stress and redox signaling. Indeed, owing to its potent redox activity, regulating local sites of H2O2
generation and trafficking is critical to determining downstream physiological and/or pathological conse-
quences. We now report the design, synthesis, and biological evaluation of Microtubule Peroxy Yellow 1 (MT-
PY1), an activity-based sensing fluorescent probe bearing a microtubule-targeting moiety for detection of H2O2
in living cells. MT-PY1 utilizes a boronate trigger to show a selective and robust turn-on response to H2O2 in
aqueous solution and in living cells. Live-cell microscopy experiments establish that the probe co-localizes with
microtubules and retains its localization after responding to changes in levels of H2O2, including detection of
endogenous H2O2 fluxes produced upon growth factor stimulation. This work adds to the arsenal of activity-
based sensing probes for biological analytes that enable selective molecular imaging with subcellular resolution.
Activity-based sensing
Fluorescent probe
Molecular imaging
Reactive oxygen species
Hydrogen peroxide
Main text
established selectivity for H2O2 detection over competing ROS and use
in living cells30, we have designed activity-based boronate fluorescent
Hydrogen peroxide (H2O2) is a central member of the reactive oxy-
gen species (ROS) family and is continually produced by foundational
cellular processes that span respiration, oxidase catalysis, protein
folding, and peroxisome activity1,2. On the other hand, dysregulation of
H2O2 triggers oxidative stress and damage cascades that are implicated
in aging3 and disease states, including cancer4,5, inflammation6,7, dia-
betes8, and neurodegeneration9. In the context of H2O2 as a physiolog-
ical signal10,11, controlled and localized generation of this ROS occurs in
response to various stimuli such as growth factors, cytokines, and neu-
rotransmitters12–15, where membrane-bound NADPH oxidases play a
pivotal role in generating H2O2 fluxes in confined cellular spaces to react
probes for monitoring H2O2 with varying excitation/emission colors31
,
reagents with increased sensitivity for visualizing endogenous H2O2 at
signaling levels32,33, two-color dyes for ratiometric H2O2 detection34,35
,
bifunctional probes for organelle-specific H2O2 detection36,37, cell-
trappable sensors for intracellular H2O2 signaling and identification of
peroxide channels and peroxide-dependent neurogenesis38,39, and more
recently tandem activity-based sensing/labeling for identifying trans-
cellular H2O2 signaling in microglia-neuron co-cultures40. Beyond
acting as a general H2O2 caging group for fluorophores, the versatility of
boronate triggers for H2O2 detection also enables development of the
Peroxy Caged Luciferin family of bioluminescent H2O2 reporters based
on caged luciferins41,42, histochemical analysis with Peroxymycin-1, a
with downstream targets16–18
.
To meet the need for identifying and characterizing the diverse
sources and functions of H2O2 as a transient redox messenger, we19–21
and others22–27 have developed molecular probes for selective moni-
toring of H2O2 to selectively disentangle its contributions from other
ROS. In particular, our laboratory has advanced the use of H2O2-medi-
ated boronate oxidation for H2O2 detection19,21 as part of a larger pro-
gram in activity-based sensing for selective monitoring of biological
analytes28,29. Since our initial report of Peroxyfluor-1 (PF1) that
puromycin-based H2O2 detection reagent43, and a caged radiotracer for
44
positron emission tomography (PET) imaging of H2O2
.
Against this backdrop, a key challenge to studying H2O2 signaling is
the transient and localized nature of H2O2 fluxes. Indeed, traditional
boronate fluorescent probes are diffusible before and after analyte
detection, which can limit spatial resolution in monitoring localized
H2O2 fluxes. To address this issue, we have reported SNAP Peroxy Green
1 (SNAP-PG1) and Peroxy Green 1 Fluoromethyl (PG1-FM) reagents that
* Corresponding author at: Department of Chemistry and Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
E-mail address: chrischang@berkeley.edu (C.J. Chang).
https://doi.org/10.1016/j.bmcl.2021.128252
Received 27 May 2021; Received in revised form 30 June 2021; Accepted 3 July 2021
Available online 7 July 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S. Jia and C.J. Chang
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128252
moiety, Peroxy Yellow 1 (PY1), has already been reported in a previous
paper from our laboratory (Supplementary Figure S1)33. The synthesis
of MT-PY1 is depicted in Scheme 1. The Boc-group on docetaxel was
removed by treatment with formic acid, and the dicarboxylic linker was
introduced with TSTU coupling to afford 2. The Fmoc-protected H2O2
sensing motif 3 shared the same initial steps with Mito-PY1. However,
the amide formation step with TSTU was incompatible in the presence of
dibenzofulvene as a byproduct of Fmoc-deprotection. This byproduct
was removed by using tris(2-aminoethyl)amine (TAEA) as both depro-
tection reagent and dibenzofulvene scavenger50, which enables facile
purification of the deprotected secondary amine for coupling with 3 to
afford MT-PY1.
