Photoactivatable Fluorogens by Intramolecular C-H Insertion of Perfluoroaryl Azide

Article abstract of DOI:10.1021/acs.joc.9b02050

Molecules, capable of fluorescence turn-on by light, are highly sought-after in spatio-temporal labeling, surface patterning, monitoring cellular and molecular events, and high-resolution fluorescence imaging. In this work, we report a fluorescence turn-on system based on photoinitiated intramolecular C-H insertion of azide into the neighboring aromatic ring. The azide-masked fluorogens were efficiently synthesized via a cascade nucleophilic aromatic substitution of perfluoroaryl azides with carbazoles. The scaffold also allows for derivatization with biological ligands, as exemplified with d-mannose in this study. This photoinitiated intramolecular transformation led to high yields, high photo-conversion efficiency, and well-separated wavelengths for photoactivation and fluorescence excitation. The mannose-derivatized structure enabled spatio-temporal activation and showed high contrast and signal amplification. Live cell imaging suggested that the mannose-tagged fluorogen was transported to the lysosomes.

Full text of DOI:10.1021/acs.joc.9b02050

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Article
Photoactivatable Fluorogens by Intramolecular
C-H Insertion of Perfluoroaryl Azide
Sheng Xie, Giampiero Proietti, Olof Ramstrom, and Mingdi Yan
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b02050 • Publication Date (Web): 07 Oct 2019
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Photoactivatable Fluorogens by Intramolecular C-H Insertion of
Perfluoroaryl Azide
Sheng Xie,*,†,‡ Giampiero Proietti,‡ Olof Ramström,*,‡,§, and Mingdi Yan*,§
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†
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan
‡
University, Changsha 410082, P. R. China; Department of Chemistry, KTH - Royal Institute of Technology, Teknikringen
36, SE-100 44 Stockholm, Sweden; Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854,
USA; Department of Chemistry and Biomedical Sciences, Linnaeus University, SE-39182 Kalmar, Sweden.
§

*E-mail: shengxie@hnu.edu.cn, olof_ramstrom@uml.edu, mingdi_yan@uml.edu
ABSTRACT: Molecules capable of fluorescence turn-on by light are highly sought-after in spatio-temporal labeling, surface
patterning, monitoring cellular and molecular events, and high-resolution fluorescence imaging. In this work, we report a fluorescence
turn-on system based on photo-initiated intramolecular insertion of azide into the neighboring aromatic ring. The azide-masked
fluorogens were efficiently synthesized via a cascade nucleophilic aromatic substitution of perfluoroaryl azides with carbazoles. The
scaffold also allows for derivatization with biological ligands, as exemplified with D-mannose in this study. This photo-initiated
intramolecular transformation led to high yields, high photo-conversion efficiency, and well-separated wavelengths for photo-
activation and fluorescence excitation. The mannose-derivatized structure enabled spatio-temporal activation, and showed high
contrast and signal amplification. Live cell imaging suggested that the mannose-tagged fluorogen was transported to the lysosomes.
INTRODUCTION
designs are mostly modular and can be readily tailored for
different applications.
Fluorescence imaging has become an essential tool to study
biological events both in vitro and in vivo. Photoactivatable
fluorogens, molecules that become florescent upon light
irradiation, enable turn-on fluorescence as well as spatial and
temporal control, and have thus emerged as an important class
One such photo-sensitive group to mask fluorophores in the
dark state is the azide. In this case, the fluorescence can be
switched on upon transformation of the azide, for example, by
2
3
17-21
24
H
2
S reduction, photo-reduction,
or ‘click’ conjugation.
of fluorophores finding unique applications in surface
In particular, the photochemical azide (1)-to-amine (2)
1,2
patterning, in situ labeling,
macromolecules, and high resolution fluorescence imaging
such as PALM (photo-activated localization microscopy). In
tracking cells and
conversion has been demonstrated to be a successful strategy in
3
17-21
PALM by Moerner and coworkers (Scheme 1a).
The
4-7
photochemical azide-to-amine reduction reaction is mediated
by the aryl nitrene after extrusion of nitrogen, which then
these applications, as the photo-controllable fluorogenic
transformation dictates the quality of the imaging, it is
2
5,26
undergoes hydrogen abstraction.
This high spatial resolution
important that the photochemical reaction is robust and yields
was attributed to an efficient azide-to-amine transformation
(87%) in the film, and thus higher signal-to-noise compared to
4
clean transformations.
For practical reasons, other
2
1
requirements are also necessary for these fluorogens, including
modular synthesis, straightforward ligand functionalization,
biocompatibility with minimum perturbation of the biological
system, high signal-to-noise ratio, high photo-stability, minimal
susceptibility to environmental changes, specific labeling
ability, and good compatibility with microscopy techniques
non-fluorinated aryl azide derivatives. A potential issue with
this protocol is the random insertion of the fluorinated nitrene
into nearby molecular structures, as for example used in
2
1
fluorogenic photo-affinity labeling. This could cause high
background noise and also result in potential perturbation of the
21
biosystem. Additionally, it is not trivial to introduce
8
(
especially confocal laser microscopy). In cell imaging, light-
functional groups on these molecules for subsequent
conjugation.
controlled fluorescent proteins are highly popular, but suffer
from limited spectral variation, photo-bleaching, and
Nitrenes generated by thermolysis or photolysis of azides can
9
environmental sensitivity. In this regard, small organic photo-
also react with neighboring C-H bonds to form N-heterocycles,
activatable compounds have become an increasingly important
27,28
for example, in the synthesis of carbazoles.
