A General Strategy Toward Highly Fluorogenic Bioprobes Emitting across the Visible Spectrum

Article abstract of DOI:10.1021/jacs.7b05920

A general approach toward highly fluorogenic probes across the visible spectrum for various analytes offers significant potential for engineering a wide range of bioprobes with diverse sensing and imaging functions. Here we show a facile and general strategy that involves introducing a new fluorogenic mechanism in boron dipyrromethene (BODIPY) dyes, based on the principle of stimuli-triggered dramatic reduction in the electron-withdrawing capabilities of the meso-substituents of BODIPYs. The fluorogenic mechanism has been demonstrated to be applicable in various BODIPYs with emission maxima ranging from green to far red (509, 585, and 660 nm), and the synthetic strategy allows access to a panel of highly fluorogenic bioprobes for various biomolecules and enzymes (H₂O₂, H₂S, and protease) via introducing specific triggering motifs. The potency of the general design strategy is exemplified by its application to develop a mitochondria-targeting far-red probe capable of imaging of endogenous H₂O₂ in living cells.

Full text of DOI:10.1021/jacs.7b05920

Subscriber access provided by ECU Libraries
Article
A General Strategy Toward Highly Fluorogenic
Bioprobes Emitting across the Visible Spectrum
Haoliang Chen, Xiujing He, Meihui Su, Wenhao Zhai, Hao Zhang, and Changhua Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05920 • Publication Date (Web): 27 Jun 2017
Downloaded from http://pubs.acs.org on June 27, 2017
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 free 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 accessible to all
readers and 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.
Journal of the American Chemical Society 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 20
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
A General Strategy Toward Highly Fluorogenic Bioprobes Emitting
across the Visible Spectrum
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
Haoliang Chen, Xiujing He, Meihui Su, Wenhao Zhai, Hao Zhang, and Changhua Li*
State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Functional Polymer Materials, Ministry of
Education, College of Pharmacy, and Collaborative Innovation Center of Chemical Science and Engineering
(Tianjin), Nankai University, Tianjin 300071, P. R. China
ABSTRACT: A general approach toward highly fluorogenic probes across the visible spectrum for various analytes
offers significant potential for engineering a wide range of bioprobes with diverse sensing and imaging functions. Here
we show a facile and general strategy by introducing a new fluorogenic mechanism in boron dipyrromethene (BODIPY)
dyes, based on the principle of stimuli-triggered dramatic reduction in the electron-withdrawing capabilities of the meso-
substituents of BODIPYs. The fluorogenic mechanism has been demonstrated to be applicable in variable BODIPYs
with emission maxima ranging from green to far red (509, 585, and 660 nm), and the synthetic strategy allows to ac-
cess a panel of highly fluorogenic bioprobes for various biomolecules and enzymes (H2O2, H2S, and protease) via in-
troducing specific triggering motifs. The potency of the general design strategy is exemplified by its application to de-
velop a mitochondria-targeting far red probe capable of imaging of endogenous H2O2 in living cells.
quires electron or energy transfer between donors and
acceptors, rendering them difficult to be applied in a set
of BODIPYs with different emission wavelengths.
■
INTRODUCTION
Fluorescence imaging techniques have emerged as
powerful tools for noninvasive monitoring biological ana-
lytes and processes in real time with high spatiotemporal
resolution.1 Despite the diverse palette of available fluo-
rescence probes that are essential for fluorescence im-
aging, there is still a demand for more specific, sensitive,
and versatile probes, especially fluorogenic bioprobes.2
A unified design strategy toward highly fluorogenic bi-
oprobes sensitive and selective for variable analytes of
interest with tunable emission maxima throughout the
visible spectral region provides significant potential to
engineer a broad range of bioprobes with desired sens-
ing and imaging capabilities for biological research and
clinical needs.
trigger
O-/NH2
509 nm
green
O
N
O
N
Triggering
Analyte
Self-
Immolation
O
N
O
N
O
N
O
N
B
585 nm
orange
F
F
R1
R2
B
B
R1
R2
R1
R2
F
F
-
F
F
BODIPY-COO
highly
Bioprobe
[intermidiate]
nonfluorescent
H2O2 (ROS)
fluorescent (3)
660 nm
far red
H2S (signaling molecule)
PGA (protease)
(1)
(2)
small biomolecules
enzymes
As one class of highly fluorescent dyes, the boron di-
