A visible and near-infrared light activatable diazocoumarin probe for fluorogenic protein labeling in living cells

Article abstract of DOI:10.1021/jacs.0c08068

Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazocoumarin compounds that can be photoactivated by visible (430-490 nm) and nearinfrared light (800 nm) irradiation to photo-uncage reactive carbene intermediates, which could subsequently undergo an insertion reaction with concomitant fluorescence "turned on". With these new molecules in hand, we have developed a new approach for rapid, selective, and fluorogenic labeling of endogenous protein in living cells. By using CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as hypoxia-induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for introducing external reporters with "click chemistries". We believe that this approach of fluorescence labeling of endogenous protein by bioorthogonal photoirradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology.

Full text of DOI:10.1021/jacs.0c08068

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
A Visible and Near-Infrared Light Activatable Diazo-Coumarin
Probe for Fluorogenic Protein Labeling in Living Cells
Sheng-Yao Dai, and Dan Yang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c08068 • Publication Date (Web): 01 Sep 2020
Downloaded from pubs.acs.org on September 1, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
A Visible and Near-Infrared Light Activatable Diazo-Coumarin Probe
for Fluorogenic Protein Labeling in Living Cells
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Sheng-Yao Dai, Dan Yang*
Supporting Information Placeholder
ABSTRACT: Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin
dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex
intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazo-coumarin compounds
that can be photo-activated by visible (430‒490 nm) and near-infrared light (800 nm) irradiation to photo-uncage reactive carbene
intermediates, which could subsequently undergo insertion reaction with concomitant fluorescence “turned-on”. With these new molecules
in hand, we have developed a new approach for rapid, selective and fluorogenic labeling of endogenous protein in living cells. By using
CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell
imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as
hypoxia-induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling
can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for
introducing external reporters with “click chemistries”. We believe that this approach of fluorescence labeling of endogenous protein by
bioorthogonal photo-irradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology.
been demonstrated that purified peptides or proteins can be labeled
in vitro by metallocarbenoid with the use of dirhodium(II)-
metallopeptides21-29 and ruthenium(II)-porphyrin30 catalysts.
Reactive carbene intermediates can also be photochemically
generated from diazo and diazirine compounds to label proteins in
cells for photoaffinity protein profiling.31 However, their activation
requires UV irradiation that is detrimental to biomolecules and
cells via DNA damage32-34 and generation of reactive oxygen
species,35 which hindered the subsequent live-cell applications.
How to design new diazo compounds for specific protein
modification in live cells in a bioorthogonal manner is still
challenging.
Here we present a new photochemical approach by connecting a
diazo group to an extended π conjugation system, such as a
fluorophore, which allows the excitation wavelength of the diazo
group to be drastically red-shifted from 254 nm36-37 to visible light
region and enables specific intracellular protein labeling in a
biocompatible manner without click-chemistry (Scheme 1a). Such
a protein modification strategy presents several advantages. Firstly,
compared to methods utilizing diazirine,38-39 benzophenone,40
arylazide,41-44 tetrazole45-50 and α‑ketoamide,51 the use of long-
wavelength (400‒800 nm) irradiation can avoid the cytotoxicity of
INTRODUCTION
Chemical modification of proteins is a powerful method for
protein
engineering
and
conjugation
to
construct
biopharmatheuticals.1-3 The ability to selectively label proteins,
particularly in living cells, is critical for characterizing protein
function, localization and dynamics, but is challenging for
chemical biology and drug development.4-7 Precise protein labeling
can be achieved by genetic engineering approaches, such as fusion
with fluorescent proteins, self-labeling tags8 (e.g. SNAP-tag,9
CLIP-tag10 and HaloTag11) or incorporation of unnatural amino
acids (UAAs) by amber codon suppression.12 However, the
considerably large fusion proteins (19‒33 kDa) may perturb the
functions of POIs. The challenges of UAA incorporation in
mammalian cells also hindered its wide applications.13 Most
importantly, these methods can only be applied to cell types that
are amenable to genetic manipulation. Direct chemical
modification of endogenous proteins with small molecules is a
prevailing avenue, as it minimally perturbs protein functions owing
to the small sizes and bypasses the need for genetic manipulation.
However, small molecule-based live-cell protein labeling was only
achieved by limited examples including ligand-directed chemistry
developed by Hamachi group and others.14-19 Their strategies rely
on nucleophilic residues on proteins to react with the electrophilic
moiety of affinity-based probes, which may intrinsically
compromise the labeling efficiency due to restricted amino-acid
coverage and competing hydrolysis reaction. Developing new
chemistries for selective modification of native endogenous
proteins has thus become one of the most important challenges.
Carbene-mediated insertion reactions have been demonstrated to
be versatile methods in protein labeling because of their ability to
modify a broad scope of residues. One of the most well-known
carbene precursors are diazo compounds, which have recently been
employed extensively in chemical biology.20 For instance, it has
UV light.
Secondly,
copper(I)-catalyzed
azide‒alkyne
cycloaddition (CuAAC) or other reactions that are often required
to introduce fluorescence reporters can be obviated, which allows
real-time report of labeled proteins. Furthermore, photochemical
method provides rapid and non-invasive controls for labeling with
spatiotemporal precision.52-55
In our studies, we first analyzed the photolysis reaction of diazo-
coumarins under visible light irradiation and further optimized the
structure by introducing a trifluoromethyl substituent adjacent to
the diazo group to improve the carbene insertion propensity.
Covalent modification of purified proteins by diazo-coumarin can
be accomplished with mild irradiation of blue LED lamps. It was
1
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 9
confirmed that a range of amino acids can be covalently modified.
(19%). While an aldehyde product 1cresulting from the carbene
oxidation by oxygen62 was isolated in 35% yield, the major product
was found to be a non-fluorescent ring expansion product 1e
(38%),63 formed by Wolff rearrangement of the carbene
intermediate to an allene64 followed by methanol addition. This
process is undesirable since the allene intermediate has a long
lifetime and preferentially reacts with nucleophiles to give non-
fluorescent products.
1
2
3
4
5
6
7
8
Directed by small-molecule ligands, labeling of specific proteins
was further advanced into living cells. Notably, the protein labeling
in cells was found to be rapid, mild and specific. The coumarin
fluorescence was recovered upon photo-irradiation to facilitate the
instant report of labeled proteins in a variety of analyses, including
SDS-PAGE, confocal microscopy and flow cytometry.