Consistent with
1
0
11,12
class of fluorogens, including caged dyes,
photo-activated
the studies of many non-fluorinated aryl azides, nitrenes
generated in these cases also led to a range of other products,
for example, those via ring rearrangement pathways. It has
and fluorogenic reaction-based platforms (e.g., those
1
3
accomplished by the tetrazole-alkene cycloaddition reaction,
29
2
,14,15
photocyclodehydrogenation,
dehydrogenation, azide-to-amine photo-reduction,
photo-oxidative
been extensively studied and shown that the introduction of
fluorine atoms ortho to the aryl azide increases the lifetime and
inhibits ring-expansion rearrangement of the photo-generated
singlet nitrenes, leading to higher yields of the C-H and N-H
insertion products compared to the non-fluorinated aryl azide
16
17-21
and
22
reversible isomerization ). In these systems, a photo-sensitive
group is used to mask the fluorescence of the fluorogen, for on-
demand removal or transformation by a photo-controlled
reaction to generate the fluorophore. These reaction-based
29
analogs. We have developed a series of perfluoroaryl azides
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Scheme 1. Photo-activatable PFAA fluorogens via a) functional groups (R) on the PFAAs. As an example, a
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hydrogen abstraction,
and b) intramolecular insertion
carbohydrate, D-mannose, was introduced onto the fluorogen
and was used to evaluate the performance of this conjugate in
cell imaging.
(this work).
a)
NC
NC
NC
NC
F
F
F
F
CN
F
F
F
CN
h
O
O
N3
Hydrogen abstraction
H2N
RESULTS AND DISCUSSION
Synthesis of PFAA fluorogens
F
1
, dark
2, fluorescent
The photoactivatable PFAA fluorogens were synthesized
following a straightforward protocol via nucleophilic aromatic
substitution in two steps from the commercially available
starting material methyl pentafluorobenzoate 5 (Scheme 2).
Refluxing 5 and sodium azide in acetone/water afforded PFAA
b)
R
R
h
F
N
F
N
0
1
2
3
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0
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0
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0
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9
0
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7
8
9
0
Intramolecular insertion
44
N3
HN
3
, dark
4, fluorescent
6
in 95% yield. When PFAA 6 and carbazole 7 were mixed in
o
DMSO in the presence of Cs
aryl azides were formed exclusively in high isolated yields
products 8, 86%; 9, 75%; 10, 82%). The conversion was much
2 3
CO at 50 C for 8 h, di-substituted
(PFAAs) as a general class of heterobifunctional coupling
reagents to immobilize molecules onto surfaces and
nanomaterials by photocoupling.
(
30,31
Herein, we demonstrate a
slower when other solvents such as DMF, MeCN or THF were
used (Table 1). This is likely due to the excellent solvation
new fluorescence turn-on strategy based on the intramolecular
C-H insertion of perfluoroaryl nitrenes into neighboring
aromatic rings (Scheme 1b). While the PFAAs (3) are non-
fluorescent, the fluorescence can be turned on by
photochemical conversion to the corresponding fluorescent
carbazole structure (4). The reaction is efficient and modular,
where additional molecules can be conjugated through
+
ability of DMSO for the Cs cation, which is known to enhance
3
2
the basicity of cesium salts. Other bases were also tested
(
(
Cs
Table 1). For weaker bases like K
24 h) was needed and the yield (75%) was lower than with
CO . In the case of NEt , complete transformation of the
2 3
CO , longer reaction time
2
3
3
Scheme 2. Synthesis of (a) PFAA fluorogens 8-10, and (b) mannose-derivatized PFAA fluorogen 12.
HO
HO
HO
OH
O
R
a)
b)
O
O
O
N
O
O
O
O
O
O
O
O
N
F
F
F
F
F
F
F
F
F
F
[
a]
[b]
[c]
N
F
F
[d]
R
R
8
HN
O
H
N
N
N
F
N
N3
F
N3
N3
8
9
R= H,
R= Br,
86%
75%
N
F
R
R
N3
5
6
95%
7
10 R= OMe, 82%
11 70%
12 55%
R
Conditions: [a] compound 5 (40 mmol), NaN
(
aminoethyl α-D-mannopyranoside, DMF, in dark, r.t., 24 h.
3
(52 mmol), acetone/water 2:1 (90 mL), reflux, 6 h; [b] compound 6 (0.4 mmol), compound 7
o
o
1.1 mmol), Cs CO (2.0 mmol), DMSO (5 mL), 50 C, 8 h; [c] i). NaOH, MeOH, r.t., 5 h; ii). NHS, EDAC, DCM, 40 C, 48 h. [d] 2-
2
3
Table 1. Optimization of reaction conditions.
O
OMe
F
O
OMe
Nu
O
OMe
Nu
H
N
F
F
Base
F
F
F
+
+
Solvent
F
F
Nu
F
Nu=
N3
N3
N3
N
6
7a
8a
8
entry
base
solvent
time/temp.
8 (%)b
97 (86c)
76
8:8ab
n.d.
n.d.
14:1
8:1
1
2
3
4
5
6
7
8
9
Cs
Cs
Cs
Cs
2
2
2
2
CO
CO
CO
CO
3
DMSO
12 h / 25 °C
12 h / 25 °C
12 h / reflux
24 h / reflux
72 h / 80 °C
24 h / 25 °C
12 h / 25 °C
12 h / 25 °C
3 h / 25 °C
3
3
3
DMF
MeCNa
THF
44
37
NEt
3
DMSO
DMSO
DMSO
DMSO
MeCNa
17
3:1
K
NaHe
t-BuOKe
t-BuOKe
2
CO
3
75
n.d.