With the MT-PY1 probe in hand, we first evaluated its fluorescence
turn-on behavior for activity-based sensing of H2O2 in in vitro experi-
ments. We incubated the probe at 37 ◦C in aqueous media buffered to
neutral pH for 1 h and observed a negligible measurable fluorescence
response. In contrast, after incubation with 100 μM H2O2, we observed a
12-fold turn-on response within 1 h (Figure 2a), which is comparable
with previously published boronate probes for activity-based sensing of
H2O2. This result suggests that the introduction of docetaxel as the
localizing moiety does not interfere with the activity-based sensing re-
action between H2O2 and boronic ester. We further tested the response
of MT-PY1 to other biologically relevant reactive oxygen species (ROS)
and nitrogen species (RNS) (Figure 2b). The results show that MT-PY1
reacts primarily with H2O2, which is in agreement with its non-
targeted analog PY1 and other previously reported boronate-based
H2O2 probes. The only exception is peroxynitrite (ONOOꢀ ), which is
not surprising given that some boronic esters also react with this
RNS51,52. Indeed, we note that despite the fact that the relative in vitro
reactivity of boronates with peroxynitrite can be comparable or faster
than with hydrogen peroxide in aqueous buffer, in biological contexts
one must consider that ONOOꢀ is a highly reactive species that exists in
cells with exceedingly short lifetimes of 10–20 ms and estimated con-
centrations in the sub-nanomolar range53, whereas local H2O2 concen-
trations can approach micromolar levels54. As such, we advocate for the
use of a straightforward control experiment in cells and other biological
Figure 1. Structures of (a) microtubule-targeting molecule docetaxel; (b)
mitochondria-localizing H2O2 probe Mito-PY1, and (c) microtubule-localizing
H2O2 probe MT-PY1.
covalently label H2O2-responsive dyes onto intracellular proteins to
limit probe diffusion37,40. Along these lines, we now report the design,
synthesis, and evaluation of Microtubule Peroxy Yellow 1 (MT-PY1), a
unique H2O2-responsive probe that localizes to microtubules that form
part of the cytoskeleton and can retain its spatial localization before and
after reporting on changes in H2O2 levels.
Inspired by reports that conjugate fluorophores to taxoids as a
microtubule-targeting group for conventional45,46 and super-reso-
lution47–49 imaging of the cytoskeleton in living cells, along with
leveraging our laboratory’s previous work on a modular rhodol scaffold
in the development of a mitochondrial-targeted H2O2 probe, Mito-
PY136, we designed and synthesized MT-PY1 by linking a boronate
rhodol to the primary amine of a Boc-deprotected docetaxel via a suberic
acid linker (Figure 1a). The analogous H2O2 probe without a targeting
Scheme 1. Synthesis of MT-PY1 and its fluorescence turn-on reaction upon activity-based sensing of H2O2.
2

S. Jia and C.J. Chang
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128252
models with a nitric oxide synthase (NOS) inhibitor. This type of
experiment can distinguish between peroxide-dependent and
peroxynitrite-dependent signals using boronate reagents, as the former
will be insensitive to NOS inhibition whereas the latter will be sensitive
to blocking of an NO source.
We next evaluated the MT-PY1 reagent in cell imaging experiments
by first testing whether the probe is able to localize onto microtubule
structures. As expected, MT-PY1 showed an even distribution in the
cytosol, visualizing a network of filaments that resembles the cytoskel-
eton outside the nucleus (Figure 3a). Moreover, images of a dividing cell
also displayed a structure with the shape of a spindle apparatus with
chromosomes in the center (Figure 3b). To confirm the subcellular
localization of the MT-PY1 probe, we performed dual-color imaging
experiments with cells that were transfected with mCherry-tubulin that
assemble to form mCherry-decorated microtubules. Images of these cells
after staining with MT-PY1 are shown in Figure 3c. Since taxoids show
limited binding to free tubulin and only associate with assembled mi-
crotubules, cells in the MT-PY1 channel show a well-defined filament
structure. In contrast, due to the different expression levels of mCherry-
tubulin, some cells showed free, unassembled tubulin which light up the
entire cytoplasm with less pronounced filament features. For cells that
show primarily microtubule mCherry signal (outlined by dotted lines in
Figure 3c), we observed strong colocalization between MT-PY1 in the
green channel and mCherry-labeled microtubule in the red channel with
a Pearson correlation coefficient of 0.92. Moreover, the probe is not
cytotoxic at the low doses needed for imaging, with no observable in-
hibition effect even at 4 μM over 24 h incubation, which is well above
the typical dose/time for an imaging experiment (Supplementary Figure
S3). This result is in agreement with observations of reduced cytotox-
icity in previously reported docetaxel-fluorophore conjugates compared
to free docetaxel55 and is likely due to the increase of probe hydro-
Figure 2. In vitro characterization of MT-PY1. (a) Fluorescence emission
phobicity that can alter cell permeability and microtubule affinity49
.
spectrum of 5 μM MT-PY1 in 25 mM HEPES buffer pH 7.4 (bottom), and its
turn-on response after treatment with 100
μ
M H2O2 at 37 ◦C for 5, 15, 30, 45
Taken together, the imaging results establish that the taxoid moiety
behaves as expected to assemble itself onto microtubule structures in
and 60 min. (b) Fluorescence responses of 5 μM MT-PY1 to 100 μM of various
reactive oxygen and nitrogen species at 37 ◦C after 5, 15, 30, 45 and 60 min
of incubation.