Furthermore, in situ two-photon labeling was achieved in cells with
a near-infrared laser, providing an exciting potential for achieving
protein labeling with spatiotemporal control. Collectively, we
envision that diazo-coumarin probes can be exploited as a new
photochemical tool for live-cell protein modifications to facilitate
the future biological studies, and also provide useful templates for
the rational design of next-generation protein modification
reactions.
Scheme 2. Photolysis of 2 (30 µM) in methanol upon blue LED
irradiation and the isolated yields of photolysis products.
H
H
OH
H
O
OH
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
O
O
N
O
O
N
O
O
N
O
H
1
N
1a, 2.3%
1b, 3.3%
1c, 35%
MeOH
H
OMe
O
MeO
N
O
Scheme 1. (a) Schematic illustration of light-induced
fluorogenic labeling of protein-of-interest (POI). (b) Photo-
induced carbene insertion reaction of diazo-coumarins 1 and 2.
N
O
N
O
O
1d, 19%
1e, 38%
Since trifluoromethyl substituent can prevent intramolecular
rearrangement of carbene intermediate generated from
trifluoromethylaryldiazirine,65 we designed and synthesized a
trifluoromethyl analog 2 (Scheme S2). Compound 2 was found to
have a longer absorption maximum positioned at 408 nm
(extinction coefficient ε of 2259 M−1cm−1, Table S1) and was non-
fluorescent (quantum yield Φfl of 0.003). When 2 was subjected to
blue LED irradiation, the peak at 408 nm only underwent a slight
blue-shift and that at ~300 nm dropped sharply. This indicates the
coumarin chromophore was preserved, whereas the diazo moiety
that usually absorbs below 300 nm underwent decomposition. A
significant fluorescence enhancement of 135-fold was also
detected (Figure 1b,c). Subsequent analyses (Figure 1d,e) revealed
that most products were fluorescent, including insertion products
2b (17%) and 2d (43%; Φfl of 0.22), ketone product 2a (15%), and
cyclized66 byproduct 2c (10%) (Figure S2, Table S1). Most
importantly, no ring-expansion was found.
RESULTS AND DISCUSSION
Photochemical Characterization of Diazo-coumarins Under
Blue Light. In the course of surveying different combinations of
diazo group and fluorophores, we found that 4-
diazomethylcoumarins56-59 were used as precursors for photo-
releasing of compounds,60 but the photochemistry of those diazo
compounds has been unexplored. We hypothesize that diazo-
coumarin compounds can be activated by photo-irradiation with
long-wavelength light to “uncage” reactive carbene species for
covalent modification of proximal proteins (Scheme 1b). To test
whether diazo-coumarins can be photo-activated by visible light,
we synthesized compound 1 according to procedures reported in
the literature (Scheme S1).58 It was found that a large portion of the
longest absorption peak fell into the visible light region (> 400 nm)
(Figure S1a). Unlike ordinary coumarins, the fluorescence of 1 was
found to be quenched by the diazo group, presumably via internal
charge transfer (ICT) .61 Consequently, photolysis of 1 was first
carried out in methanol by irradiation with a household blue LED
lamp (430‒490 nm). Upon irradiation, changes in absorption and
fluorescence spectra were observed (Figure S1a–b), indicating a
photochemical transformation of 1 under blue light as we
anticipated. It is highly conceivable that a carbene intermediate is
formed and trapped by methanol. The reaction mixture was
analyzed by LCMS, and multiple products were observed (Figure
S1c). To identify these products, a large scale photolysis reaction
was performed. The photolysis mixture was found to include
several solvent insertion products (Scheme 2, Figure S1d), such as
the methanol C‒H insertion product 1a (2.3%), the water insertion
product 1b (3.3%) and the methanol O‒H insertion product 1d
Figure 1. Photo-chemical characterizations of diazo-coumarins.
(a)‒(b) Change in absorption and emission spectra of 2 (30 µM) in
methanol upon blue LED irradiation. λex = 400 nm. (c) Change in
fluorescence intensity at λem = 484 nm upon blue LED irradiation.
(d) HPLC analysis of 2 before and after photolysis. (e) Isolated
yields of photolysis products.
Reactivity and Stability of Diazo-coumarins in Biologically
Relevant Conditions. Encouraged by these results, we performed
the blue light-prompted reaction of model protein bovine serum
albumin (BSA) with 1 or 2. The fluorogenic property of coumarin
facilitated the visualization of proteins during in-gel fluorescence
2
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
scanning without the need for “click” chemistry. While both
with 3 followed by blue LED irradiation (Figure 3b). As visualized
by the resulting SDS-PAGE, labeling of CA-II by 3 was furnished
in both concentration (Figure 3c, EC50: 2.48 µM) and time (Figure
3d) dependent manner, and was diminished by competition with an
excess of CBS (Figure 3e, IC50: 182 µM). An enzyme activity assay
was performed, which showed that 3 has an IC50 value comparable
to that of CBS (Figure 3f, IC50 of 3: 0.90 µM, IC50 of CBS: 2.14
µM). These results showed that the binding property of the
sulfonamide was not disrupted despite its modest interaction with
CA-II. Further, LC/MS/MS analysis revealed that 3 labeled His2
and Trp4 residues of the N-terminal peptide of CA-II (Figure 3g,
Figure S4). Both residues were located at the entrance of the ligand-
binding pocket (Figure 3h), suggesting that labeling is induced by
the specific interaction between CA-II and 3. Consistently,
exclusive labeling of 29 kDa CA-II could also be observed in HeLa
cell lysates spiked with the recombinant protein (Figure S5a).
1
2
3
4
5
6
7
8
compounds can covalently label BSA at high concentrations, 2
appeared to be more efficient (Figure 2a) than 1. Through tryptic
digestion and tandem mass spectrometry (LC/MS/MS) analysis
(Figures 2b, c, and S3), 35 and 8 modification sites by 1 and 2 were
identified on BSA, respectively. It was found that although the two
compounds modified similar regions, 2 labeled neutral and polar
side-chains without strong preference, suggesting the formation of
a reactive carbene intermediate. However, 1 additionally reacted
with a large number of nucleophilic residues, such as His, Lys, Ser,
Arg, Thr and Tyr, indicative of the generation of ketene
intermediate along with the reactive carbene, which could
potentially lead to non-specific labeling67 in a more complex
environment. These results suggested that 2 is superior to 1 in terms
of sensitivity and selectivity in protein labeling. Since diazo groups
are prone to decomposition, especially towards thiols68 and acids,69
the stability of 2 was further examined. When 2 was incubated with
an equal amount of reduced glutathione (GSH), an intracellularly
abundant nucleophile and reductant, in phosphate buffer for 24 h in
dark, no reaction was observed by NMR analysis (Figure 2d). In
addition, as shown in Figure 2e, 2 was stable across a wide range
of pH; the fluorescence increased only when the compound was
exposed to light.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Reactivity and stability of diazo-coumarins. (a) SDS-
PAGE of BSA (15 µM) labeled by 1 and 2 of various
concentrations. (b)‒(c) Bar chart showing the amino acid labeling
frequencies of 1 and 2 obtained from tryptic peptide LC/MS/MS
analysis of BSA. (d) Comparison of 1H NMR spectra of 2 (5 mM)
before and after 24 h incubation in sodium phosphate buffer (10
mM), pH 7.4 (D2O)/CD3OD 1:1, in the dark at room temperature,
in the presence of reduced glutathione (5 mM). (e) Fluorescence
intensity of 2 (λex = 400 nm, λem = 510 nm) in sodium phosphate
buffer of various pH values incubated in dark or under blue LED
irradiation as indicated.