-d
-d
53
68 (52c)
16
7:1
b
a
Conditions: PFAA 6 (0.4 mmol, 1 equiv.), carbazole 7a (0.8 mmol), base (1.0 mmol), solvent (5 mL). Solvent (20 mL). Determined by
1
9
c
d
e
F-NMR. Isolated yield. Compound 8a not detected. Base (0.4 mmol). n.d., compound 8a not determined.
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azide could not be accomplished even at higher temperature,
much shorter wavelength at ~ 420 nm (Fig. S3) whereas product
13 emitted at 550 nm (Fig. 2c).
1
2
3
4
5
6
7
8
9
and the reaction time needed to be substantially extended (17%
yield at 80 °C after 72 h). Stronger bases, such as NaOH and t-
BuOK, gave faster conversions but lower isolated product
yields. In these cases, significant amounts of dark-colored by-
products containing the carbazole moiety were observed,
together with the corresponding aniline from PFAA 6.
Scheme 3. Reactions of compound 8: a) photo-initiated
intramolecular C-H insertion; and b) reduction to aniline.
a)
O
OMe
F
O
OMe
F
N
F
N
F
The reaction occurs through two sequential substitutions of
the aromatic fluorine by the carbazole nitranion. Interestingly,
the mono-substituted product 8a was detected only in trace
amount (Table 1), even when a large excess of PFAA 6 (8 eq.)
h
MeOH, r.t., 4 h
N
N
N3
HN
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
13, 68% (> 90%)
3
3
8
was used. In the mono-substituted product, the large planar
carbazole group is expected to be nearly perpendicular to the
aryl ring due to steric repulsion. This strongly decreases the
conjugative donation of the non-bonding electrons on
b)
O
OMe
F
O
OMe
N
F
10% Pd/C, H2
THF, r.t., 5 h
N
F
F
3
4
nitrogen. As a result, the carbazole moiety activates the C-F
bond in the para-position. This para-favored double
substitution pattern is frequently observed in the nucleophilic
N
N
N3
NH2
8
14, 90%
3
5,36
aromatic substitution of polyfluoroaromatics.
Conditions: a) Compound 8 (100 mg) in MeOH (120 mL), N
2
The carboxyl group on the PFAA fluorogen can be further
modified, thus allowing convenient incorporation of this
photoactivatable fluorogen into other molecules and
atmosphere, 350 nm (Rayonet photochemical reactor), 4 h, isolated
yield (NMR yield); b) compound 8 (200 mg) in THF (1 mL), Pd/C
(34 mg, 0.032 mmol) in MeOH (2 mL), H , r.t., 5 h, isolated yield.
2
3
0
materials. Here, we installed an α-D-mannopyranosyl (Man)
group on compound 8 by first converting the methyl ester to an
activated NHS ester (11) followed by addition of an amine-
The photo-activation of the PFAA fluorogens was monitored
by UV-vis and fluorescence spectroscopy, and the results are
shown in Fig. 1 (compound 8) and Fig. S2 (compounds 9-10).
The increased absorption at 378 nm was a result of formation
of product 13 (Fig. 1a), attributed to the increased conjugation
in the product (Scheme 3a). The process was also accompanied
by a decrease of absorption at 328 nm which is from the intact
carbazole moiety (Fig. 1a). The absorption profile could be
fitted to a typical first-order kinetic model, which gave a half-
life (t1/2) of approximately 12 seconds (Fig. 1b). While
compound 8 showed negligible fluorescence, the emission of
compound 13 at 550 nm was more than 200 times higher than
for compound 8 (Fig. 1c). This demonstrates a high contrast
between the on and off states of the fluorogen, a property that
is critically important in imaging where fluorescence from the
excited product needs to be easily distinguished from the
precursor to give clear images and high signal-to-noise ratios.
Moreover, the Stokes shift of fluorescent product 13 was more
than 170 nm. The irradiation wavelength for compound 8 (300
nm) and the excitation wavelength for product 13 (378 nm)
were well separated, which is convenient for practical
operation.
3
7
derivatized Man structure (Scheme 2b). The resulting
compound 12 showed much improved solubility in water and
was subsequently used for cell imaging.
Fluorescence turn-on studies
The solutions of compounds 8-12 were minimally fluorescent,
but all displayed visible fluorescence after irradiation with a
handheld UV lamp. Compound 8 was chosen as the model
fluorogen to study the photochemical conversion. Upon
irradiating compound 8 in MeOH at 350 nm for 4 h, a clean
conversion of compound 8 to product 13 was observed (> 90%
by 1H NMR) (Scheme 3a). After purification by flash column
chromatography, product 13 was isolated as the only
fluorescent compound in 68% yield. The product was
characterized by NMR and HRMS (Figs. S19-S21). To further
confirm that the product was formed via the intramolecular
insertion rather than hydrogen abstraction from neighboring
molecules to give 14, compound 14 was independently
synthesized by reducing compound
8
by catalytic
hydrogenation (Scheme 3b). In addition to the differences in
NMR and IR (Figs. S23-S26), compound 14 fluoresced at a
Figure 1. Characterization of the photo-activated fluorescence turn-on process of compound 8 by UV-vis and fluorescence spectroscopy. a)
Absorption changes of compound 8 during photo-activation. These are difference spectra obtained by subtraction the initial spectrum without
light-activation. Direction of arrows indicates increase in irradiation time. b) Changes in the absorption at 328 nm and 376 nm vs. irradiation
time. Lines drawn to aid visualization. c) Fluorescence spectra before and after 350 nm irradiation for 200 s, excitation wavelength: 378 nm.
