Dual-site lysosome-targeted fluorescent probe for separate detection of endogenous biothiols and SO2 in living cells

Article abstract of DOI:10.1039/c8tb01152d

Biothiols and SO₂ play crucial roles in many physiological and pathological processes. To unravel their complicated interrelationship and cellular cross-talk, it would be highly desirable to develop single-molecule fluorescent probes that can selectively detect biothiols and SO₂via different emission channels. Here, a novel chromenylium derivative, BPO-Py-diNO₂, based on the rational design of dual recognition sites for biothiols and SO₂ selectively and sensitively responded to biothiols with near-infrared fluorescence, and to SO₂ with green fluorescence. The emission shift for the two channels was 170 nm. BPO-Py-diNO₂ was selectively enriched in lysosomes. It could also be used to evaluate dual-channel imaging of endogenous biothiols and SO₂ in living HeLa cells, and it could be used for monitoring the mutual interconversion of biothiols and SO₂.

Full text of DOI:10.1039/c8tb01152d

View Article Online
View Journal
Journal of
Materials Chemistry B
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: X. Yu, M. Wu, Y.
Wang and Y. Liu, J. Mater. Chem. B, 2018, DOI: 10.1039/C8TB01152D.
This is an Accepted Manuscript, which has been through the
Volume
4 Number 1 7 January 2016 Pages 1–178
Royal Society of Chemistry peer review process and has been
accepted for publication.
Journal of
Materials Chemistry B
Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Please do not adjust margins
Journal of Materials Chemistry B
Page 1 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
Dual-Site Lysosome-Targeted Fluorescent Probe for Separate
Detection of Endogenous Biothiols and SO2 in Living Cells
MingꢀYu Wu*a, Yue Wanga, YanꢀHong Liu b, XiaoꢀQi Yu*b
Received 00th January 20xx,
Accepted 00th January 20xx
Biothiols and SO2 play crucial roles in many physiological and pathological processes. To unravel their complicated
interrelationship and cellular crossꢀtalk, it would be highly desirable to develop singleꢀmolecule fluorescent probes that can
DOI: 10.1039/x0xx00000x
www.rsc.org/
selectively detect biothiols and SO2 via different emission channels. Here,
a novel chromenylium derivative
BPO-Py-diNO2 based on the rational design of dual recognition sites for biothiols and SO2 selectively and sensitively
responded to biothiols with nearꢀinfrared fluorescence, and to SO2 with green fluorescence. The emission shift for the two
channels was 170 nm. BPO-Py-diNO2 was selectively enriched in lysosomes. It could also be used to evaluate dualꢀchannel
imaging of endogenous biothiols and SO2 in living HeLa cells, and be used for monitoring the mutual interconversion of
biothiols and SO2.
Yin developed the first coumarinꢀbased, dualꢀsite fluorescent probe
for imaging the metabolism of Cys to SO2 in living cells.8a
However, these probes had drawbacks such as: (1) short
excitation and emission wavelengths, (2) limited shifts in
emission wavelengths for the different channels that resulted in
significant spectral overlaps in fluorescence imaging.
Introduction
In physiological and pathological processes, reactive sulfur species
(RSS) such as biothiols and SO2 are critical components.1 The
biothiols including cysteine (Cys), homocysteine (Hcy), and
glutathione (GSH) are small sulfhydrylꢀcontaining amino acids
which are essential molecular of RSS2 and can regulate the
oxidative stress, control signal transduction, chelate with metal
The lysosome is an essential organelle responsible for the
degradation and recycling of macromolecules.11 RSS are closely
associated with proteolysis in the lysosome.12 For example, GSH
stabilizes lysosome membranes, and Cys stimulates albumin
degradation in liver lysosomes.13 Therefore, fluorescent probes that
respond to biothiols and SO2 within the lysosome are of particular
ions and so forth.3 As
a
signal transmitter,4 SO2 exhibits
bioactivities especially in the regulation of cardiovascular
function in synergy with NO and lowering of blood pressure.5
Abnormal SO2 concentrations are associated with symptoms of
neurological disorders, cardiovascular diseases, and lung cancer.6
Although SO2 can be generated endogenously from biothiols
via biosynthetic pathways,7 it would be highly valuable to
better understand their relationship and cellular crossꢀtalk.
Therefore, the establishment of convenient detection methods for
both biothiols and SO2 are desirable.8 Fluorescence probes exhibit
high selectivity and sensitivity for nonꢀinvasive imaging of
biological molecules, and exhibit high spatial and temporal
resolution in realꢀtime.9 However, the development of
singleꢀmolecule fluorescent probe bearing dual recognition sites with
distinct responses and different emission channels for biothiols and
SO2 has been challenging.