Labeling of Recombinant CA-II with Blue Light. The
affinity-based approach was then employed to achieve specific
protein labeling using small-molecule ligands. Carbonic
anhydrase-II (CA-II, 29 kDa) was chosen as a target for selective
labeling as it has been widely adopted in studies of affinity-based
probes.70 CA-II can be reversibly inhibited by aromatic primary
sulfonamides,71 such as 4-carboxybenzene-sulfonamide (CBS) (Kd
~ 3.2 µM).72 Accordingly, we designed and synthesized 3 (Figure
3a, Scheme S2) by modifying one of the ethyl substituents on the
7-amino group to connect CBS through a simple amide linker. In
vitro labeling of recombinant CA-II was then performed by mixing
Figure 3. Fluorescence labeling of recombinant CA-II with blue
light. (a) Structures of 4-carbonxybenzenesulfonamide (CBS) and
3. (b) Schematic illustration of blue LED light-induced labeling of
recombinant CA-II. (c)‒(e) SDS-PAGE of recombinant CA-II (1.7
µM) labeled by 3 (10 µM in (d) and (e)) under blue LED
irradiation. (f) Enzymatic activity assay of CA-II catalyzed the
hydrolysis of p-nitrophenyl acetate. Error bar indicates SD, n = 3.
(g) Mass spectrum of modified CA-II tryptic peptide by 3. (h)
Crystal structure of CA-II showing Zn2+ ion and the two labeled
residues.
Labeling of Endogenous CA-II in Living Cells. Our ultimate
goal is to label endogenously expressed proteins in living cells.
Therefore, labeling was attempted with live MCF7 human breast
cancer cells73 and HCT116 human colorectal cancer cells,74 which
endogenously express CA-II. After incubation with 3, cells were
irradiated with the blue LED lamp (Figure 4a). Subsequently, cell
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 9
lysates were harvested and separated by SDS-PAGE to visualize
the fluorescent signal. strong single fluorescent band
observed in the cytosolic region of HCT116 cells (Figure 4d) and
1
2
3
4
5
6
7
8
A
MCF7 cells (Figure S6a). In addition, the fluorescent signal was
diminished when CBS was added to compete with 3. When a
washout step was performed prior to the irradiation, minimal
labeling was observed (Figure S6b). The fluorescence signals were
clearly excluded from the nucleus that was stained by the red
nucleus probe (NucRed), which was consistent with the reported
cytosolic distribution of CA-II.75 Immunofluorescence staining
showed that the signal from 3 was highly co-localized with that of
CA-II antibody (Figure S6c). Moreover, the fact that the
fluorescence signal of 3 was retained after cell permeabilization is
also evidence that the covalent bonding was established in response
to light.
By using fluorescence-activated cell sorting (FACS) analysis,
the fluorescence “turn-on” effect and competition can be
quantitatively assessed (Figure 5a). As recent work has
demonstrated the assessment of the expression level of the
cannabinoid receptors by tandem live-cell photoaffinity labeling
and click chemistry on fixed cells,76 we envisage that such an
assessment can be carried out in living cells with our approach. As
shown in Figure 5b, the strongest specific labeling for CA-II was
obtained in MCF7 cells, followed by HCT116 cells, whereas HeLa
cells showed minimal labeling. This result suggests that the
expression level of CA-II should follow this order: MCF7 >
HCT116 > HeLa, which was validated by Western blot analysis
(Figure 5c).
corresponds to CA-II was observed in SDS-PAGE of MCF7 cells,
but was blocked by CBS co-treatment (Figure 4b, lanes 3 and 4).
A weaker band was observed without irradiation (Figure 4b, lane
2), suggesting that a minor reaction occurred in the dark. Western
blot analysis confirms that CA-II was evenly expressed among the
four groups and matched the molecular weight of fluorescent
bands. Similar results were obtained in HCT116 cells (Figure
S5b,c). Surprisingly, in the live-cell time-dependent experiments,
the labeling was found to complete within 2 mins under irradiation
(Figure S5d). The faster labeling in living cells is presumably
attributed to the enriched concentrations of 3 and the intracellular
proteins when confined within cells. When the CA-II expression of
MCF7 cells was knocked down by siRNA, the in-gel fluorescent
signal disappeared (Figure 4c). Taken altogether, these results
convincingly demonstrated the visible-light-induced specific and
rapid labeling of CA-II by diazo-coumarin in living cells.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. FACS analysis of CA-II expression in different cell
types. (a) FACS analysis of cells treated with: (i) 3 (5 µM), blue
LED irradiation; (ii) 3 (5 µM) and CBS (100 µM), blue LED
irradiation; (iii) 3 (5 µM), no irradiation; (iv) untreated. (b) Relative
CA-II signals obtained from FACS analysis in MCF7, HCT116 and
HeLa cells. (c) Western blot of CA-II in three cell lines.
Labeling of CA-IX under Hypoxia-mimetic Condition. CA-
IX is a membrane-associated isoform of CAs, which is markedly
overexpressed in hypoxic tumor cells.77 As it plays a pivot role in
malignant progression, especially in regulating pH homeostasis
under acidic extracellular matrix, CA-IX is thus regarded as a
valuable biomarker as well as an attractive target in cancer
therapy.78 The expression of CA-IX is modulated by hypoxia-
inducible factor 1 (HIF-1), which under normoxia condition is
hydroxylated at proline residues by prolyl hydroxylase domain-
containing protein 2 (PHD2) and subsequently ubiquitinated by
Von Hippel–Lindau (VHL) E3 ligase for proteasome degradation
(Figure 6a).79 Since oxygen-sensing of PHD2 relies on its catalytic
iron(II) center, iron chelator deferoxamine mesylate (DFO) can
inhibit PHD2 to stabilize HIF-1, thus acting as a hypoxia-mimetic
agent.80 In our experiment, HeLa cells, which have no CA-II
Figure 4. Fluorescence labeling of endogenously CA-II in cells. (a)
Schematic illustration of live-cell labeling. (b) SDS-PAGE of
MCF7 live-cell labeling by 3 (10 µM) in the presence/absence of
CBS (200 µM). (c) SDS-PAGE and western blot of MCF7 live-cell
labeling by 3 (10 µM) after CA-II knockdown by siRNA. (d) Live-
cell confocal imaging of HCT116 cells treated with 3 (5 µM),
followed by blue LED irradiation. 3: λex = 405 nm, NucRed: λex
=
633 nm. Scale bar = 10 µm.