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The photo-activation of compounds 8 and 12 were
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additionally evaluated by fluorescence microscopy. In the
experiment shown in Fig. 2, both compounds were deposited on
the same cover slip to keep the conditions consistent, and the
right half of the sample was then irradiated at 312 nm for 30 s
(
Fig. 2a). The irradiated areas showed the anticipated
fluorescence, yellow for compound 8 and green for compound
2, respectively (right panel inserts), and the un-irradiated areas
1
remained dark (left panel inserts). The fluorescence intensity of
the regions after irradiation were ~ 30 and ~14 times higher than
those before irradiation for compounds 12 and 8, respectively
Figure 2. Characterization of the dynamic fluorescence turn-on
processes of compounds 8 (C8) and 12 (C12) by confocal
fluorescence microscopy. (a) Schematic of the prepared sample and
lambda-mode micrographs of the four regions of interest showing
the photo-activation of compounds 8 and 12. Stock solutions of
compounds 8 and 12 (10 mM in ethanol) were dropped on a glass
coverslip in two separate regions (top: compound 8, bottom:
compound 12) which were covered with smaller coverslips (shown
as smaller circles) and then sealed with nail polish. The right half
of the sample (pink color) was irradiated for 30 s using a UV table
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figs. 2b and 2c), demonstrating that the photo-activation could
be spatially resolved and the fluorescence turn-on was of good
contrast. Additionally, the emission profiles of the fluorescence
spectra after irradiation (black curves, Figs. 2b and 2c) were the
same as those before irradiation (green curves). It can therefore
be concluded that the two fluorogens underwent the same
photon-promoted reaction despite the difference in the
substitution pattern on the PFAA structure.
(Spectroline Ultraviolet Transilluminator TVC-312R/F, 312 nm).
The photophysical and photochemical parameters of the
azide-masked fluorogens, together with the previously reported
perfluorophenyl azide-derivatized dihydrofuran fluorophore 1
are displayed in Table 2. Compared to compound 1, compounds
(b, c) Fluorescence spectra of compounds 12 and 8 before and after
photoactivation. The green curves were the red curves amplified at
0x and 14x, respectively. A fiber-coupled LED 300 nm (Thorlabs
3
M300F2, ca 250 mA with a spot size ca 3 mm) was used to irradiate
the sample.
8
-10 showed shorter absorption wavelengths for the fluorogen
(
λabs, azide) and the product (λabs, p), but comparable conversions
and quantum yields (F, except for10). The fluorescence of the
irradiated product was the strongest for compound 9 and was
much weaker in the case of compound 10. The photo-
conversion quantum yield (p) measures the probability of the
azide transformation for each photon absorbed. Compared to
compound 1 (0.017), compounds 8-10 displayed higher values
Cell imaging
The PFAA fluorogen was then evaluated in in vitro imaging
using human umbilical vein endothelial (HUVEC) cells (Fig.
3
). In the experiment, Man-derivatized PFAA fluorogen 12 was
cultured at a concentration of 10 µM with HUVEC cells for 4
h. The cells were then irradiated by LED (300 nm), focused on
the spot of the focal area of the microscope objective (Fig. 3a),
and the process was monitored by confocal fluorescence
microscopy. Fig. 3b is a representative merged micrograph after
switching on the LED for 3 min. The temporal development of
the bright-field (dashed lines) and fluorescence (solid lines)
signals of four regions of interest (ROIs) are shown in Fig. 3c
of 
the carbazole moiety also influenced 
OMe) being the highest at 0.62. Masked fluorogens with high
photo-conversion efficiency enable photoactivation at lower
p
by a factor of at least 10 (0.18-0.62). The substituent on
p
, with compound 10 (R
=
3
8
illumination intensity, leading to less photo-damage. These
results demonstrate that the PFAA fluorogens possess excellent
photoactivatable fluorescence switch-on properties.
(LED switched on at t = 1 min). The fluorescence from the cells
increased with the photo-activation time. This observation is
consistent with the fluorescence micrographs collected at 0, 2,
4
and 6 min (Fig. 3d).
Table 2. Photophysical and photochemical parameters of the azide fluorogens.
abs, azide (nm)a
-1
λ
abs, p (nm) b
-1 -1
λ
c
Yieldd
(%)
Fe
t1/2f
(s)
g
λ
fl

p
Azide
-1
{λ
p
}h
{ϵ
max (M cm )}
{ϵmax (M cm )}
(nm)
(%)
0.017i
4
{
3
{
3
{
07
463
1 k
8
578
87
80
95
90
0.62
0.51
0.76
0.11
85i
26,700}
28
{20,000}
376
{385 nm}
0.18j
550
538
581
15j
12,400}
45
{22,890}
378
{350 nm}
0.46j
10j
9
13,830}
{15,670}
394
{350 nm}
0.62j
358
13,830}
1
0
6j
{
{16,200}
{350 nm}
a
b
c
Peak absorbance and molar absorptivity of azide fluorogen. Peak absorbance and molar absorptivity of product. Peak fluorescence
d
e
emission of product. By NMR, showing the percentage of the starting azide transformed into the amine product. Fluorescence quantum
yield of the corresponding product after photo-activation. Photoconversion half-life. Photoconversion quantum yield of azide. Wavelength
used to photoactivate azide fluorogen. Light intensity: 1.1 mW cm . Light intensity: 0.46 mW cm . Data from reference 21. Measurements
f
g
h
i
-2
j
-2 k
for compounds 8-10 were performed in ethanol at 10 μM concentration.
ACS Paragon Plus Environment

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The Journal of Organic Chemistry
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0
Figure 3. In situ photo-activation of compound 12 incubated with HUVEC cells. a) Schematic illustration of experimental setup. b) Merged
bright-field and fluorescence micrograph 3 min after switching on the UV LED (Fiber-Coupled LED 300 nm, Thorlabs M300F2). Images
were obtained on a confocal fluorescence microscope (Zeiss LSM780). The transmission of the radiation of 300 nm from the UV LED
through the microscopic objective was very limited so the built-in laser (405 nm) was used to generate the bright-field micrographs for
calibrating the fluorescence acquisition. c) Photon counts of four representative regions of interests (ROIs) on the sample. Solid lines:
fluorescence signals, dashed lines: bright-field signals. d) Fluorescence micrographs at 0, 2, 4, 6 min of irradiation. Blue color is the pseudo
color, showing the fluorescence channel (410–695 nm), while the bright field was generated by the built-in excitation laser of 405 nm.