Recently, a carbazoleꢀbased dualꢀsite fluorescent probe was
reported for selective detection of biothiols and SO2 in live cells.10
interest.14 To this end,
a lysosomeꢀtargeted singleꢀmolecule
fluorescence probe for detecting both biothiols and SO2 with
relatively long emission wavelengths and a large wavelength
shift was developed.
Results and discussion
Rational design and synthesis of probes
a
The design strategy for the probes was as depicted in Scheme 1.
Chromenylium is a fluorophore that exhibits large Stokes shifts,
substantial quantum yields, and photostability under
physiological conditions.9a,15 It contains a carbonꢀcarbon double
bond linked to oxonium that provides a latent nucleophilic
addition site for SO2. When the conjugation is broken, green
fluorescence is emitted. At the same time, when the phenolic
hydroxyl group of chromenylium is protected by pyridine
derivative with strong electronꢀwithdrawing groups, its
fluorescence is quenched. However, the protecting group can be
cleaved by thiols to allow nearꢀinfrared fluorescence.16
Therefore,
the
chromenylium
was
modified
with
nitroꢀsubstituted pyridine for the synthesis of three different
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 2 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
probes BPO-Py-diNO2
,
BPO-Py-3-NO2, and BPO-Py-5-NO2
,
reaction with 500
ꢀ
M sulfite, the absorption peaks shifted
to respond to biothiols and SO2 with distinct fluorescent signals.
towards shorter wavelength (Fig. 1a), and the emission intensity
at 495 nm increased significantly (Fig. 1b). This confirmed that the
conjugated system was broken by SO2. Meanwhile, the colour
changed from purple to pale yellow under visible light and
green fluorescence increased under 365ꢀnm excitation from an
ultraꢀviolet (UV) lamp (Fig. 1a). In the same system, after
treatment with 500
ꢀM GSH, the absorption peak of
BPO-Py-diNO2 redꢀshifted from 555 nm to 566 nm, and the
absorption intensity increased (Fig. 1a). The emission intensity
at 665 nm was also enhanced (Fig. S1). In the meantime, the
colour of the solution changed to dark purple (Fig. 1a).
Therefore, BPO-Py-diNO2 could detect SO2 and biothiols via
two different emission channels, and exhibited two advantages
for bioimaging over other reported probes8a, 10: (1) nearꢀinfrared
detection for biothiols, (2) a 170 nm shift in fluorescence
emission from the two different channels (Fig. 1b).
Scheme 1 Design and structures of probes for biothiols and SO2.
The synthesis route for the target molecules is illustrated in
Scheme 2. It required four steps as follows. BPOH was
synthesized as reported previously.17 Briefly, FriedelꢀCrafts
acylation reaction of 3ꢀ(diethylamino)phenol with phthalic
anhydride yielded compound
cyclohexanone in a twoꢀstep cascade reaction to yield oxonium
Then, the condensation reaction between
4ꢀhydroxybenzaldehyde with compound yielded BPOH
Finally, BPO-Py-diNO2 BPO-Py-3-NO2, and BPO-Py-5-NO2
3
,
which reacted with
5.
Fig. 1 (a) Absorption spectra of BPO-Py-diNO2 (10 ꢁM)
before and after interacting with 500 ꢁM SO2 and GSH. Insert:
corresponding colour and fluorescence changes under visible
light and a 365 nm UV lamp. (b) Normalized fluorescence
spectra of BPO-Py-diNO2 before and after interacting with SO2
or GSH at different emission channels. Excitation wavelength
for green channel (λex) = 390 nm. Slit: 5 nm/5 nm. Excitation
wavelength for nearꢀinfrared channel (λex) = 556 nm. Slit: 10
nm/10 nm.
5
.
,
were obtained by a nucleophilic substitution reaction of BPOH
with 2ꢀchloropyridine derivatives. The probes were
1
characterized by H NMR, 13C NMR, and high resolution mass
spectrometry (see supporting information).
BPO-Py-3-NO2 and BPO-Py-5-NO2 exhibited similar
absorption and emission spectral changes when reacted with
SO2, but not for biothiols (Figs. S2 and S3). As shown in Figs.
S4 and S5, there were almost no fluorescence changes when
biothiols
were
added.