pyrromethene (BODIPY) family has been widely em-
ployed as scaffold for functional fluorogenic bioprobes
due to their outstanding spectroscopic and photophysical
properties including excellent environmental stability,
large extinction coefficients, and sharp fluorescence
spectrum with high quantum yield.3 Most importantly, the
wide versatility of synthetic routes to BODIPYs allows
the facile synthesis of fluorophores with desired emission
maxima ranging from cyan to far red region. The excel-
lent features of BODIPY make it a viable candidate for a
universal scaffold to access diverse fluorogenic probes
with various photophysical properties. The key to estab-
lishing such a platform was the identification of a fluoro-
genic mechanism that could be applied in BODIPYs with
emission maxima across the visible region. However, at
present, most BODIPY-based fluorogenic probe designs
are based on the principle of PeT4 or FRET5 that re-
(1) Across the visible spectrum (509 nm, 585 nm, 660 nm)
(2) Versatile synthetic strategy(for small biomolecules& enzymes)
(3) Highly fluorogenic (133 ~ 320-fold enhancement)
Figure 1. Detection mechanism of the highly fluorogenic
BODIPY-based bioprobes with tunability in both emitting
wavelength and sensing analytes.
Recently, the fluorescence properties of BODIPYs
were found to be highly dependent on the electronic
properties of the meso-substituents of the dye.6 For ex-
ample, enzymatic hydrolysis of highly electron-
withdrawing meso-ester groups to electron-donating me-
so-carboxylate anions induces dramatic enhancement of
fluorescence at 550 nm in aqueous solution.6a Recogniz-
ing that the fluorescence turn on is merely due to the
dramatic reduction in the electron-withdrawing capabili-
ties of the meso-substituents, we hypothesized that this
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 20
“ester-to-carboxylate”
conversion-based
fluorogenic
COOH-FR) in phosphate buffer solution (PBS; 10 mM,
pH 7.4), respectively (Figure S1). Notably, the emission
1
2
3
4
5
6
7
8
mechanism could be potentially applied in various BOD-
IPYs across the entire visible spectrum. On the other
hand, it is known that carboxyl groups (-COOH) could be
modularly caged by a self-immolating linker via formation
of an ester bond and selectively decaged in response to
specific stimuli of interest.7 We envisioned that by incor-
porating of self-immolative chemistry, meso-COOH-
substituted BODIPYs can be modularly derivatized to
afford a palette of fluorogenic probes for variable ana-
lytes of interest throughout the visible spectrum. As de-
picted in Figure 1, in the presence of analyte of interest,
the nonfluorescent meso-ester-substituted bioprobes
would be converted to highly labile intermediates, fol-
lowed by the linker self-immolation to form meso-
carboxylate-substituted BODIPYs with strong fluores-
cence emission.
intensities of the three synthesized BODIPY-COOHs are
almost constant irrespective of solution pH in the physio-
logically relevant pH range 4-9 (Figure S2-S4).6a,8 Fol-
lowing a facile synthetic route shown in Scheme S1, the
BODIPY-COOHs were reacted with H2O2-responsive
arylboronate triggers,9 trigger-HP, to afford the corre-
sponding three H2O2 probes, i.e. P-HP-G, P-HP-O, and
P-HP-FR.
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
a)
R
R
O
N
N
O
N
N
O
N
N
F
F
F
F
F
F
B
O(CH2CH2O)3CH3
O(CH2CH2O)3CH3
B
B
HO
HO
HO
R =
R
B-COOH-G
B-COOH-O
B-COOH-FR
b)
O
O
O
■
RESULTS AND DISCUSSION
O
O
O
B
B
B
R
R
Applying the Proposed Fluorogenic Mechanism in
BODIPYs Emitting throughout the Visible Spectrum.
To test our hypothesis that the proposed fluorogenic
mechanism could be applied in BODIPY fluorophores
with tunable emission maxima, we synthesized three
typical meso-carboxyl-substituted BODIPY-COOH fluor-
ophores (Figure 2a and supporting information), which
exhibit emission maxima at 509 nm (green, B-COOH-G),
585 nm (orange, B-COOH-O), and 660 nm (far red, B-
O
O
N
F
O
O
N
N
O
N
N
F
F
F
B
B
B
F
F
N
O
R
P-HP-G
P-HP-O
P-HP-FR
Figure 2. Chemical structures of (a) three typical meso-
carboxyl-substituted BODIPYs, B-COOH-G, B-COOH-O,
and B-COOH-FR, and (b) the corresponding three H2O2
probes P-HP-G, P-HP-O, and P-HP-FR.
Figure 3. Fluorescence emission spectra recorded for 0.5 μM solution of (a) P-HP-G, (b) P-HP-O, and (c) P-HP-FR upon in-
cubating with H2O2 (100 μM) for varying time intervals (0−60 min) at 37 C; inset in each figure shows the time-dependent
increases in normalized fluorescence intensities at 509, 585, and 660 nm, respectively. (d) Optical photographs recorded un-
der ambient light (top panel) and UV (365 nm) light (bottom panel) for P-HP-G, P-HP-O, and P-HP-FR in the absence (left) or
presence (right) of H2O2 after 60 min.
o
ACS Paragon Plus Environment