Encouraged by the above results, we next tested the PFAA
fluorogens for cell imaging using both compound 8 and
compound 12. Cultured HUVEC cells were incubated with each
compound (10 µM) for 4 h, after which the cells were washed
with fresh growth medium, and were then subjected to
photoactivation (312 nm) for 30 s. Minimal fluorescence was
observed for cells incubated with 8 or 12 (Fig. 4). After photo-
activation, strong fluorescence was seen and the fluorescence
was distributed in the entire cell except the nucleus. This is
consistent with our previous study which showed high cellular
tolerance of PFAA. After 24 h, the fluorescence remained
strong across the whole cells for compound 8, whereas
compound 12 seems to be dissipated from the cells. The
difference in the mode of action between compound 8 and
compound 12 warrants future investigation.
the locations of compound 12 and lysosome. It is clear from the
merged image that compound 12 was localized in the lysosome.
Figure 5. Fluorescence micrographs of C12–treated HUVEC cells
co-stained with LysoTracker 633 (4 h) taken at different excitation.
Excitation lasers: 633 nm 0.7000% and 405 nm 3.0%. Filters: 491-
618 nm; Lysotracker 633: 642-695 nm. Both yellow and red are
pseudo-colors.
4
3
CONCLUSIONS
In summary, we have developed azide carbazole derivatives as
masked fluorogens, which undergo photo-activated
fluorescence turn-on resulting from nitrene-mediated
intramolecular C-H insertion. The photochemical reaction was
clean, fast and highly efficient. These compounds exhibited
high signal amplification and contrast. This new class of azide-
masked fluorogens was efficiently prepared by nucleophilic
aromatic substitution of perfluoroaryl azides with carbazole.
The structure could be further derivatized or modified to
introduce functional groups or ligands. The photochemical
conversion is compatible with bioimaging, and the process
could be conveniently monitored by fluorescence microscopy.
The versatile system could find utilities in other imaging
applications that require spatio-temporal controls.
Figure 4. Fluorescence micrographs of HUVEC cells incubated
with 10 µM compound 12 (C12) or 8 (C8) for 4 h before and after
UV activation. 15 µL of the stock solution of compound 12 or 8 (1
mM in 99.5% ethanol, analytical grade) was injected into the cell
culture (1.5 mL) to obtain the final concentration of 10 µM. The
‘before UV’ images show the pseudo color of the fluorescence
channel (410-695 nm). The ‘after UV’ images show both the
pseudo color and the true color of the fluorescence. The ‘24 hr’
image shows the true color of the fluorescence.
EXPERIMENTAL SECTION
General methods. Reagents and solvents were used as received
from Sigma Aldrich, Alfa Aesar, Fluka, and Merck. Thin-layer
chromatography was conducted using TLC silica gel 60 F254
(
Merck Co.), visualized with ultraviolet light. Oil bath was used
1
13
19
to control the reaction at high temperature. H-, C- and F-
NMR data were recorded on Bruker Ascend 400 or DMX 500
TM
To identify the location of the fluorogen, cells were co-stained
with LysoTracker 633, a fluorophore that freely permeates cell
membranes but is retained in acidic subcellular compartments,
such as the lysosome. Fig. 5 are the confocal images showing
instruments. Chemical shifts are reported as  values (ppm)
1
3
with (residual) solvent as internal reference. All C NMR
1
9
spectra were recorded as proton-decoupled data. F NMR
signals were referenced to hexafluorobenzene ( = -161.75 in
ACS Paragon Plus Environment

The Journal of Organic Chemistry
) unless noted otherwise. High-
d ):  3.22 (s, 3H), 7.59 (m, 4H), 7.71 (t, 4H, J = 8.5 Hz), 8.63
Page 6 of 9
CDCl
3
or -162.65 in DMSO-d
6
6
HH
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
resolution electrospray ionization (HRMS-ESI) mass
spectrometry data were obtained from Proteoomika
tuumiklabor at the University of Tartu, Estonia, or from the
Mass Spectrometry Lab at the University of Illinois at Urbana-
Champaign. Infrared spectra (IR) were recorded on a
ReactIRTM IC10 (Mettler Toledo Co.) for liquid samples, or a
SPECTRUM 2000 (Perkin Elmer) for solid samples in the ATR
mode. UV-vis absorption spectra were measured on a
PerkinElmer Lambda 750 spectrophotometer with 2 nm slit
width. Fluorescence spectra were recorded on the Varian Cary
Eclipse Fluorescence Spectrophotometer. Quantum yields were
(m, 4H); 13C{H} NMR (101 MHz, DMSO-d ):  52.9, 112.5,
6
112.8, 113.5, 113.6, 116.3, 116.4, 117.1, 117.2, 123.9, 124.1,
129.6, 129.8, 133.1, 139.2, 139.5, 149.2, 151.2, 152.5, 154.6,
19
160.8; F NMR (375 MHz, DMSO-d
6
):  -122.3 d F, JFF
=
13.0 Hz), -131.6 (d, 1F, JFF = 13.0 Hz); HRMS-ESI FTICR
analyzer: Calcd. for C32H16Br F N O [M+H] : 855.8006,
4 2 5 2
+
found 855.8010; IR (ATR) see Supporting Information.