Due to the unique fluorogenic property of the diazo-coumarin
probe 3 and biocompatibility of the labeling condition, live-cell
imaging of the intracellular CA-II can be performed, which is
otherwise difficult to achieve with other methods. After the
treatment of 3 with blue LED irradiation, strong fluorescence was
4
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
expression, were cultured under normal or hypoxia-mimetic
cells were imaged. As shown in Figure 7c, stronger fluorescence
signals were detected in cells exposed to NIR light, indicating that
two-photon activated labeling of CA-II can be achieved in live
cells.
1
2
3
4
5
6
7
8
conditions (treated with DFO). From the western blot result shown
in Figure 6b, it can be observed that CA-IX was upregulated as a
result of HIF-1 accumulation. After that, these cells were treated
with 3 and followed by irradiation with blue LED light. The
resulting live-cell confocal images showed that substantial signals
accumulated on the plasma membrane in cells treated with DFO,
while untreated cells displayed minimal signal (Figure 6c). The
majority of the signals could be displaced by co-treatment of CBS.
This evidently indicated that 3 could fluorescently label the
membrane-bound CA-IX under hypoxia-mimetic condition. Our
results demonstrated that our visible light activated probe could be
used for labeling proteins that are dynamically expressed under
specific conditions, which is otherwise hard to be accomplished
with overexpression system of genetically modification approach.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. In situ two-photon labeling and imaging of CA-II. (a)
Change in emission spectra of 2 (30 µM) upon two-photon
photolysis with a mode-locked Ti:sapphire 800 nm femtosecond
pulses focused laser at 200 mW. Emission was measured at λex
=
400 nm. (b) Fluorescence intensity (λex = 400 nm, λex = 484 nm)
measured at 30 min after irradiation with focused or unfocused
laser beam of varying laser power. (c) Schematic illustration of
two-photon induced labeling of CA-II with 3 (5 µM) in living cells
and confocal imaging of MCF7 cells before and after two-photon
activation with 800 nm. Scale bar = 20 µm.
Figure 6. Fluorescence labeling of CA-IX in cells under hypoxia-
mimetic condition. (a) Schematic illustration of inducible CA-IX
expression under hypoxia or treatment by DFO. (b) Western blot
of HeLa cells cultured under normal or hypoxia-mimetic (DFO,
100 µM for 24 h) conditions. (c) Live-cell confocal imaging of HeLa
cells cultured under normal or hypoxia-mimetic (DFO, 100 µM for
24 h) conditions. Cells were treated with 3 (5 µM) in the
presence/absence of CBS (100 µM), followed by blue LED
irradiation. 3: λex = 405 nm. Scale bar = 10 µm.
In situ Two-photon Labeling and Imaging. Since 7-
aminocoumarin has been applied in two-photon uncaging.81 we
wonder if two-photon protein labeling can be performed. In
principle, the ability of two-photon activation would potentially
lead to reduced photo-toxicity, better spatial control, and deeper
tissue penetration.82 We, therefore, tested the feasibility of two-
photon activation of 2 to form carbene with the use of near-infrared
(NIR) light to initiate the reaction.83-84 When 2 was exposed to the
femtosecond pulses of an 800 nm focused laser beam, a similar
photo-chemical response to that of blue light irradiation was
observed (Figure 7a). Dependence of the fluorescence increase on
the focused laser power was recorded, however, no reaction
occurred when the laser was unfocused (Figure 7b). These results
suggest that 2 can be two-photon activated by NIR light. As a
proof-of-concept experiment, in situ two-photon labeling and
imaging of CA-II was performed in living cells with 3 (Figure 7c).
In this experiment, MCF7 cells were treated with 3 and the cell
location was first tracked with NucRed under confocal microscopy.
Next, only selected cells were irradiated with a laser at 800 nm of
the microscope. Followed by the washing of excess probes, the
Figure 8. Fluorescence labeling of mCherry-eDHFR in HeLa cells.
(a) Chemical Structures of TMP and 4. (b) Live-cell confocal
imaging of HeLa cells with or without transient expression of
mCherry-eDHFR, treated with 4 (5 µM) and TMP (50 µM) after
5
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 9
blue LED irradiation. 4: λex = 405 nm, mCherry: λex = 561 nm.
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
Scale bar = 10 µm.
We thank Dr. Ming-De Li (Shantou University) and Prof. David
Philip (The University of Hong Kong) for help in the two-photon
photolysis experiment setup. We thank Dr. Fang-Fang Shen and
Dr. Xiang David Li (The University of Hong Kong) as well as Dr.
Nai-Kei Wong (The Second Hospital Affiliated to Southern
University of Science and Technology) for helpful advice on
experiments and critical reading of the manuscript. We thank The
University of Hong Kong Li Ka Shing Faculty of Medicine Faculty
Core Facility for support in confocal microscopy and flow
cytometry. We thank Dr. Ruijun Tian (Southern University of
Science and Technology) for assistance in LC/MS/MS experiment.
This work was supported by The University of Hong Kong, the
University Development Fund, the Morningside Foundation, and
the Hong Kong Research Grants Council under the Area of
Excellence Scheme (AoE/P-705/16).
Labeling of eDHFR in Living Cells. To demonstrate that our
labeling strategy can be applied to other target proteins, 4 was
synthesized by incorporating trimethoprim (TMP, Kd ~ 10 nM) as
a ligand moiety (Figure 8a),85 which can selectively bind E. coli
dihydrofolate reductase (eDHFR). HeLa cells were transfected
plasmid DNA to overexpress eDHFR fused with a red fluorescent
protein (mCherry-eDHFR). The transfection of this fusion protein
helps to confirm the co-localization with diazo-coumarin in living
cells under confocal microscopy. When the cells were treated with
4 (5 µM) and irradiated with blue light, co-localization of the probe
signal and mCherry was observed in live-cell imaging (Figure 8b).