Page 3 of 20
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
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 4. UV-vis absorption spectra recorded for 0.5 µM solution of (a) P-HP-G, (b) P-HP-O, and (c) P-HP-FR upon incubat-
ing with H2O2 (100 µM) for varying time intervals at 37 oC. HPLC chromatograms of (d) P-HP-G, (e) P-HP-O, and (f) P-HP-FR
without treatment of H2O2 (top); with H2O2 (200 equivalent) treatment for different time as indicated at 37 C (middle); and the
corresponding meso-carboxylate-substituted BODIPY-COOH only (bottom). The samples were analyzed by HPLC using an
isocratic elution program with a solvent system of methanol/deionized water (95/5, V/V) at a flow rate of 1.0 mL/min with de-
tection wavelength at 500, 570, and 656 nm for P-HP-G, P-HP-O, and P-HP-FR, respectively.
o
Table 1. Photophysical Properties of All BODIPY-
COOHs and Bioprobes
The fluorescence quantum yields of the meso-
carboxylate-substituted BODIPY-COOHs and corre-
sponding meso-ester-substituted H2O2 probes were
measured in PBS as shown in Table 1. Gratifyingly, we
found that the fluorescence quantum yields of all three
BODIPY-COOHs are significantly higher than the corre-
sponding meso-ester-substituted H2O2 probes, indicating
that the “ester-to-carboxylate” conversion should turn on
the fluorescence of the BODIPYs possessing emission
maxima that range from green to far red wavelengths.
Thus, the successful conversion of meso-ester groups to
meso-carboxylate groups via self-immolation upon
treatment with special analytes becomes pivotal to the
design strategy shown in Figure 1.
e
labs
(nm)
lem
(nm)
compound
Ffl (%)
(M-1cm-1)
132800
123200
86000
B-COOH-Ga
B-COOH-Oa
B-COOH-FRa
495
565
640
509
585
660
52
43
11
P-HP-Gb
P-HP-Oc
P-HP-FRa
513
595
688
109700
103000
57100
509
585
660
0.12
0.24
0.053
Most attention has been paid to the triggered release
of phenol or amine groups via self-immolative chemistry,
while relatively few reports on the triggered release of
carboxyl groups.7,9,10 To assess the feasibility of the fluo-
rogenic probes, P-HP-G, P-HP-O, and P-HP-FR (0.5
µM) were treated with H2O2 in PBS at 37 oC. All the three
P-HP probes underwent significant fluorescence en-
hancement (P-HP-G: 320-fold; P-HP-O: 220-fold; P-HP-
FR: 133-fold) upon addition of 100 µM H2O2 within 60
min (Figure 3a-c). Concomitantly, the color of the three
P-HP-FR-tppc
688
57200
660
0.057
P-HS-FRa
P-PGA-FRa
688
688
58000
58600
660
660
0.062
0.042
aData were collected in 10 mM PBS (pH 7.4) at 37 oC. bData
were collected in 10 mM PBS (pH 7.4; 8% v/v DMSO) at 37
c
oC. Data were collected in 10 mM PBS (pH 7.4; 1% v/v
DMSO) at 37 oC.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 20
probe solutions changes obliviously as shown in Figure Mitochondria-targeting Far Red Probe for Live Cell
1
2
3
4
5
6
7
8
3d. The H2O2-induced fluorescence turn-on can also be
visualized by naked eye, as evidenced by the transition
from non-fluorescence to the intense emission (Figure
3d). The absorption spectra showed obvious blue-shifts
during the incubation due to the H2O2-induced “ester-to-
carboxylate” conversion (Figure 4a-c). HPLC analysis of
the assay solution further confirmed the complete con-
version to meso-COOH-substituted products in the pres-
ence of H2O2 as shown in Figure 4d-f. All the three P-HP
probes are highly stable in the absence of H2O2 (Figure
S5-S7). As shown in Figure 5a and S8, the fluorescence
intensities have an excellent linear relationship with the
concentrations of H2O2 in 0-50 µM. The detection limit
was calculated to be as low as 0.15 µM, 0.20 µM, 0.25
µM, for P-HP-G, P-HP-O, and P-HP-FR, respectively.
Collectively, the above experiments clearly demonstrat-
ed that the proposed fluorogenic mechanism (Figure 1)
can be applied in several BODIPYs, to produce highly
sensitive fluorogenic H2O2 probes across the visible
spectrum. It was also demonstrated that the three P-HP
probes are highly selective for H2O2 over other ROSs
and some typical physiological nucleophiles (Figure 5b).
Imaging of H2O2. Since the overproduction of H2O2 is
concerned with several diseases and mitochondria are
the primary sources of H2O2,9a,11 our next goal was to
design a far red probe that can be specifically localized
in mitochondria and evaluate its performance in cell im-
aging of endogenous H2O2. It is well-known that lipophilic
triphenylphosphonium cation (tpp) moieties can specifi-
cally localize in mitochondria4b,12. We then synthesized a
tpp functionalized far red probe, P-HP-FR-tpp (Figure 6
and supporting information). As shown in Figure 7a and
S9a, this P-HP-FR analogue maintained the enhance-
ment in fluorescence of P-HP-FR while possessing a
mitochondria-targeting moiety, making it well-suited for
selective monitoring endogenous H2O2 in mitochondria
of living cells. The stability of P-HP-FR-tpp was as-
sessed by incubating it in PBS in the absence of H2O2.
Only small fluorescence enhancement (2.2-fold) was
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
o
observed after incubation for 1.5 h at 37 C (Figure 7a
and S9b). To examine the selectivity, the probe (0.5 µM)
was treated with various potentially interfering species,
including ROSs and physiological nucleophiles under
standard assay conditions. As shown in Figure 7b, vari-
ous species up to 100 µM, including ·OH, ClO-, tBuO·,
1O2, Cys, GSH, and H2S induced no obvious changes in
fluorescence intensities. In addition, the low cytotoxicity
of P-HP-FR-tpp was also demonstrated by methyl thia-
zolyltetrazolium (MTT) assay (Figure 7c).
a)
P-HP-G
P-HP-O
P-HP-FR
30
20
10
0
O
O
O
O
mitochondria-
targeting moiety
B
PPh3Br
N
N
N
O
O
N
N
F
F
B
R2 = 0.99986 (G)
R2 = 0.99987 (O)
R2 = 0.99962 (FR)
O
O
O
O
O
O
O
O
P-HP-FR-tpp
0
10
20
30
40
50
Figure 6. Chemical structure of lipophilic triphenylphospho-
nium cation (tpp) functionalized H2O2 probe, P-HP-FR-tpp.
[H2O2] (µM)
b)
We next tested P-HP-FR-tpp for visualization of en-
dogenous H2O2 in living cells. A H2O2-insensitive meso-
ester-substituted BODIPY dye, B-TEG4-Me, was synthe-
sized as a control compound (see supporting infor-
mation). It is quite stable in the absence or presence of
H2O2 (Figure S10). HeLa cells were then incubated with
B-TEG4-Me and P-HP-FR-tpp. After incubation for 30
min, control cells treated with B-TEG4-Me showed no
detectable fluorescence (Figure 7d), whereas the fluo-
rescence of P-HP-FR-tpp treated cells was visible but
weak (Figure 7e). This should be ascribed to the endog-
enous H2O2-induced partial conversion to B-COOH-FR.
HeLa cells were then pretreated with phorbol-12-
myristate-13-acetate (PMA; an intracellular H2O2 stimula-
tor13) for 1 h before incubation with P-HP-FR-tpp to pro-
mote the intracellular H2O2 production. Compared to un-
treated cells, CLSM images revealed a significant in-
crease of red channel emission intensities for PMA-
pretreated cells (Figure 7f). Quantitative analysis re-
vealed that red emission intensities increased 4-fold (un-
treated) and 13-fold (PMA-pretreated) for P-HP-FR-tpp
incubated HeLa cells, respectively, as compared to B-
P-HP-G
P-HP-O
P-HP-FR
30
20
10
0
Figure 5. (a) H2O2 concentration dependence of normalized
fluorescence intensities after incubating 0.5 μM of (●) P-HP-
G, (○) P-HP-O, or (□) P-HP-FR with H2O2 (0-50 μM) for 30
min at 37 oC. (b) Fluorescence response of P-HP-G (green),
P-HP-O (orange), or P-HP-FR (red) toward various ROSs
and some physiological nucleophiles.
ACS Paragon Plus Environment