Figure 3. Confocal microscopy images of MT-PY1 localization in HeLa cells.
Figure 4. Fluorescence responses of MT-PY1 to exogenous addition of H2O2 in
Cells were incubated with 1 μ
M MT-PY1 for 15 min at 37 ◦C prior to imaging.
living cells. HeLa cells were incubated with 1 μ
M MT-PY1 for 0.5 h at 37 ◦C,
(a) A HeLa cell in metaphase. (b) A HeLa cell in interphase. Cell nucleus is
stained by Hoechst 33342 in (a) and (b). (c) HeLa cells expressing mCherry-
tubulin stained with MT-PY1. Enlarged image is shown in Supplementary
followed by treatment with (a) vehicle control or (b) 100
0.5 h; quantification is shown in (c). Scale-bar: 20 m.
μ
M H2O2 at 37 ◦C for
μ
Figure S2. Scale-bar: 10 μm.
3

S. Jia and C.J. Chang
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128252
Figure 5. Fluorescence imaging of endogenous H2O2 generated by the EGF signaling pathway in live A431 cells. A431 cells incubated with 1
37 ◦C were treated with (a) vehicle control, (b) 100 ng/mL EGF, (c) 100 ng/mL EGF and 500
M L-NAME or (d) 100 ng/mL EGF and 50 M PD15305 for 30 min at 37
◦C and imaged. (e) Zoomed-in images of A431 stained with MT-PY1 showing the high spatial resolution of this fluorescence probe. (f) Quantification of fluorescence
intensity of a-d shown as mean ± s.d. Scale-bar: 50 m.
μM MT-PY1 for 0.5 h at
μ
μ
μ
living cells with negligible toxicity, which endows the MT-PY1 H2O2
probe with the ability to localize to microtubules.
signaling events. In addition to further applications of MT-PY1 and
related reagents in deciphering the contributions of H2O2 to redox
biology, this work provides a starting point for the design of a broader
palette of activity-based sensing probes that utilize taxoid-targeting
moieties to minimize probe diffusion before and after analyte detection.
We then evaluated the ability of MT-PY1 to respond to changes in
H2O2 levels in living cells with exogenous incubation of ROS. Indeed,
after loading live HeLa cells with MT-PY1, treatment with 100 μM H2O2
resulted in a significant fluorescence enhancement (Figure 4). More
importantly, the probe retained its microtubule-localizing pattern after
its turn-on response to H2O2, which establishes that the MT-PY1 reagent
can sense changes in H2O2 localized to microtubules.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
After establishing that MT-PY1 is suitable for monitoring changes in
levels of H2O2 in cells with spatial resolution proximal to microtubules,
we moved forward to testing the response of this activity-based sensing
reagent to endogenous H2O2 produced by growth factor stimulation. We
used A431 cells, a cancer cell line with high expression of the epidermal
growth factor (EGF) receptor, as a biological model for endogenous
peroxide production generated by EGF treatment. Upon treatment of
MT-PY1-loaded A431 cells with EGF (100 ng/mL), we observed a sig-
nificant fluorescence turn-on response (Figure 5a,b), which is in the
same range with PY1 under similar conditions with higher amounts of
EGF added (500 ng/mL). Moreover, when we treated the cells with both
EGF and L-NAME, a nitric oxide synthase (NOS) inhibitor that can reduce
generation of peroxynitrite and other RNS in cells, we observed a
comparable fluorescence intensity signal compared to cells treated with
EGF only (Figure 5c). This result indicates that peroxynitrite, which
showed reactivity with MT-PY1 in in vitro assays, does not contribute to
the observed turn-on response of the H2O2 probe in this biological
context. In contrast, when we treated cells with EGF and PD15305, an
EGF receptor inhibitor, we observed similar fluorescence to the un-
treated cells (Figure 5d), further confirming that the turn-on effect was
indeed resulted from endogenous H2O2 generated in the EGF signaling
pathway. In contrast to the punctate staining of PY1 in A431 cells, a
higher-magnification image showed filament-localization pattern of the
MT-PY1 probe, correlating with the microtubule-localizing property of
this dye that give rise to even and spatially-resolved distribution in
cytoplasm (Figure 5e).
Acknowledgments
We thank the NIH (GM139465, GM79465, and ES28096) and Agilent
for supporting this work. C.J.C. is a CIFAR Fellow. We thank Prof.
Shixian Lin for help with mentoring and technical support in the early
stages of this project.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128252.
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