While the cells without transfection did not show any staining, the
addition of TMP also predictably perturbed the labeling in
transfected cells.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
REFERENCES
In the investigation described above, we have demonstrated that
diazo-coumarin can be activated by visible light to “photo-uncage”
reactive carbene intermediate, which can subsequently undergo
insertion reaction with biomolecules. Through detailed chemical
analysis, the structure of diazo-coumarin was optimized by the
introduction of a trifluoromethyl group to improve the carbene
insertion propensity. Moreover, we showed that efficient visible
light photo-activation and fluorogenic property of diazo-coumarin
can be exploited as a biorthogonal tool in endogenous protein
labeling. As a result, proteins-of-interest can be covalently
modified after mild blue LED irradiation, with instant fluorescence
report bypassing the need to perform “click” chemistry. We
demonstrated the applications of diazo-coumarin in affinity
labeling of endogenous proteins in living cells using CA-II as a
model protein. Remarkable labeling speed and specificity of
endogenous CA-II has been achieved, which enables reliable
visualization of localization by live-cell imaging and quantitative
assessment of expression level among different cell types by flow
cytometry. Live-cell imaging also allowed monitoring of protein
dynamics as proved by hypoxia-induced CA-IX accumulation.
Furthermore, diazo-coumarin can be photo-activated by two-
photon excitation, which allows spatiotemporally controlled
protein labeling in living cells with the least phototoxic NIR light.
As a further illustration, this strategy can also be successfully
applied to the labeling of eDHFR. We believe that this photo-
chemical approach should expand the chemical repertoire of the
covalent labeling of biomolecules in living cells to facilitate
biological discoveries.
1. Hoyt, E. A.; Cal, P. M. S. D.; Oliveira, B. L.; Bernardes, G. J.
L., Contemporary approaches to site-selective protein
modification. Nat. Rev. Chem. 2019, 3, 147-171.
2. Krall, N.; da Cruz, F. P.; Boutureira, O.; Bernardes, G. J. L., Site-
selective protein-modification chemistry for basic biology and drug
development. Nat. Chem. 2015, 8, 103-113.
3. Boutureira, O.; Bernardes, G. J., Advances in chemical protein
modification. Chem. Rev. 2015, 115, 2174-95.
4. Tamura, T.; Hamachi, I., Chemistry for Covalent Modification
of Endogenous/Native Proteins: From Test Tubes to Complex
Biological Systems. J. Am. Chem. Soc. 2019, 141, 2782-2799.
5. Amaike, K.; Tamura, T.; Hamachi, I., Recognition-driven
chemical labeling of endogenous proteins in multi-molecular
crowding in live cells. Chem. Commun. 2017, 53, 11972-11983.
6. Kubota, R.; Hamachi, I., Protein recognition using synthetic
small-molecular binders toward optical protein sensing in vitro and
in live cells. Chem. Soc. Rev. 2015, 44, 4454-71.
7. Zhang, G.; Zheng, S.; Liu, H.; Chen, P. R., Illuminating
biological processes through site-specific protein labeling. Chem.
Soc. Rev. 2015, 44, 3405-17.
8. Wang, Z.; Ding, X.; Li, S.; Shi, J.; Li, Y., Engineered
fluorescence tags for in vivo protein labelling. RSC Adv. 2014, 4,
7235-7245.
9. Juillerat, A.; Gronemeyer, T.; Keppler, A.; Gendreizig, S.; Pick,
H.; Vogel, H.; Johnsson, K., Directed Evolution of O6-
Alkylguanine-DNA Alkyltransferase for Efficient Labeling of
Fusion Proteins with Small Molecules In Vivo. Chem. Biol. 2003,
10, 313-317.
10. Gautier, A.; Juillerat, A.; Heinis, C.; Corrêa, I. R.; Kindermann,
M.; Beaufils, F.; Johnsson, K., An Engineered Protein Tag for
Multiprotein Labeling in Living Cells. Chem. Biol. 2008, 15, 128-
136.
11. Los, G. V.; Encell, L. P.; McDougall, M. G.; Hartzell, D. D.;
Karassina, N.; Zimprich, C.; Wood, M. G.; Learish, R.; Ohana, R.
F.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.;
Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D. H.; Bulleit, R. F.;
Wood, K. V., HaloTag: A Novel Protein Labeling Technology for
Cell Imaging and Protein Analysis. ACS Chem. Biol. 2008, 3, 373-
382.
12. Lang, K.; Chin, J. W., Cellular incorporation of unnatural
amino acids and bioorthogonal labeling of proteins. Chem. Rev.
2014, 114, 4764-806.
ASSOCIATED CONTENT
Supporting Information
Supporting figures, tables, experimental procedures, synthesis,
characterization of compounds, LC/MS/MS spectra and NMR
spectra.
AUTHOR INFORMATION
Corresponding Author
Dan Yang ‒ Morningside Laboratory for Chemical Biology,
Department of Chemistry, The University of Hong Kong,
Pokfulam Road, Hong Kong, China; Email: yangdan@hku.hk
13. Lin, X.; Yu, A. C.; Chan, T. F., Efforts and Challenges in
Engineering the Genetic Code. Life (Basel) 2017, 7.
14. Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.;
Nishikawa, Y.; Fujisawa, A.; Hamachi, I., Rapid labelling and
covalent inhibition of intracellular native proteins using ligand-
Authors
Sheng-Yao Dai ‒ Morningside Laboratory for Chemical
Biology, Department of Chemistry, The University of Hong
Kong, Pokfulam Road, Hong Kong, China
6
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
directed N-acyl-N-alkyl sulfonamide. Nat. Commun. 2018, 9,
1870.
33. McMillan, T. J.; Leatherman, E.; Ridley, A.; Shorrocks, J.;
Tobi, S. E.; Whiteside, J. R., Cellular effects of long wavelength
UV light (UVA) in mammalian cells. J. Pharm. Pharmacol. 2008,
60, 969-76.
34. Barlev, A.; Sen, D., DNA's Encounter with Ultraviolet Light:
An Instinct for Self-Preservation? Acc. Chem. Res. 2018, 51, 526-
533.
35. Bai, X.; Huang, Y.; Lu, M.; Yang, D., HKOH-1: A Highly
Sensitive and Selective Fluorescent Probe for Detecting
Endogenous Hydroxyl Radicals in Living Cells. Angew. Chem. Int.
Ed. 2017, 56, 12873-12877.
36. Chowdhry, V.; Vaughan, R.; Westheimer, F. H., 2-diazo-3,3,3-
trifluoropropionyl chloride: reagent for photoaffinity labeling.
Proc. Natl. Acad. Sci. U.S.A 1976, 73, 1406-1408.