Page 5 of 20
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
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. (a) Time evolution of normalized emission intensities at 660 nm recorded for P-HP-FR-tpp (0.5 μM) in the absence
(◇) or presence (○) of H2O2 (100 μM). (b) Fluorescence response of P-HP-FR-tpp toward various ROSs and some physiologi-
cal nucleophiles. (c) Cell viability of HeLa cells in the presence of P-HP-FR-tpp with indicated concentrations determined by
MTT. Error bars represent mean ± SD (n = 5). (d-f) Confocal fluorescence imaging of endogenous H2O2 with B-TEG4-Me (as
control) or P-HP-FR-tpp in HeLa cells. Cells were treated with 0.2 μM Mito-Tracker Green (MTG) and 0.5 μM B-TEG4-Me (d)
or P-HP-FR-tpp (e,f) for 15 min, then incubating for 30 min at 37 C; note that cells were pretreated (d,e) without or (f) with
PMA (1 μg/mL) for 1 h. Scale bar: 20 μm. (g) Normalized probe (red) channel emission intensities quantified from CLSM im-
o
ages. (h) Scatter plot of red and green pixel intensities of Hela cells shown in the right panel of (f).
TEG4-Me incubated HeLa cells (Figure 7g). In addition,
the red-channel fluorescence showed excellent overlay
with green-channel fluorescence of Mito-Tracker Green
(MTG), with Pearson’s correlation coefficient at 0.86 ±
0.04 (evaluated by ImageJ software), indicating the
probes specifically localized in mitochondria of the HeLa
cells (Figure 7h).
abled us to prepare a H2S probe (P-HS-FR) containing
H2S-sensitive benzyl azide as triggerable motif14 (Figure
8a and supporting information), which is very stable in
PBS (Figure S11). Upon addition of 100 µM of NaHS (a
standard source of H2S), the fluorescence intensity of P-
HS-FR increased by up to nearly 227-fold before reach-
ing a plateau after 150 min incubation (Figure 9a). HPLC
analysis of the assay solution confirmed the full conver-
sion to B-COOH-FR in the presence of NaSH (Figure
9b). The high selectivity of P-HS-FR for H2S over other
biological species including glutathione (GSH) and cyste-
ine was also demonstrated (Figure 9c and S12). Fluo-
rescence titration experiments indicated an excellent
linear relationship with the concentrations of NaSH in 0-
Assessment of the Generality of the Proposed
Strategy. Having established that the fluorogenic mech-
anism can work in a set of BODIPYs, and demonstrated
the feasibility of P-HP-FR-tpp as mitochondria-targeting
fluorogenic bioprobe for endogenous H2O2 in living cells,
we then evaluated the generality of the proposed strate-
gy (Figure 1). The flexibility of the synthetic strategy en-
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 20
Finally, we expanded the design strategy for highly
50 µM with detection limit of 0.19 µM (Figure 9d and
S13).
1
2
3
4
5
6
7
8
fluorogenic probe sensing of protease that is known to
be upregulated in several diseases such as cancer,
rheumatoid arthritis, and neurodegenerative disorders.15
A water soluble far red probe, P-PGA-FR, for penicillin G
amidase (PGA; selective as a model protease) with phe-
nyl acetamide as triggerable motif16 was synthesized as
a paradigm (Figure 8b and supporting information). Hav-
ing demonstrated its stability in PBS (Figure S14a), we
then investigated the capability of P-PGA-FR in detection
of PGA activity. As shown in Figure 10 and S14b-d, upon
gradually increasing PGA levels (0-10 U/mL), fluores-
cence intensities at 660 nm of the decaged B-COOH-FR
increased more abruptly. Specifically, the enzyme-
induced conversion was finished within 60 min in the
presence of 10 U/mL PGA. Together, the above results
indicated that meso-carboxylate-substituted BODIPYs
could be modularly derivatized to generate variable high-
ly sensitive fluorogenic probes for biomolecules and en-
zymes by facilely replacing the trigger moiety of interest
(Figure 1 and Scheme S1).
a)
O
O
O
O
O
O
O
O
N3
H2S trigger
N
N
O
O
F
F
B
O
O
O
O
O
O
O
O
9
P-HS-FR
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
Ph
b)
O
O
O
O
PGA trigger
O
O
O
O
O
NH
N
F
O
B
F
N
O
O
O
O
O
O
O
O
O
P-PGA-FR
Figure 8. Chemical structures of (a) hydrogen sulfide probe,
P-HS-FR, and (b) PGA probe, P-PGA-FR.
Figure 9. Fluorescence emission spectra recorded for 0.5 µM solution of P-HS-FR upon incubating with NaSH (100 µM) for
varying time intervals (0−180 min) at 37 C; inset shows the time-dependent increases in normalized fluorescence intensities
at 660 nm. (b) HPLC chromatograms of P-HS-FR without treatment of NaSH (top); with NaSH (200 equivalent) treatment for
different time as indicated at 37 C (middle); and B-COOH-FR only (bottom). The samples were analyzed by HPLC using an
isocratic elution program with a solvent system of methanol/deionized water (95/5, V/V) at a flow rate of 1.0 mL/min with de-
tection wavelength at 656 nm. (c) Fluorescence response of P-HS-FR toward various biologically relevant species. (d) NaSH
concentration dependence of normalized emission intensities at 660 nm after incubating P-HS-FR (0.5 μM) with NaSH (0-50
μM) for 60 min at 37 oC.
o
o
ACS Paragon Plus Environment