Thus,
BPO-Py-3-NO2
and
BPO-Py-5-NO2 could only detect SO2 efficiently. From their
structures, it can be inferred that sufficiently strong
electronꢀwithdrawing groups must be present for biothiols to
break the ether bond.
Fluorescent responses of probes to SO2
As shown in Figs. S6–S8, the 495ꢀnm fluorescent intensity of
all three probes changed little when the pH was increased from
Scheme 2 Synthesis route of fluorescent probes.
4.0 to 10.0. However, upon addition of 500
ꢀM Na2SO3, the
BPO-Py-diNO2 fluorescent intensity increased when the pH
increased from 4.0 to 8.0. When the pH was increased to 10.0,
the fluorescent intensity decreased slightly. In contrast, the
fluorescent intensity of BPO-Py-3-NO2 and BPO-Py-5-NO2
Optical response to biothiols and SO2
An absorption maximum was observed at 555 nm for
BPO-Py-diNO2 in a 10ꢀmmol phosphate buffered saline (PBS)
solution containing 20% dimethylsulfoxide (DMSO). After
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 2
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 3 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
increased when the pH was increased from 4.0 to 7.0; further Fluorescent responses of BPO-Py-diNO2 to biothiols
increases to 10.0 decreased the fluorescent intensity. Therefore,
pH 8.0 was optimal for BPO-Py-diNO2 in the following
experiments, while pH 7.0 was optimal for BPO-Py-3-NO2 and
The addition of GSH to BPO-Py-diNO2 in PBS/DMSO (8/2, v/v,
pH 8.0) induced significant fluorescent emission centred at 665 nm
with excitation at 556 nm. As shown in Fig. 3a, the 665ꢀnm
BPO-Py-5-NO2
In Fig. 2a and 2b, the emission intensity at 495 nm increased
gradually with the addition of Na2SO3. For BPO-Py-diNO2
600 M Na2SO3 led to an intensity enhancement factor of 12.6,
.
fluorescence intensity increased with GSH concentration over
the range 0–100 M. The detection limit for GSH was 202 nM
ꢀ
,
(Fig. 3b). The selectivity of BPO-Py-diNO2 for reactive oxygen
ꢀ
ꢀ
species (NaClO, H2O2, TBHP), reactive nitrogen species (NO2
and NO3 ), reactive sulfur species (SO42ꢀ, SO32ꢀ, H2S, Cys, HCy,
with quantum yields increasing from 0.003 to 0.130 (Table S1).
The linear detection range was 20ꢀ200 ꢀM (Fig. 2b), with a 150 nM
detection limit. For BPO-Py-3-NO2, 200 ꢀM Na2SO3 increased
the fluorescent intensity by a factor of 47, with a 60 nM
detection limit (Figs. S9 and S10).
ꢀ
and GSH), and amino acids were investigated in the nearꢀinfrared
wavelength region. Fluorescence enhancements were observed only
for biothiols (Fig. 3c), which induced the only colour change from
purple to dark purple (Fig. 3d). Thus, BPO-Py-diNO2 exhibited
excellent selectivity for biothiols at 665 nm.
Fig. 2 Fluorescence responses of BPO-Py-diNO2 to SO2. (a)
Fluorescence spectra of BPO-Py-diNO2 (10 ꢁM) after addition
of various concentrations of Na2SO3 (0ꢀ600 ꢁM). (b) Linear fit
of BPO-Py-diNO2 fluorescence intensity changes with Na2SO3
at 495 nm. (c) and (d) BPO-Py-diNO2 (10 ꢁM) fluorescence
spectrum and intensity changes at 495 nm after interacting with
500 ꢁM amino acids, reactive oxygen species, reactive nitrogen
species, and reactive sulfur species. Excitation wavelength (λex)
= 390 nm. Slit: 5 nm/5 nm. 1. BPO-Py-diNO2, 2. Ala, 3. Arg.
4. Asp, 5. Glu, 6. Gly, 7. His, 8. Ile, 9. Leu, 10. Lys, 11. Met,
12. Phe, 13. Pro, 14, Ser, 15. Val, 16. H2O2, 17, NaClO, 18.
Fig. 3 Fluorescent responses of BPO-Py-diNO2 to GSH. (a)
Fluorescence spectra of BPO-Py-diNO2 (10 ꢁM) after addition
of various concentrations of GSH (0ꢀ100 ꢁM). (b) Linear fit of
fluorescence intensity changes of BPO-Py-diNO2 with GSH at
665 nm. (c) Fluorescence spectrum changes of BPO-Py-diNO2
(10 ꢁM) interacting with 500 ꢁM amino acids, reactive oxygen
species, reactive nitrogen species, and reactive sulfur species.