37. Singh, A.; Thornton, E. R.; Westheimer, F. H., The Photolysis
of Diazoacetylchymotrypsin. J. Biol. Chem. 1962, 237, 3006-3008.
38. Tomohiro, T.; Morimoto, S.; Shima, T.; Chiba, J.; Hatanaka,
Y., An Isotope-Coded Fluorogenic Cross-Linker for High-
Performance Target Identification Based on Photoaffinity
Labeling. Angew. Chem. Int. Ed. 2014, 53, 13502-5.
1
2
3
4
5
6
7
8
15. Matsuo, K.; Nishikawa, Y.; Masuda, M.; Hamachi, I., Live-Cell
Protein Sulfonylation Based on Proximity-driven N-Sulfonyl
Pyridone Chemistry. Angew. Chem. Int. Ed. 2018, 57, 659-662.
16. Yamaguchi, T.; Asanuma, M.; Nakanishi, S.; Saito, Y.;
Okazaki, M.; Dodo, K.; Sodeoka, M., Turn-ON fluorescent affinity
labeling using a small bifunctional O-nitrobenzoxadiazole unit.
Chem. Sci. 2014, 5, 1021-1029.
17. Fujishima, S. H.; Yasui, R.; Miki, T.; Ojida, A.; Hamachi, I.,
Ligand-directed acyl imidazole chemistry for labeling of
membrane-bound proteins on live cells. J. Am. Chem. Soc. 2012,
134, 3961-4.
18. Tsukiji, S.; Miyagawa, M.; Takaoka, Y.; Tamura, T.; Hamachi,
I., Ligand-directed tosyl chemistry for protein labeling in vivo. Nat.
Chem. Biol. 2009, 5, 341-3.
19. Hughes, C. C.; Yang, Y.-L.; Liu, W.-T.; Dorrestein, P. C.;
Clair, J. J. L.; Fenical, W., Marinopyrrole A Target Elucidation by
Acyl Dye Transfer. J. Am. Chem. Soc. 2009, 131, 12094-12096.
20. Mix, K. A.; Aronoff, M. R.; Raines, R. T., Diazo Compounds:
Versatile Tools for Chemical Biology. ACS Chem. Biol. 2016, 11,
3233−3244.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
39. Tomohiro, T.; Yamamoto, A.; Tatsumi, Y.; Hatanaka, Y., [3-
(Trifluoromethyl)-3H-diazirin-3-yl]coumarin as
a
carbene-
21. Ohata, J.; Ball, Z. T., A Hexa-rhodium Metallopeptide Catalyst
for Site-Specific Functionalization of Natural Antibodies. J. Am.
Chem. Soc. 2017, 139, 12617-12622.
22. Martin, S. C.; Vohidov, F.; Wang, H.; Knudsen, S. E.; Marzec,
A. A.; Ball, Z. T., Designing Selectivity in Dirhodium
Metallopeptide Catalysts for Protein Modification. Bioconjug.
Chem. 2017, 28, 659-665.
23. Vohidov, F.; Coughlin, J. M.; Ball, Z. T., Rhodium(II)
metallopeptide catalyst design enables fine control in selective
functionalization of natural SH3 domains. Angew. Chem. Int. Ed.
2015, 54, 4587-91.
24. Minus, M. B.; Liu, W.; Vohidov, F.; Kasembeli, M. M.; Long,
X.; Krueger, M. J.; Stevens, A.; Kolosov, M. I.; Tweardy, D. J.;
Sison, E. A. R.; Redell, M. S.; Ball, Z. T., Rhodium(II) Proximity-
Labeling Identifies a Novel Target Site on STAT3 for Inhibitors
with Potent Anti-Leukemia Activity. Angew. Chem. Int. Ed. 2015,
54, 13085-13089.
25. Kundu, R.; Ball, Z. T., Rhodium-catalyzed cysteine
modification with diazo reagents. Chem. Commun. 2013, 49, 4166-
8.
26. Chen, Z.; Vohidov, F.; Coughlin, J. M.; Stagg, L. J.; Arold, S.
T.; Ladbury, J. E.; Ball, Z. T., Catalytic protein modification with
dirhodium metallopeptides: specificity in designed and natural
systems. J. Am. Chem. Soc. 2012, 134, 10138-45.
27. Popp, B. V.; Ball, Z. T., Proximity-driven metallopeptide
catalysis: Remarkable side-chain scope enables modification of the
Fos bZip domain. Chem. Sci. 2011, 2, 690-695.
28. Chen, Z.; Popp, B. V.; Bovet, C. L.; Ball, Z. T., Site-specific
protein modification with a dirhodium metallopeptide catalyst.
ACS Chem. Biol. 2011, 6, 920-5.
29. Popp, B. V.; Ball, Z. T., Structure-Selective Modification of
Aromatic Side Chains with Dirhodium Metallopeptide Catalysts. J.
Am. Chem. Soc. 2010, 132, 6660-6662.
30. Ho, C.-M.; Zhang, J.-L.; Zhou, C.-Y.; Chan, O.-Y.; Yan, J. J.;
Zhang, F.-Y.; Huang, J.-S.; Che, C.-M., A Water-Soluble
Ruthenium Glycosylated Porphyrin Catalyst for Carbenoid
Transfer Reactions in Aqueous Media with Applications in
Bioconjugation Reactions. J. Am. Chem. Soc. 2010, 132, 1886-
1894.
31. Ge, S.-S.; Chen, B.; Wu, Y.-Y.; Long, Q.-S.; Zhao, Y.-L.;
Wang, P.-Y.; Yang, S., Current advances of carbene-mediated
photoaffinity labeling in medicinal chemistry. RSC Advances 2018,
8, 29428-29454.
32. Ridley, A. J.; Whiteside, J. R.; McMillan, T. J.; Allinson, S. L.,
Cellular and sub-cellular responses to UVA in relation to
carcinogenesis. Int. J. Radiat Biol. 2009, 85, 177-95.
generating photocross-linker with masked fluorogenic beacon.
Chem. Commun. 2013, 49, 11551-3.
40. Murale, D. P.; Hong, S. C.; Yun, J.; Yoon, C. N.; Lee, J. S.,
Rational design of a photo-crosslinking BODIPY for in situ protein
labeling. Chem. Commun. 2015, 51, 6643-6.
41. Wang, Y.; Hu, L.; Xu, F.; Quan, Q.; Lai, Y.-T.; Xia, W.; Yang,
Y.; Chang, Y.-Y.; Yang, X.; Chai, Z.; Wang, J.; Chu, I. K.; Li, H.;
Sun, H., Integrative approach for the analysis of the proteome-wide
response to bismuth drugs in Helicobacter pylori. Chem. Sci. 2017,
8, 4626-4633.