Page 7 of 20
Journal of the American Chemical Society
oprobes in both emission wavelength and sensing ana-
lytes for biological research and clinical needs.
a)
1
2
3
4
5
6
7
8
P-PGA-FR
lem = 660 nm
1600
1200
800
400
0
■
ASSOCIATED CONTENT
120 min
0 min
Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
9
Experimental details, imaging methods, supplemen-
tary UV/vis and fluorescence spectra, HPLC chroma-
tograms, and NMR spectra (PDF)
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
650
700
750
800
Wavelength (nm)
■
AUTHOR INFORMATION
Corresponding Author
b) 400
*chli@nankai.edu.cn
Notes
The authors declare no competing financial interest.
300
200
100
0
■
ACKNOWLEDGMENT
Financial support from Nankai University, National Natural
Science Foundation of China (NSFC) projects (Grant no.
51673101 and 81601590), and Natural Science Foundation
of Tianjin of China (Grant no. 15JCZDJC65800) is gratefully
acknowledged.
[PGA]
■
REFERENCES
0
30
60
90
120
(1) (a) Germain, R. N.; Robey, E. A.; Cahalan, M. D. Science
2012, 336, 1676-1681. (b) Pittet, M. J.; Weissleder, R. Cell
2011, 147, 983-991.
(2) (a) Garland, M.; Yim, J. J.; Bogyo, M. Cell Chem. Biol.
2016, 23, 122-136. (b) Zhu, H.; Fan, J. L.; Du, J. J.; Peng, X. J.
Acc. Chem. Res. 2016, 49, 2115-2126. (c) Zhang, R. R.;
Schroeder, A. B.; Grudzinski, J. J.; Rosenthal, E. L.; Warram, J.
M.; Pinchuk, A. N.; Eliceiri, K. W.; Kuo, J. S.; Weichert, J. P.
Nat. Rev. Clin. Oncol. 2017, 14, 347-364.
(3) (a) Kowada, T.; Maeda, H.; Kikuchi, K. Chem. Soc. Rev.
2015, 44, 4953-4972. (b) Loudet, A.; Burgess, K. Chem. Rev.
2007, 107, 4891-4932. (c) Ulrich, G.; Ziessel, R.; Harriman, A.
Angew. Chem., Int. Ed. 2008, 47, 1184-1201. (d) Boens, N.;
Leen, V.; Dehaen, W. Chem. Soc. Rev. 2012, 41, 1130-1172.
(e) Grossi, M.; Morgunova, M.; Cheung, S.; Scholz, D.; Conroy,
E.; Terrile, M.; Panarella, A.; Simpson, J. C.; Gallagher, W. M.;
O'Shea, D. F. Nat. Commun. 2016, 7, 10855.
(4) (a) Belzile, M. N.; Godin, R.; Durantini, A. M.; Cosa, G. J.
Am. Chem. Soc. 2016, 138, 16388-16397. (b) Shie, J. J.; Liu,
Y. C.; Lee, Y. M.; Lim, C.; Fang, J. M.; Wong, C. H. J. Am.
Chem. Soc. 2014, 136, 9953-9961. (c) Krumova, K.; Greene, L.
E.; Cosa, G. J. Am. Chem. Soc. 2013, 135, 17135-17143.
(5) (a) Liu, H. W.; Zhang, X. B.; Zhang, J.; Wang, Q. Q.; Hu,
X. X.; Wang, P.; Tan, W. H. Anal. Chem. 2015, 87, 8896-8903.
(b) Zhao, C. C.; Zhang, X. L.; Li, K. B.; Zhu, S. J.; Guo, Z. Q.;
Zhang, L. L.; Wang, F. Y.; Fei, Q.; Luo, S. H.; Shi, P.; Tian, H.;
Zhu, W. H. J. Am. Chem. Soc. 2015, 137, 8490-8498.
Time (min)
Figure 10. (a) Fluorescence emission spectra recorded for
0.5 µM P-PGA-FR in PBS (10 mM, pH 7.4) after the addi-
tion of 5 U/mL PGA for varying time intervals (0−120 min) at
o
37 C. (b) Time-dependent increases in normalized emis-
sion intensity at 660 nm recorded for P-PGA-FR (0.5 μM)
under varying concentrations of PGA (from bottom to top: 0,
1, 2, 5, 10 U/mL).
■
CONCLUSION
In conclusion, with the aim of developing a general
strategy toward highly fluorogenic bioprobes across the
visible spectrum for diverse analytes, we designed and
successfully synthesized a set of BODIPY-based bi-
oprobes with the meso-carboxyl moieties caged by stim-
uli-responsive self-immolative linker via an ester bond.
As proof-of-concept examples, we established that the
meso-ester moieties can be converted to meso-