(d) Colour and fluorescence changes of BPO-Py-diNO2 (10
ꢀ
ꢀ
TBHP, 19. NO3 , 20. NO2 , 21. SO42ꢀ, 22. Cys, 23. Hcy, 24.
ꢀ
M) upon addition of 500 ꢁM of different species under visible
light and 365ꢀnm excitation. Excitation wavelength (λex) = 556
nm. Slit: 10 nm/10 nm. 1. BPO-Py-diNO2, 2. Ala, 3. Arg. 4.
Asp, 5. Glu, 6. Gly, 7. His, 8. Ile, 9. Leu, 10. Lys, 11. Met, 12.
Phe, 13. Pro, 14, Ser, 15. Val, 16. H2O2, 17, NaClO, 18. TBHP,
GSH, 25. H2S, 26. SO2.
Fig. 2c and 2d revealed that sulfite led to large fluorescence
spectral and intensity changes. However, reactive oxygen
ꢀ
ꢀ
19. NO3 , 20. NO2 , 21. SO42ꢀ, 22. Cys, 23. Hcy, 24. GSH, 25.
ꢀ
species (NaClO, H2O2, TBHP), reactive nitrogen species (NO2
H2S, 26. SO2.
ꢀ
and NO3 ), other reactive sulfur species (SO42ꢀ, H2S, Cys, HCy,
and GSH), and amino acids produced negligible changes,
except for the slight fluorescence intensity increase induced by
H2S. Fig. 3d revealed colour changes under visible and 365 nm
light before and after the addition of various compounds. Only
Sensing mechanisms
Compound 5 (Scheme 2) had similar fluorescence spectra with
probes interacting with SO2 (Fig. S15), which indicated that it had a
similar conjugated structure to the reaction products. Then1H NMR
experiments following the addition of Na2SO3 to the
BPO-Py-5-NO2 in DMSOꢀd6/D2O (V/V = 3:2) displayed that the
SO2 induced
a strong green fluorescence enhancement.
BPO-Py-3-NO2 and BPO-Py-5-NO2 exhibited much better
selectivity for SO2 relative to BPO-Py-diNO2 (Figs. S11–S14).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 4 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
original carbon carbon double bond as singlet at 8.31 ppm shifted contrast. The fluorescence image of BPO-Py-diNO2 in the red
upfield to 5.38 ppm as doublet, which could be attributed to the channel (Fig. 5a) overlapped well with the LyT in green channel
2−
addition of SO3
to the carbon carbon double bond of (Fig. 5b, Pearson's correlation coefficient of 0.91). However, poor
BPO-Py-5-NO2 (Fig. S16). The transformation from overlap was observed between BPO-Py-diNO2 (Fig. 5f) and MT
BPO-Py-diNO2 to BPOH was also confirmed by photophysical (Fig. 5g, Pearson's correlation coefficient of 0.66). These results
spectral changes, as reported previously 18
. These results verified that indicated that BPO-Py-diNO2 could specifically target lysosomes
BPO-Py-diNO2 detected SO2 and biothiols via two reaction sites.
in living HeLa cells.
To examine fluorescence imaging of biothiols and SO2 in
living HeLa cells (Figs. 6, S19 and 20), the cells were incubated
with BPO-Py-diNO2. After 30 min, there was no fluorescence
in the green channel (Fig. 6a), but strong fluorescence in the red
channel (Fig. 6b). When the HeLa cells were preꢀtreated with
Nꢀethylmaleimide (NEM, an intracellular thiol scavenger) for
Cytotoxicity and fluorescence imaging in living cells
Cytotoxicity was examined with a standard cell counting kitꢀ8
(
CCKꢀ8) assay with HeLa cells at different concentrations (Figs. 4,
S17 and S18). BPO-Py-diNO2 exhibited favourable
biocompatibility relative to two other probes. In Fig. 4, 70% of
the cells survived when incubated in 10
for 24 h.
ꢀM BPO-Py-diNO2
30 min and further incubated with BPO-Py-diNO2
, a
significant fluorescence decrease in the red channel was
observed (Fig. 6f), indicating that BPO-Py-diNO2 was
detecting intracellular biothiols. At the same time, there was a
fluorescence increase in the green channel (Fig. 6e) relative to
Fig. 6a, which indicated that endogenous SO2 was produced
when the biothiols were consumed by NEM. In addition, when
the HeLa cells were preꢀtreated with NEM for 30 min, and
further incubated with BPO-Py-diNO2 and Na2SO3
successively, fluorescence increases were observed both in the
green channel (Fig. 6i) and in the red channel (Fig. 6j). This
indicated potential conversion from SO2 to biothiols in living
cells. Therefore, the results indicated that BPO-Py-diNO2 was
cytomembraneꢀpermeable and could be used to image
endogenous thiols and SO2 from two different emission
channels. Moreover, as a singleꢀmolecule fluorescent probe,
BPO-Py-diNO2 could be applied to monitor the mutual
interconversions of SO2 and biothiols from each other in living
cells.