42. Chao, A.; Jiang, N.; Yang, Y.; Li, H.; Sun, H., A Ni-NTA-based
red fluorescence probe for protein labelling in live cells. J. Mater.
Chem. B 2017, 5, 1166-1173.
43. Lai, Y. T.; Chang, Y. Y.; Hu, L.; Yang, Y.; Chao, A.; Du, Z.
Y.; Tanner, J. A.; Chye, M. L.; Qian, C.; Ng, K. M.; Li, H.; Sun,
H., Rapid labeling of intracellular His-tagged proteins in living
cells. Proc Natl Acad Sci U S A 2015, 112, 2948-53.
44. Chiba, K.; Asanuma, M.; Ishikawa, M.; Hashimoto, Y.; Dodo,
K.; Sodeoka, M.; Yamaguchi, T., Specific fluorescence labeling of
target proteins by using a ligand-4-azidophthalimide conjugate.
Chem. Commun. 2017, 53, 8751-8754.
45. Tian, Y.; Lin, Q., Genetic encoding of 2-aryl-5-
carboxytetrazole-based protein photo-cross-linkers. Chem.
Commun. 2018, 54, 4449-4452.
46. Tian, Y.; Jacinto, M. P.; Zeng, Y.; Yu, Z.; Qu, J.; Liu, W. R.;
Lin, Q., Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-
Selective Protein Photo-Cross-Linking. J. Am. Chem. Soc. 2017,
139, 6078-6081.
47. Cheng, K.; Lee, J. S.; Hao, P.; Yao, S. Q.; Ding, K.; Li, Z.,
Tetrazole-Based Probes for Integrated Phenotypic Screening,
Affinity-Based Proteome Profiling, and Sensitive Detection of a
Cancer Biomarker. Angew. Chem. Int. Ed. 2017, 56, 15044-15048.
48. Zhao, S.; Dai, J.; Hu, M.; Liu, C.; Meng, R.; Liu, X.; Wang, C.;
Luo, T., Photo-induced coupling reactions of tetrazoles with
carboxylic acids in aqueous solution: application in protein
labelling. Chem. Commun. 2016, 52, 4702-5.
49. Li, Z.; Qian, L.; Li, L.; Bernhammer, J. C.; Huynh, H. V.; Lee,
J. S.; Yao, S. Q., Tetrazole Photoclick Chemistry: Reinvestigating
Its Suitability as
a Bioorthogonal Reaction and Potential
Applications. Angew. Chem. Int. Ed. 2016, 55, 2002-6.
50. Herner, A.; Marjanovic, J.; Lewandowski, T.; Marin, V. L.;
Patterson, M. J.; Miesbauer, L.; Ready, D.; Williams, J.;
Vasudevan, A.; Lin, Q., 2-Aryl-5-carboxytetrazole as a New
Photoaffinity Label for Drug Target Identification. J. Am. Chem.
Soc. 2016, 138, 14609-14615.
7
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
51. Ota, E.; Usui, K.; Oonuma, K.; Koshino, H.; Nishiyama, S.;
based detection of human carbonic anhydrase II and IX. Med.
Chem. Commun. 2016, 7, 2045-2062.
1
2
3
4
5
6
7
8
Hirai, G.; Sodeoka, M., Thienyl-Substituted alpha-Ketoamide: A
Less Hydrophobic Reactive Group for Photo-Affinity Labeling.
ACS Chem. Biol. 2018, 13, 876-880.
52. Ankenbruck, N.; Courtney, T.; Naro, Y.; Deiters, A.,
Optochemical Control of Biological Processes in Cells and
Animals. Angew. Chem. Int. Ed. 2018, 57, 2768-2798.
53. Sato, S.; Morita, K.; Nakamura, H., Regulation of target protein
knockdown and labeling using ligand-directed Ru(bpy)3
photocatalyst. Bioconjug Chem 2015, 26, 250-6.
54. Sato, S.; Nakamura, H., Ligand-directed selective protein
modification based on local single-electron-transfer catalysis.
Angew. Chem. Int. Ed. 2013, 52, 8681-4.
55. Geri, J. B.; Oakley, J. V.; Reyes-Robles, T.; Wang, T.;
McCarver, S. J.; White, C. H.; Rodriguez-Rivera, F. P.; Parker, D.
L.; Hett, E. C.; Fadeyi, O. O.; Oslund, R. C.; MacMillan, D. W. C.,
Microenvironment mapping via Dexter energy transfer on immune
cells. Science 2020, 367, 1091.
71. Supuran, C. T., How many carbonic anhydrase inhibition
mechanisms exist? J. Enzyme Inhib. Med. Chem. 2016, 31, 345-60.
72. Li, G.; Liu, Y.; Liu, Y.; Chen, L.; Wu, S.; Liu, Y.; Li, X.,
Photoaffinity labeling of small-molecule-binding proteins by
DNA-templated chemistry. Angew. Chem. Int. Ed. 2013, 52, 9544-
9.
73. Mallory, J. C.; Crudden, G.; Oliva, A.; Saunders, C.;
Stromberg, A.; Craven, R. J., A novel group of genes regulates
susceptibility to antineoplastic drugs in highly tumorigenic breast
cancer cells. Mol. Pharmacol. 2005, 68, 1747-56.
74. Hulikova, A.; Aveyard, N.; Harris, A. L.; Vaughan-Jones, R.
D.; Swietach, P., Intracellular carbonic anhydrase activity
sensitizes cancer cell pH signaling to dynamic changes in CO2
partial pressure. J. Biol. Chem. 2014, 289, 25418-30.
75. Supuran, C. T., Carbonic Anhydrases - An Overview. Curr.
Pharm. Des. 2008, 14, 603-614.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
56. Ito, K.; Sawanobori, J., 4-Diazomethyl-7-Methoxycoumarin as
a New Type of Stable Aryldiazomethane Reagent. Synth. Commun.
1982, 12, 665-671.
76. Soethoudt, M.; Stolze, S. C.; Westphal, M. V.; van Stralen, L.;
Martella, A.; van Rooden, E. J.; Guba, W.; Varga, Z. V.; Deng, H.;
van Kasteren, S. I.; Grether, U.; AP, I. J.; Pacher, P.; Carreira, E.
M.; Overkleeft, H. S.; Ioan-Facsinay, A.; Heitman, L. H.; van der
Stelt, M., Selective Photoaffinity Probe That Enables Assessment
of Cannabinoid CB2 Receptor Expression and Ligand Engagement
in Human Cells. J. Am. Chem. Soc. 2018, 140, 6067-6075.