carboxylate upon stimuli (H2O2, H2S, PGA), and the con-
version induces a significant reduction in the electron-
withdrawing capabilities of the meso-substituents of
BODIPYs, leading to the dramatic enhancement of fluo-
rescence emission. Moreover, the fluorogenic mecha-
nism has been successfully applied in various BODIPYs
emitting throughout the visible spectrum (509, 585, 660
nm). Further application to the visualization of endoge-
nous H2O2 in mitochondria was also validated for P-HP-
FR-tpp in living cells. Given the generality of the highly
fluorogenic mechanism and the versatility of the synthet-
ic strategy, we envisage that the concept disclosed here-
in could be exploited for customization of diverse bi-
(6) (a) Kim, S.; Kim, H.; Choi, Y.; Kim, Y. Chem. Eur. J.
2015, 21, 9645-9649. (b) Kim, S.; Bouffard, J.; Kim, Y. Chem.
Eur. J. 2015, 21, 17459-17465.
(7) (a) Qiu, N. S.; Liu, X. R.; Zhong, Y.; Zhou, Z. X.; Piao, Y.;
Miao, L.; Zhang, Q. Z.; Tang, J. B.; Huang, L.; Shen, Y. Q. Adv.
Mater. 2016, 28, 10613-10622. (b) Song, C. C.; Ji, R.; Du, F.
S.; Liang, D. H.; Li, Z. C. ACS Macro Lett. 2013, 2, 273-277.
(8) (a) Ni, Y.; Zeng, L. T.; Kang, N. Y.; Huang, K. W.; Wang,
L.; Zeng, Z. B.; Chang, Y. T.; Wu, J. S. Chem. Eur. J. 2014, 20,
2301-2310. (b) Palao, E.; Slanina, T.; Muchova, L.; Solomek,
T.; Vitek, L.; Klan, P. J. Am. Chem. Soc. 2016, 138, 126-133.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 20
(9) (a) Lippert, A. R.; De Bittner, G. C. V.; Chang, C. J. Acc.
Imanishi, H.; Nakada, K.; Honma, Y.; Hayashi, J. I. Science
2008, 320, 661-664.
(12) Lv, W.; Zhang, Z.; Zhang, K. Y.; Yang, H. R.; Liu, S. J.;
Xu, A. Q.; Guo, S.; Zhao, Q.; Huang, W. Angew. Chem., Int.
Ed. 2016, 55, 9947-9951.
(13) (a) Hanna, R. D.; Naro, Y.; Deiters, A.; Floreancig, P. E.
J. Am. Chem. Soc. 2016, 138, 13353-13360. (b) Kim, E. J.;
Bhuniya, S.; Lee, H.; Kim, H. M.; Cheong, C.; Maiti, S.; Hong,
K. S.; Kim, J. S. J. Am. Chem. Soc. 2014, 136, 13888-13894.
(14) Henthorn, H. A.; Pluth, M. D. J. Am. Chem. Soc. 2015,
137, 15330-15336.
Chem. Res. 2011, 44, 793-804. (b) Karton-Lifshin, N.; Albertaz-
zi, L.; Bendikov, M.; Baran, P. S.; Shabat, D. J. Am. Chem.
Soc. 2012, 134, 20412-20420. (c) Karton-Lifshin, N.; Segal, E.;
Omer, L.; Portnoy, M.; Satchi-Fainaro, R.; Shabat, D. J. Am.
Chem. Soc. 2011, 133, 10960-10965. (d) Deng, Z. Y.; Qian, Y.
F.; Yu, Y. Q.; Liu, G. H.; Hu, J. M.; Zhang, G. Y.; Liu, S. Y. J.
Am. Chem. Soc. 2016, 138, 10452-10466.
(10) (a) Wu, X. F.; Li, L. H.; Shi, W.; Gong, Q. Y.; Ma, H. M.
Angew. Chem., Int. Ed. 2016, 55, 14728-14732. (b) Alouane,
A.; Labruere, R.; Le Saux, T.; Schmidt, F.; Jullien, L. Angew.
Chem., Int. Ed. 2015, 54, 7492-7509. (c) Roth, M. E.; Green,
O.; Gnaim, S.; Shabat, D. Chem. Rev. 2016, 116, 1309-1352.
(11) (a) Finkel, T.; Serrano, M.; Blasco, M. A. Nature 2007,
448, 767-774. (b) FRuehauf, J. P.; Meyskens, F. L. Clin. Can-
cer. Res. 2007, 13, 789-794. (c) Houstis, N.; Rosen, E. D.;
Lander, E. S. Nature 2006, 440, 944-948. (d) Ishikawa, K.;
Takenaga, K.; Akimoto, M.; Koshikawa, N.; Yamaguchi, A.;
1
2
3
4
5
6
7
8
9
(15) Lopez-Otin, C.; Bond, J. S. J. Biol. Chem. 2008, 283,
30433-30437.
(16) Sitnikov, N. S.; Li, Y.; Zhang, D.; Yard, B.; Schmalz, H.-
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
G. Angew. Chem., Int. Ed. 2015, 54, 12314-12318.
ACS Paragon Plus Environment