Fig. 4 Cell viability of BPO-Py-diNO2 in a standard CCKꢀ8
assay in living Hela cells for 24 h. The experiment was repeated
three times (±S.D.).
Fig. 5 Coꢀlocalization of BPO-Py-diNO2 with LyT (aꢀe) and MT
(fꢀj). (a) Image of BPO-Py-diNO2
image of LyT ( ) in the green channel, (c) bright field image (d)
merged red, green, and bright field, (e) coꢀlocalization of a and b. (f)
Image of BPO-Py-diNO2 10 ) in the red channel, (g) image of
MT ( ) in the green channel, (h) bright field image (i) merged
(10 ꢀM) in the red channel, (b)
1 ꢀM
(
ꢀM
1 ꢀM
red, green, and bright field, (j) coꢀlocalization of f and g. For the
green channel: λex = 488 nm, emission wavelength λem = 510ꢀ600
nm. For the red channel: λex = 560 nm, λem = 620ꢀ720 nm.
Fig. 6 Confocal images of biothiols and SO2 in living HeLa cells.
(aꢀd) HeLa cells incubated with 10 ꢁM BPO-Py-diNO2; (eꢀh) HeLa
cells preꢀtreated with 50 ꢁM NEM and then incubated with 10 ꢁM
BPO-Py-diNO2; (iꢀl) HeLa preꢀtreated with 50 ꢁM NEM and then
incubated with 10 ꢁM BPO-Py-diNO2 and Na2SO3 (500 ꢁM)
successively. For the green channel: λex = 405 nm, λex = 460−530
nm. For the red channel: λex = 560 nm, λem = 620−720 nm.
In coꢀlocalization experiments, lysosomeꢀtargeting LyT or
mitochondrialꢀtargeting MT green fluorescent dyes were used for
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 4
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 5 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
dichloromethane (DCM) to DCM/methanol(MeOH) (10/1,
Conclusions
V/V). About 9.4 g of purple solid compound
(62% yield).
5
was obtained
In summary, three chromenyliumꢀbased probes for sensing
biothiols and SO2 via two separate emission channels were
synthesised and evaluated. BPO-Py-3-NO2 and BPO-Py-5-NO2
exhibited high selectivity and sensitivity only for SO2, with a
Compound
5
(1.72
g,
3.60
mmol) and
4ꢀhydroxybenzaldehyde (0.53 g, 4.32 mmol) were mixed in 40
o
ml AcOH, and heated to 90 C overnight under nitrogen. The
detection limit as low as 66 nM. In contrast, BPO-Py-diNO2
,
solvent was then removed under reduced pressure in a rotary
evaporator. The crude product was dissolved in 50 ml of
dichloromethane, washed with water (50 ml) three times, and
dried with sodium sulfate. The crude product was concentrated
and purified via chromatography on a silicaꢀgel column with
DCM to DCM/MeOH (10/1, V/V) to get 1.35 g BPOH as a
dark purple solid (65% yield).
could detect with high selectivity both biothiols and SO2 from the
two different channels separated by a 170 nm emission wavelength
shift. Preliminary biological experiments indicated that
BPO-Py-diNO2 targeted lysosomes, and could be used to image
endogenous biothiols and SO2 in living HeLa cells. Furthermore, it
could be used for monitoring the mutual interconversion of
biothiols and SO2, which could provide deeper insight into those
physiological processes. This strategy could also provide a better
understanding of pathological aspects of biothiols and SO2.
About 174.0 mg (0.3 mmol) BPOH was dissolved in 10 ml
dimethyl formamide. Then 3.0 mmol 2ꢀdichloropyridine
derivatives and 207 mg (1.5 mmol) K2CO3 were added. After
o
reacting for 3–5 h at 25ꢀ50 C, the solvent was removed under
reduced pressure in a rotary evaporator. The crude product was
purified via silicaꢀgel column chromatography with DCM to
DCM/MeOH (20/1, V/V) to get a dark purple solid.