Methyl 4-azido-2,5-bis(3,6-dimethoxy-9H-carbazol-9-yl)-
3
,6-difluorobenzoate (10). Synthesized following a similar
protocol as for compound 8. Yellow solid (133 mg, 82%). R
f
=
):
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0

8
.25 (1:3 EtOAc/hexanes). H NMR (500 MHz, DMSO-d
6
2 4
determined using quinine sulfate in 0.1 M aq. H SO as
3.19 (s, 3H), 3.91 (s, 12H), 7.10 (m, 4H), 7.45 (t, 4H, JHH
reference (quantum yield: 0.55). The methods for quantitative
measurements of photophysical and photochemical parameters
were reported in supporting information.
1
3
.0 Hz), 8.60 (dd, 4H, JHH = 7.3, 2.3 Hz); C{H} NMR (101
):  52.7, 55.66, 55.69, 103.4, 103.6, 111.2,
11.3, 115.2, 115.4, 116.7, 116.9, 118.0, 118.1, 123.7, 123.9,
24.6, 124.8, 132.7, 135.57, 135.64, 148.6, 150.6, 152.4, 154.3,
MHz, DMSO-d
6
1
1
1
Methyl pentafluorobenzoate (5) and carbazole (7a) were
purchased from Alfa Aesar and were used as received. Methyl
19
54.4, 161.3; F NMR (375 MHz, DMSO-d ):  -123.5 d F,
6
4
-azido-2,3,5,6-tetrafluorobenzoate (6) was synthesized by
JFF = 13.0 Hz), -131.5 (d, 1F, JFF = 13.0 Hz); HRMS-ESI FTICR
following our previously reported protocol, and was
+
analyzer: Calcd. for C36
28 2 5 6
H F N O [M+H] : 664.2008, found
1
9
1
characterized by comparing with the F and H NMR data in
664.2009; IR (ATR) see Supporting Information.
4
4
the publication. 3,6-Dibromo-9H-carbazole (7b) and 3,6-
dimethoxy-9H-carbazole (7c) were synthesized following
reported protocols, and the identities were confirmed by
comparing with the literature H and C NMR data. 2-
Aminoethyl α-D-mannopyranoside was synthesized following
our previously reported protocol, and the identity was
Methyl
3-(9H-carbazol-9-yl)-1,4-difluoro-5H-
indolo[3,2,1-de]phenazine-2-carboxylate (13). In a 200 mL
flask, compound 8 (100 mg) was dissolved in 120 mL MeOH.
After the solution was purged with N for 10 minutes, the flask
2
1
13
45
was put under 350 nm UV inside the a Rayonet photochemical
reactor. When TLC displayed full conversion (~4 h), the
mixture was evaporated to give a light red solid. The crude was
dissolved in chloroform, and silica gel (600 mg) was added. The
crude was then purified by dry-loading column chromatography
using toluene/hexanes (140:100) as eluent to give compound 13
1
13
confirmed by comparing with the reported H and C NMR
3
7
data.
Methyl
4-azido-2,5-di(9H-carbazol-9-yl)-3,6-
difluorobenzoate (8). To a solution of compound 6 (100 mg,
0.40 mmol) in DMSO (5 mL), carbazole (180 mg, 1.07 mmol)
was added, followed by cesium carbonate (650 mg, 2.0 mmol)
in portions. The mixture was heated at 50 °C until TLC showed
a full conversion of the azide. Afterwards, the mixture was
quenched with water (8 mL) and 1 M aq. HCl (2 mL), and
further extracted with toluene (20 mL × 3 times). The combined
organic phase was then washed with water (60 mL) and brine
1
as yellow flakes (68 mg, 68%). H NMR (500 MHz, DMSO-
d
7
6
):  3.15 (s, 3H), 6.50 (d, 1H, JHH = 7.5 Hz), 6.93 (t, 1H, JHH
=
.5 Hz), 7.32 (m, 6H), 7.46 (m, 3H), 7.79 (t, 1H, JHH = 7.4 Hz),
8.06 (d, 1H, JHH = 7.8 Hz), 8.23 (d, 1H, JHH = 7.8 Hz), 9.39 (br,
1
3
1H, NH); C{H} NMR (101 MHz, DMSO-d
109.8, 111.3, 111.4, 112.2, 114.4, 114.6, 120.4, 120.5, 120.6,
21.3, 122.3, 122.9, 124.4, 124.9, 126.1, 126.3, 130.0, 130.8
6
):  52.3, 107.1,
1
(
60 mL), and dried over MgSO
crude was purified by flash column chromatography
(toluene/hexanes 3:2, R = 0.22) to give compound 8 (180 mg)
as a light-yellow solid foam (86% yield). H NMR (500 MHz,
DMSO-d ):  3.17 (s, 3H), 7.38 (m, 4H), 7.53 (m, 4H), 7.61
(t, 4H, JHH = 7.3 Hz), 8.29 (t, 4H, JHH = 7.3 Hz); C{H} NMR
101 MHz, DMSO-d ):  52.7, 110.3, 110.5, 116.7, 116.9,
17.7, 117.9, 120.5, 120.6, 120.9, 121.0, 123.0, 123.2, 124.9,
26.3, 126.5, 140.2, 140.4, 148.7, 151.2, 152.3, 154.8, 161.1;
4
. After removal of toluene, the
1
9
(
m), 131.3, 136.3, 140.3, 141.7, 143.6, 143.8, 145.8, 161.8; F
NMR (375 MHz, DMSO-d
6
):  -121.8 t F, JFF = 7.7 Hz), -
f
1
42.6 (d, 1F, JFF = 9.4 Hz); HRMS-ESI FTICR analyzer: Calcd.