Page 9 of 20
Journal of the American Chemical Society
1
2
3
4
5
6
7
trigger
O-/NH2
509 nm
green
O
N
O
N
Triggering
Analyte
Self-
Immolation
O
N
O
N
O
N
O
N
B
585 nm
orange
F
F
R1
R2
B
B
R1
R2
R1
R2
F
F
-
F
F
BODIPY-COO
highly
8
9
Bioprobe
[intermidiate]
nonfluorescent
H2O2 (ROS)
fluorescent
660 nm
far red
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
H2S (signaling molecule)
PGA (protease)
enzymes
small biomolecules
9
ACS Paragon Plus Environment

O-/NH2
Journaltroigfgtehre American Chemical Society
Page 10 of 20
509 nm
green
O
O
Triggering
Analyte
Self-
Immolation
O
N
O
N
O
N
O
N
1
2
3
4
5
6
7
8
9
10
N
N
B
585 nm
orange
F
F
R1
R2
B
B
R1
R2
R1
R2
F
F
-
F
F
BODIPY-COO
highly
Bioprobe
[intermidiate]
nonfluorescent
H2O2 (ROS)
fluorescent (3)
660 nm
far red
H2S (signaling molecule)
PGA (protease)
(1)
(2)
11
12
13
14
small biomolecules
enzymes
(1) Across the visible spectrum (509 nm, 585 nm, 660 nm)
15
16
ACS Paragon Plus Environment
(2) Versatile synthetic strategy(for small biomolecules& enzymes)
17
18
(3) Highly fluorogenic (133 ~ 320-fold enhancement)

a)
Page 11 of 20 Journal of the American Chemical Society
R
R
O
N
N
N
N
O
N
N
1O
2
F
F
F
F
F
F
B
O(CH2CH2O)3CH3
O(CH2CH2O)3CH3
B
B
HO
HO
HO
R =
3
4
R
B-COOH-G
B-COOH-O
B-COOH-FR
5
6
7
b)
8
9
O
O
O
10
O
O
O
B
B
B
R
R
11
12
13
14
15
16
17
O
O
N
F
O
N
N
O
O
N
N
F
F
F
B
B
B
F
F
N
O
ACS Paragon Plus Environment
R
P-HP-G
P-HP-O
P-HP-FR

a)
b)
Journal of the American Chemical Society
Page 12 of 20
210
0
300
4000
6000
4000
2000
0
1
2
3
4
5
6
7
8
0
0
30
60
0
30
60
Time (min)
Time (min)
2000
0
P-HP-G
lem = 509 nm
P-HP-O
lem = 585 nm
500
540
580
620
570
620
670
720
9
10
11
12
Wavelength (nm)
Wavelength (nm)
13
c)
d)
14
15
16
17
18
19
20
21
22
23
24
25
26
120
0
1000
500
0
0
30
60
Time (min)
P-HP-FR
lem = 660 nm
ACS Paragon Plus Environment
640
690
740
790
Wavelength (nm)
P-HP-G
P-HP-O P-HP-FR