Experimental
Materials and general instruments
BPO-Py-diNO2: 71.6 mg (32% yields). 1H NMR (400 MHz,
CDCl3), 9.28 (d, 1H, J = 2.4 Hz), 9.21 (d, 1H, J = 2.4 Hz), 9.02ꢀ9.00
(m, 3H), 8.25 (d, 1H, J = 6.4 Hz), 8.10 (s, 1H), 7.75ꢀ7.08 (m, 2H),
7.39 (d, 2H, J = 8.4 Hz), 7.24 (d, 1H, J = 7.6 Hz), 7.13 (d, 2H, J =
6.8 Hz), 3.37 (q, 4H, J = 6.8 Hz), 3.00 (br, 2H), 2.52ꢀ2.38 (m, 2H),
1.88ꢀ1.85 (m, 2H), 1.35 (t, 4H, J = 6.8 Hz). 13C NMR (125 MHz,
CDCl3), 171.4, 161.4, 160.0, 159.6, 157.2, 154.3, 148.7, 147.9,
141.5, 135.6, 135.3, 135.1, 135.0, 133.5, 133.5, 133.3, 133.0, 131.9,
131.8, 131.4, 131.2, 129.3, 123.4, 121.6, 118.6, 118.5, 96.9, 49.0,
28.3, 27.1, 23.0, 13.2. HRMS (ESI): m/z [M]+ calcd for C36H31N4O8:
647.2136; found 647.2140.
BPO-Py-3-NO2: 82 mg (39% yields). 1H NMR (400 MHz,
CDCl3), 9.09 (d, 1H, J = 2.8 Hz), 8.52 (dd, 1H, J = 2.8 Hz, J = 8.8
Hz), 7.99 (d, 1H, J = 7.6 Hz), 7.68 (t, 1H, J = 7.2 Hz), 7.58 (t, 1H, J
= 7.2 Hz), 7.50 (d, 2H, J = 8.4 Hz), 7.42 (s, 1H), 7.27 (d, 1H, J = 7.6
Hz), 7.21 (d, 2H, J = 8.4 Hz), 7.09 (d, 1H, J = 9.2 Hz), 6.52 (d, 1H, J
= 8.8 Hz), 6.45 (d, 1H, J = 2.4 Hz), 6.38 (dd, 1H, J = 2.8 Hz, J = 8.8
Hz), 3.39 (q, 4H, J = 6.8 Hz), 2.88ꢀ2.84 (m, 1H), 2.70ꢀ2.66 (m, 1H),
2.11ꢀ2.07 (m, 1H), 1.70ꢀ1.62 (m, 3H), 1.20 (t, 4H, J = 6.8 Hz). 13C
NMR (100 MHz, CDCl3), 170.1, 166.9, 151.5, 149.4, 145.0, 135.2,
135.0, 134.5, 131.0, 129.3, 128.6, 125.0, 124.1, 123.5, 121.2, 111.5,
108.8, 108.3, 104.8, 97.3, 44.5, 27.2, 23.1, 22.4, 12.6. HRMS (ESI):
m/z [M]+ calcd for C36H32N3O6: 602.2286; found 602.2283.
BPO-Py-5-NO2: 80 mg (38% yields). 1H NMR (400 MHz,
CDCl3), 8.41ꢀ8.39 (m, 2H), 7.98 (d, 1H, J = 7.6 Hz), 7.68 (t, 1H, J =
7.6 Hz), 7.58 (t, 1H, J = 7.6 Hz), 7.49 (d, 2H, J = 8.8 Hz), 7.42 (s,
1H), 7.27ꢀ7.19 (m, 4H), 6.52 (d, 1H, J = 8.8 Hz), 6.45 (d, 1H, J = 2.4
Hz), 6.38 (dd, 1H, J = 2.8 Hz, J = 8.8 Hz), 3.39 (q, 4H, J = 6.8 Hz),
2.88ꢀ2.84 (m, 1H), 2.70ꢀ2.66 (m, 1H), 2.11ꢀ2.07 (m, 1H), 1.68ꢀ1.64
(m, 3H), 1.20 (t, 4H, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3),
170.1, 155.8, 152.4, 152.3, 151.8, 151.4, 149.4, 147.0, 135.5, 134.9,
134.7, 134.5, 130.8, 130.8, 129.3, 128.6, 127.7, 125.0, 124.4, 123.6,
121.4, 118.5, 108.9, 108.2, 104.9, 100.0, 97.3, 44.5, 27.2, 23.1, 22.4,
12.6. HRMS (ESI): m/z [M]+ calcd for C36H32N3O6: 602.2286; found
602.2285.