1
+
19 2 3 2
for C32H F N O [M] : 515.1445, found 515.1448; IR (ATR)
6

see Supporting Information.
1
3
Methyl
4-amino-2,5-di(9H-carbazol-9-yl)-3,6-
(
6
difluorobenzoate (14). To a two-necked flask charged with
Pd/C powder (34 mg, 0.032 mmol) in MeOH (2 mL) under H ,
2
compound 8 (200 mg) in THF (1 mL) was injected. The mixture
was vigorously stirred at rt. When TLC indicated full
conversion, the mixture was filtered through celite quickly and
1
1
1
9
F NMR (375 MHz, DMSO-d
6
):  -122.9 d F, JFF = 13.0
Hz), -131.2 (d, 1F, JFF = 13.0 Hz); HRMS-ESI FTICR analyzer:
Calcd. for C32
+
20 2 5 2
H F N O [M+H] : 544.1585, found 544.1587;
the organic phase was dried over MgSO
4
. Evaporation of the
IR (ATR) see Supporting Information.
organic solution gave compound 14 as a white solid (190 mg,
Methyl
4-azido-2-(9H-carbazol-9-yl)-3,5,6-
1
90% yield). H NMR (500 MHz, DMSO-d
6
):  3.05 (s, 3H),
trifluorobenzoate (8a): the mono-substituted derivative.
6
4
1
1
1
d
.52 (br, 2H), 7.33 (m, 4H), 7.42 (m, 4H), 7.50 (m, 4H), 8.27 (t,
H, JHH = 7.7 Hz); C{H} NMR (101 MHz, DMSO-d ):  51.7,
04.1, 104.2, 109.2, 109.3, 110.0, 110.1, 120.3, 120.4, 120.5,
22.9, 123.5, 123.6, 126.16, 126.19, 140.2, 140.6, 140.9 (m),
42.7, 144.6, 154.5, 156.5, 162.0; F NMR (375 MHz, DMSO-
1
Isolated in trace amount from the synthesis of compound 8. H
1
3
6
NMR (500 MHz, DMSO-d
6

):  3.19 (s, 3H), 7.23 (d, 2H, JHH
=
7.3 Hz), 7.32 (t, 2H, JHH = 7.3 Hz), 7.44 (t, 4H, JHH = 7.3 Hz),
19
7
.23 (d, 2H, JHH = 7.3 Hz). F NMR (375 MHz, DMSO-d
6
):  -
19
1
32.2 m F), -140.5 (m, 1F), -143.9 (m, 1F); HRMS-ESI
6
):  -122.7 d F, JFF = 11.4 Hz), -140.9 (d, 1F, JFF = 9.4 Hz);
+
FTICR analyzer: Calcd. for C20
H
12
F
3
N
4
O
2
[M+H] : 397.0912,
+
HRMS-ESI FTICR analyzer: Calcd. for C32
18.1680, found 518.1689; IR (ATR) see Supporting
Information.
,5-Dioxopyrrolidin-1-yl 4-azido-2,5-di(9H-carbazol-9-
22 2 3 2
H F N O [M+H] :
found 397.0917; IR (ATR) see Supporting Information.
5
Methyl 4-azido-2,5-bis(3,6-dibromo-9H-carbazol-9-yl)-
3,6-difluorobenzoate (9). Synthesized following a similar
2
protocol as for compound 8. Light yellow solid (214 mg, 75%).
yl)-3,6-difluorobenzoate (11). To a flask charged with
compound 8 (270 mg, 0.5 mmol) in MeOH (40 mL) and THF
1
R
f
= 0.61 (1:1 toluene/hexanes). H NMR (500 MHz, DMSO-
ACS Paragon Plus Environment

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The Journal of Organic Chemistry
p
12 mL), aqueous sodium hydroxide solution (20%, w/w; 10 quantum yield of the photoconversion (Ф ) was estimated by
(
2
1
1
2
3
4
5
6
7
8
9
mL) and water (5 mL) were added. The mixture was then stirred
at rt. When TCL showed full conversion (~5 h), the mixture was
the following equation:
푅푝
1
acidified with 1 M aq. HCl and extracted with CHCl
3
(3 x 60
훷 =
푝
=
푅푎푏푠
휆
mL). The solvent was evaporated under reduced pressure to
give the crude intermediate. The crude intermediate was then
dissolved in DCM (30 mL) containing N-hydroxysuccinimide
휏 퐼 휎 (
)
푝 휆
휆
ℎ푐
where p is the exponential time constant from fitting the
decaying absorption (Fig. S2b), and equals t½/ln2; I is the
irradiation intensity, measured to be 0.46 mW cm in all cases;
is the absorption cross-section; λ is the excitation
(
65 mg, 0.56 mmol) and EDAC (164 mg, 0.86 mmol), and the
mixture were stirred at rt. When TLC indicated full conversion
~48 h), the mixture was diluted with water (50 mL) and
extracted twice with DCM (2 × 30 mL). The combined organic
phase was washed with water, dried over MgSO , and
evaporated under reduced pressure. The crude was purified by
flash column chromatography using EtOAc/hexanes (1:3, R
0.13) as eluent to give compound 11 as a pale yellow solid (219
λ
-2
(
σ
λ
wavelength; ℎ is Planck’s constant; c is the speed of light.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
Cell imaging. Human umbilical vein endothelial cells
(
HUVEC) were purchased from Lonza and cultured in EGM-2
f
=
medium (EGM-2MV Basal Medium+EGM-2MV SingleQuots)
complemented with 1% penicillin/streptomycin/glutamine, and
1
mg, 70%). H NMR (500 MHz, DMSO-d
6
):  3.59 (s, 4H), 7.35
0
.1% amphotericin B. Cells of passage 2-5 were used in the
(t, 2H, JHH = 7.3 Hz), 7.41 (t, 2H, JHH = 7.3 Hz), 7.49 (t, 2H, JHH
experiments. Compounds incubated with HUVEC cells were
studied by confocal and bright-field microscopy. HUVEC cells
cultured in glass-bottom dishes were placed directly on the
microscope sample-stage incubator, and imaged using Zeiss
LSM 780 confocal microscope (Carl Zeiss, Jena, Germany)
with either C-Apochromat 40×/1.20 W Korr FCS M27
objective using multiple channels, z-stack, and lambda scan
modes. The excitation wavelength was 405 nm. TIFF
micrographs were exported from ZEN original image files,
using software ZEN 2012 (Zeiss, Germany) without
compression, and analyzed offline using MATLAB. UV
irradiations were done on a UV table: Spectroline ultraviolet
transilluminator TVC-312R/F (312 nm), or UV LED: Fiber-
Coupled LED 300 nm (Thorlabs M300F2) at ca 250 mA. Cell
imaging studies were repeated at least twice for each set of
experiments.