77. Yang, J.-S.; Lin, C.-W.; Chuang, C.-Y.; Su, S.-C.; Lin, S.-H.;
Yang, S.-F., Carbonic anhydrase IX overexpression regulates the
migration and progression in oral squamous cell carcinoma. Tumor
Biol. 2015, 36, 9517-9524.
78. Tafreshi, N. K.; Lloyd, M. C.; Bui, M. M.; Gillies, R. J.; Morse,
D. L., Carbonic Anhydrase IX as an Imaging and Therapeutic
Target for Tumors and Metastases. In Carbonic Anhydrase:
Mechanism, Regulation, Links to Disease, and Industrial
Applications, Frost, S. C.; McKenna, R., Eds. Springer
Netherlands: Dordrecht, 2014; pp 221-254.
57. Takadate, A.; Tamura, T.; Fujino, H.; Goya, S., Synthesis and
Properties of 4-Diazomethyl-7-methoxycoumarin as
a New
Fluorescent Labeling Reagent for Alchohols and Carboxyacids.
Chem. Pharm. Bull. 1982, 30, 4120-4125.
58. Ito, K.; Maruyama, J., Studies on Stable Diazoalkanes as
Potential
Fluorogenic
Reagents.
I.
7-Substituted
4-
Diazomethylcoumarins. Chem. Pharm. Bull. 1983, 31, 3012-3014.
59. Ito, K.; Maruyama, J., Studies on Stable Diazoalkanes as
Potential Fluorogenic Reagents. II.
:
Ring-Fused 4-
Diazomethylcoumarins. Chem. Pharm. Bull. 1986, 34, 390-395.
60. Ando, H.; Furuta, T.; Tsien, R. Y.; Okamoto, H., Photo-
mediated gene activation using caged RNA/DNA in zebrafish
embryos. Nat. Genet. 2001, 28, 317-25.
61. Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.;
Wang, Q., A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-
Azidocoumarins and Acetylenes. Org. Lett. 2004, 6, 4603-4606.
62. Bethell, D.; Stevens, G.; Tickle, P., The reaction of
diphenylmethylene with isopropyl alcohol and oxygen: the
question of reversibility of singlet-triplet interconversion of
carbenes. J. Chem. Soc. D 1970, 792b-794.
63. Dean, F. M.; Park, B. K., Activating groups for the ring
expansion of coumarin by diazoethane: benzoyl, pivaloyl,
arylsulphonyl, arylsulphinyl, and nitro. J. Chem. Soc., Perkin
Trans. 1 1976, 1260-1268.
64. McMahon, R. J.; Abelt, C. J.; Chapman, O. L.; Johnson, J. W.;
Kreil, C. L.; LeRoux, J. P.; Mooring, A. M.; West, P. R., 1,2,4,6-
Cycloheptatetraene: the key intermediate in arylcarbene
interconversions and related C7H6 rearrangements. J. Am. Chem.
Soc. 1987, 109, 2456-2469.
79. Benej, M.; Pastorekova, S.; Pastorek, J., Carbonic Anhydrase
IX: Regulation and Role in Cancer. In Carbonic Anhydrase:
Mechanism, Regulation, Links to Disease, and Industrial
Applications, Frost, S. C.; McKenna, R., Eds. Springer
Netherlands: Dordrecht, 2014; pp 199-219.
80. An, W. G.; Kanekal, M.; Simon, M. C.; Maltepe, E.;
Blagosklonny, M. V.; Neckers, L. M., Stabilization of wild-type
p53 by hypoxia-inducible factor 1α. Nature 1998, 392, 405-408.
81. Geißler, D.; Antonenko, Y. N.; Schmidt, R.; Keller, S.;
Krylova, O. O.; Wiesner, B.; Bendig, J.; Pohl, P.; Hagen, V.,
(Coumarin ‐4 ‐yl)methyl Esters as Highly Efficient, Ultrafast
Phototriggers for Protons and Their Application to Acidifying
Membrane Surfaces. Angew. Chem. Int. Ed. 2005, 44, 1195-1198.
82. Bort, G.; Gallavardin, T.; Ogden, D.; Dalko, P. I., From one-
photon to two-photon probes: "caged" compounds, actuators, and
photoswitches. Angew. Chem. Int. Ed. 2013, 52, 4526-37.
83. Dyer, J.; Jockusch, S.; Balsanek, V.; Sames, D.; Turro, N. J.,
Two-Photon Induced Uncaging of a Reactive Intermediate.
Multiphoton In Situ Detection of a Potentially Valuable Label for
Biological Applications. J. Org. Chem. 2005, 70, 2143-2147.
84. Urdabayev, N. K.; Popik, V. V., Wolff Rearrangement of 2-
Diazo-1(2H)-Naphthalenone Induced by Nonresonant Two-Photon
Absorption of NIR Radiation. J. Am. Chem. Soc. 2004, 126, 4058-
4059.
65. Dubinsky, L.; Krom, B. P.; Meijler, M. M., Diazirine based
photoaffinity labeling. Bioorg. Med. Chem. 2012, 20, 554-70.
66. Ito, K.; Maruyama, J., A Facile Intramolecular Cyclization of
4-Diazomethylcoumarins.
A
Convenient
Route
to
Benzopyrano[3,4-c]pyrazol-4(3H)-ones. Heterocycles 1984, 22,
1057-1059.
67. Preston, G. W.; Wilson, A. J., Photo-induced covalent cross-
linking for the analysis of biomolecular interactions. Chem. Soc.
Rev. 2013, 42, 3289-3301.
68. Takagaki, Y.; Gupta, C. M.; Khorana, H. G., Thiols and the
diazo group in photoaffinity labels. Biochem. Biophys. Res.
Commun. 1980, 95, 589-595.
69. McGrath, N. A.; Andersen, K. A.; Davis, A. K.; Lomax, J. E.;
Raines, R. T., Diazo compounds for the bioreversible esterification
of proteins. Chem. Sci. 2015, 6, 752-755.
85. Miller, L. W.; Cai, Y.; Sheetz, M. P.; Cornish, V. W., In vivo
protein labeling with trimethoprim conjugates: a flexible chemical
tag. Nat. Methods 2005, 2, 255-257.
70. Teruya, K.; Tonissen, K. F.; Poulsen, S.-A., Recent
developments of small molecule chemical probes for fluorescence-
8
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
POI
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Visibile or
NIR Light
N2
CF3
CF3
N
N
O
O
O
O
CBB
FL
9
ACS Paragon Plus Environment