Unless otherwise noted, materials were obtained from
AladdinꢀReagent, and SigmaꢀAldrich, and were used without
further purification. All solvents were dried according to
standard methods, and were either high performance liquid
chromatography grade or spectroscopic grade for optical
spectroscopic studies. Thinꢀlayer chromatography (TLC)
analyses were performed on silica gel GF 254. Column
chromatographic purifications were performed on silica gel
(HG/T2354ꢀ92). NMR spectra were obtained with a Bruker
AMXꢀ400, where 1H NMR (400 MHz) chemical shifts were
given in ppm relative to the internal reference tetramethyl
silane. 13C NMR (100 MHz or 125 MHz) chemical shifts used
CDCl3 as the internal standard. Highꢀresolution mass spectral
data were recorded on a Bruker Daltonics Bio timeꢀofꢀflight
mass spectrometer. Fluorescence excitation and emission
spectra were obtained with a Hitachi F4600 spectrofluorometer
and a 10ꢀmm quartz cuvette. UVꢀVis absorption spectra were
recorded on a Hitachi PharmaSpec UVꢀ1900 UVꢀVisible
spectrophotometer.
Synthesis of probes
To a 250ꢀml roundꢀbottom flask was added 16.5 g (100 mmol)
3ꢀdiethylamino phenol, 19.0 g (128 mmol) phthalic anhydride,
and 70 ml toluene. The mixture was heated under nitrogen to 80
oC for 10 h, 90 oC for 5 h, 100 oC for 2 h, and then 110 oC for 1
h. After cooling to room temperature, the resulting precipitate
was filtered and washed with PhMe to yield 28 g of crude
purple solid compound
without purification.
3 (89.5% yield), which was used
Freshly distilled cyclohexanone (6.6 ml, 63.7 mmol) was
added dropwise to concentrated H2SO4 (70 ml) and cooled to 0
°C. Then, compound
3 (32 mmol) was added in portions with
vigorous stirring. The reaction mixture was heated to 90 °C for
1.5 h, cooled, and then poured onto ice (300 g). Perchloric acid
(70%; 7 ml) was then added, and the resulting precipitate was
filtered and washed with cold water (100 ml). The precipitate
was purified with silicaꢀgel column chromatography with
Preparation of the test solutions
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 6 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
Probe stock solutions of BPO-Py-diNO2, BPO-Py-3-NO2, and BPO-Py-diNO2 was collected at 620–720 nm (excited at 560
BPO-Py-5-NO2 (2.0 mM) were prepared in DMSO. Stock nm) for the red channel.
solutions of anions, reactive species, and amino acids were
Cell culture and confocal imaging
prepared in deionized water at a concentration of 50 mM. Test
solutions were prepared by mixing an aliquot of stock solution
and 15 ꢁL of the probe stock solution in 3.0 mL of PBS
buffer/DMSO (8:2, V/V) solution. Absorption and emission
spectra were recorded after the test solution was incubated at
room temperature for 2 h for SO2 or 30 min for biothiols.
HeLa cells were cultured in Dulbecco’s modified Eagle
medium (DMEM) containing 10% fetal bovine serum and 1%
AntibioticꢀAntimycotic at 37 °C in a 5% CO2/95% air
incubator. For fluorescence imaging, cells (4×103 well−1) were
placed on a 6ꢀwell plate and incubated for 24 h. Before staining,
the cells were washed three times with physiological PBS.
Confocal fluorescent images were recorded with a Zeiss LSM
780 confocal laser scanning microscope. The green channel was
covered the range 460–530 nm (excitation at 405 nm). For the
Quantum Yields
The fluorescence quantum yield (Φ) was calculated using:
Φx = Φs * (As / Ax) * (Dx / Ds) *(nx2 / ns2)
where the subscripts s and x refer to the standard [fluorescein in red channel, the confocal fluorescent images were collected
0.1M NaOH (Φs = 0.85)]19 and the test samples, respectively. over the range 620–720 nm (excitation at 560 nm).
Thus, Φs and Φx were the respective quantum yields; ns and nx
were the refractive indices of the solvents used; As and Ax were
Conflicts of interest
There are no conflicts to declare.
the absorption intensities at the respective excitation
wavelengths of the standard and test samples; and Ds and Dx
were integrals of the fluorescence intensities.
CCK-8 assay for the cell cytotoxicity
Acknowledgements
Toxicity toward HeLa cells was determined with the CCKꢀ8.