=
7.3 Hz), 7.56 (t, 2H, JHH = 7.3 Hz), 7.64 (d, 2H, JHH = 7.7 Hz),
7
2
1
1
1
1
.70 (d, 2H, JHH = 7.7 Hz), 8.23 (d, 2H, JHH = 7.7 Hz), 8.31 (d,
H, JHH = 7.7 Hz); C{H} NMR (126 MHz, DMSO-d ):  25.3,
10.3, 110.6, 111.2, 111.4, 120.4, 120.7, 120.8, 121.1, 123.1,
23.2, 126.4, 126.6, 135.8, 140.2, 140.5, 149.6, 151.6, 154.0,
56.1, 156.9, 169.1; F NMR (375 MHz, DMSO-d
1
3
6
1
9
6
):  -
16.3 d F, JFF = 12.6 Hz), -130.3 (d, 1F, JFF = 12.6 Hz);
+
HRMS-ESI FTICR analyzer: Calcd. for C35
27.1592, found 627.1596; IR (ATR) see Supporting
Information.
-Azido-2,5-di(9H-carbazol-9-yl)-3,6-difluoro-N-(2-
((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-
21 2 6 4
H F N O [M+H] :
6
4
(
(hydroxymethyl)tetrahydro-2H-pyran-2-
yl)oxy)ethyl)benzamide (12). To a solution of compound 11
(110 mg, 0.18 mmol) in DMF (3 mL) in the dark, 2-aminoethyl
α-D-mannopyranoside (51 mg, 0.23 mmol) was added. The
mixture was stirred at rt for 24 h. The solvent was directly
evaporated in vacuo at rt. Afterwards, the crude was purified by
ASSOCIATED CONTENT
Supporting Information.
flash column chromatography (DCM/MeOH 11:1, R
f
= 0.1) to
give compound 11 (77 mg, 55%) as a pink solid. H NMR (400
MHz, CD
1
The synthetic scheme of 2-aminoethyl α-D-mannopyranoside,
UV-vis spectra of perfluoroaryl compounds and carbazole,
photochemical reactions of compounds 9 and 10 monitored by UV-
vis and fluorescence spectroscopy, fluorescent properties of
compound 14, and NMR spectra of the synthesized compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
3
CN):  2.82 (m, 2H), 2.94 (m, 1H), 3.14 (m, 4H), 3.20
m, 1H), 3.30 (m, 1H), 3.45 (m, 2H), 3.55 (m, 3H), 4.38 (s, 1H),
(
7
8
.25 (s, 1H, NH), 7.40 (m, 4H), 7.50 (m, 4H), 7.54 (m, 4H),
.20 (d, 2H, JHH = 7.6 Hz), 8.24 (t, 2H, JHH = 7.6 Hz); C{H}
13
NMR (101 MHz, CD
71.8, 73.2, 100.4, 110.7, 110.9, 111.0, 120.9, 121.2, 121.7,
123.7, 123.9, 124.0, 124.1, 126.9, 127.2 (m), 131.6 (m), 141.1,
3
CN):  25.7, 39.5, 62.2, 66.2, 68.2, 70.9,
1
9
1
(
1
41.4, 141.5, 149.6 (m), 152.2 (m), 154.7 (m), 160.26; F NMR
375 MHz, CD CN):  -125.1 d F, JFF = 14.0 Hz), -133.5 (d,
F, JFF = 14.0 Hz); HRMS-ESI FTICR analyzer: Calcd. for
AUTHOR INFORMATION
Corresponding Author
3
+
C
39
H
32
F
2
N
9
O
7
Na [M+Na] : 799.2290, found 799.2294; IR
*
Email: shengxie@hnu.edu.cn (S.X.).
*Email: olof_ramstrom@uml.edu (O.R.).
Email: mingdi_yan@uml.edu(M.Y.).
(ATR) see Supporting Information.
*
Quantitative measurements of photophysical and
photochemical parameters. Measurements were carried out
1
7-18, 21
ACKNOWLEDGMENT
using reported protocols,
at low concentrations to avoid
complications with aggregate formation. Molar absorption
coefficients were measured from serial dilutions with known
concentrations. Samples were prepared in MeOH unless noted
otherwise. A hand-held UV lamp (365 nm) or a Rayonet
photochemical reactor (350 nm) were used as light sources. The
intensity of the light was determined by a UV-light meter
(Sentry Optronics Corp.) before experiments. The sample
solutions were freshly prepared and quartz cuvettes were used
We thank Prof. Ying Fu for help with the fluorescence imaging.
This work was supported in part by NIH (R15GM128164 to M.Y.).
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