a)
Journalbof)the American Chemical Societyc)
P-HP-O
Page 13 of 20
P-HP-G
P-HP-FR
0 min
0.06
0 min
0 min
0.04
0.03
0.02
0.01
0.06
1
2
3
4
5
0.04
0.04
90 min
120 min
150 min
0.02
0.02
0
6
7
0
0
8
430
490
550
480
570
660
550
650
750
9
Wavelength (nm)
Wavelength (nm)
Wavelength (nm)
10
11
12
d)
e)
f)
13
P-HP-O
10 min
30 min
60 min
90 min
120 min
14
15
16
17
18
19
20
21
22
23
24
25
P-HP-G
P-HP-FR
10 min
30 min
60 min
90 min
60 min
120 min
150 min
B-COOH-G
B-COOH-O
B-COOH-FR
ACS Paragon Plus Environment
0
2
4
6
8
10
0
2
4
6
8
10
0
2
4
6
8
10
Time (min)
Time (min)
Time (min)

Joaur)nal of the American ChPeamgieca1l4Soofc2i0ety
P-HP-G
P-HP-O
P-HP-FR
30
20
10
0
1
2
3
4
5
6
7
8
R2 = 0.99986 (G)
R2 = 0.99987 (O)
R2 = 0.99962 (FR)
0
10
20
30
40
50
9
10
11
12
[H2O2] (µM)
b)
13
P-HP-G
P-HP-O
P-HP-FR
14
15
16
17
18
19
20
21
22
23
24
25
26
30
20
10
0
ACS Paragon Plus Environment

O
O
Page 15 of 20 Journal of the American Chemical Society
O
mitochondria-
targeting moiety
B
PPh3Br
O
N
N N
O
O
N
N
F
F
1
2
3
4
5
6
7
B
O
O
O
O
O
O
ACS Paragon Plus Environment
O
O
P-HP-FR-tpp

c)
a)
b)
Journal of the American Chemical Society
Page 16 of 20
w/o H2O2
150
75
0
100 µM
50
100
50
0
1
2
3
4
5
6
7
8
9
10
25
0
P-HP-FR-tpp
lem = 660 nm
0
30
60
90
0
2.5
5
10 15 20
[Probe] (µM)
Time (min)
g)
Merge
DIC
MTG
Probe
B-TEG4-Me
w/o PMA
d)
B-TEG4-Me, w/o PMA
P-HP-FR-tpp, w/o PMA
P-HP-FR-tpp, w/ PMA
11
12
13
14
15
16
17
15
10
5
P-HP-FR-tpp
w/o PMA
e)
18
0
19
20
21
22
23
24
h)
P-HP-FR-tpp
w/ PMA
f )
25
26
27
28
29
30
31
ACS Paragon Plus Environment
Probe Channel

Paa)ge 17 of 20Journal of tOhe American Chemical Society
O
O
O
O
O
N3
H2S trigger
O
O
N
N
O
O
F
F
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
B
O
O
O
O
O
O
O
O
P-HS-FR
Ph
b)
O
O
O
O
PGA trigger
O
O
O
O
O
NH
N
N
O
F
F
B
O
O
O
O
O
O
O
ACS Paragon Plus Environment
O
O
P-PGA-FR

a)
b)
Jo2u4r0nal of the American Chemical Society
Page 18 of 20
P-HS-FR
1500
1000
500
0
20 min
1
2
3
4
5
6
0
60 min
0
60 120 180
Time (min)
120 min
180 min
P-HS-FR
lem = 660 nm
7
B-COOH-FR
8
9
0
2
4
6
8
10
650
700
750
800
10
11
12
Wavelength (nm)
Time (min)
13
c)
d)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
60
P-HS-FR, lem = 660 nm
R2 = 0.9996
1500
1000
500
0
40
20
0
ACS Paragon Plus Environment
0
10
20
30
40
50
[NaSH] (µM)

a)
JPoaugrena1l9ooffth20e American Chemical Society
P-PGA-FR
lem = 660 nm
1600
1200
800
400
0
1
2
3
4
5
6
7
8
120 min
0 min
9
650
700
750
800
10
11
12
13
Wavelength (nm)
b) 400
14
15
16
17
18
19
20
21
22
23
24
25
26
27
300
200
100
0
[PGA]
ACS Paragon Plus Environment
0
30
60
90
120
Time (min)

O-/NH2
trigger
Journal of the American Chemical Society Page 20 of 20
509 nm
green
O
O
Triggering
Analyte
Self-
Immolation
O
N
O
N
O
N
O
N
1
2
3
4
5
6
7
8
9
10
11
12
13
N
N
B
585 nm
orange
F
F
R1
R2
B
B
R1
R2
-
R1
R2
F
F
F
F
BODIPY-COO
highly
Bioprobe
nonfluorescent
[intermidiate]
fluorescent
660 nm
far red
H2O2 (ROS)
H2S (signaling molecule)
ACS Paragon Plus Environment
PGA (protease)
enzymes
small biomolecules