About 7000 cells per well were seeded into 96ꢀwell plates and
cultured overnight for a 70%–80% cell confluence. The
medium was then replaced with 50 ꢁL of fresh medium, and
This work was supported by the National Natural Science
Foundation of China (21708030) and the Fundamental
Research Funds for the Central University (2682016CX102).
We also thank the Comprehensive Training Platform of
Specialized Laboratory, College of Chemistry, Sichuan
University for laser scanning confocal imaging.
50ꢀꢁL aliquots of BPO-Py-3-NO2
, BPO-Py-5-NO2, and
BPO-Py-diNO2 at various concentrations were added to
achieve a final volume of 100 ꢁL. Twentyꢀfour hours later,
10ꢀꢁL CCKꢀ8 mixed in 90 ꢁL PBS was added to each well for
additional 1 h incubation. The absorbance was measured in an
ELISA plate reader (Model 550, BioRad) at a wavelength of
450 nm. The metabolic activity of the treated cells was
determined relative to untreated cell controls taken as 100%
activity.
Notes and references
1
2
3
S. Y. Zhang, C.ꢀN. Ong, H.ꢀM. Shen, Cancer Lett., 2004,
208, 143–153.
C. Hwang, A. J. Sinskey, H. F. Lodish, Science, 1992, 257,
1496–1502.
(a) G. I. Giles, K. M. Tasker, C. Jacob, Free Radical Biol.
Med., 2001, 31, 1279ꢀ1283. (b) M. Kemp, Y.ꢀM. Go, D. P.
Jones, Free Radical Biol. Med., 2008, 44, 921ꢀ937. (c) M. T.
V. Ishanina, M. Libiad, R. Banerjee, Nat. Chem. Biol., 2015,
11, 457ꢀ464.
Detection limit
The detection limit was calculated based on fluorescence
titration of 10ꢀµM probes. To determine the signalꢀtoꢀnoise
ratio, the probe emission intensity without Na2SO3 was
measured ten times to determine the standard deviation of
blanks. A linear relationship between the fluorescence intensity
and the concentrations of Na2SO3 or GSH were obtained. The
detection limit was then equal to 3σbi/m, where σbi was the
standard deviation of the blank measurements, m was the slope
of the intensity vs. sample concentration (signalꢀtoꢀnoise ratio
of 3:1).20
4
5
6
7
D. Liu, Y. Huang, D. Bu, A. D. Liu, L. Holmberg, Y. Jia, C.
Tang, J. Du, H. Jin, Cell Death Dis., 2014, 5, e1251.
(a) J. Li, Z. Meng, Nitric Oxide, 2009, 20, 166ꢀ174. (b) J.
Li, R. Li, Z. Meng, Eur. J. Pharmacol., 2010, 645, 143ꢀ150.
N. Sang, Y. Yun, H. Li, L. Hou, M. Han and G. K. Li,
Toxicol. Sci., 2010, 114, 226ꢀ236.
(a) X. Li, F. W. Bazer, H. Gao, W. Jobgen, G. A. Johnson, P.
Li, J. R. McKnight, M. C. Satterfield, T. E. Spencer, G. Wu,
Amino Acids, 2009, 37, 65–78. (b) O. W.Griffith, Methods
Enzymol., 1987, 143, 366ꢀ376.
Co-localization experiments
8
9
(a) Y. Yue, F. Huo, P. Ning, Y. Zhang, J. Chao, X. Meng, C.
Yin. J. Am. Chem. Soc., 2017, 139, 3181−3185. (b) B. Xu, H.
Zhou, Q. Mei, W. Tang, Y. Sun, M. Gao, C. Zhang, S. Deng,
Y. Zhang, Anal. Chem., 2018, 90, 2686ꢀ1691.
HeLa cells were incubated with BPO-Py-diNO2 (10 µM) in
physiological saline for 30 min at 37 °C, and the cells were
washed with PBS three times. Then 1.0ꢀµM Lyso Tracker
Green or Mito Tracker Green was added and coꢀincubated for
another 30 min. Cell imaging was performed after washing the
cells with physiological saline three times. Lyso Tracker Green
or Mito Tracker Green fluorescence was collected at 510–600
nm (excited at 488 nm) for the green channel and
(a) H. Chen, B. Dong, Y. Tang and W. Lin, Acc. Chem. Res.
,
2017, 50, 1410ꢀ1422. (b) J.ꢀT. Hou, W. X. Ren, K. Li, J. Seo,
A. Sharma, X.ꢀQ. Yu, J. S. Kim, Chem. Soc. Rev., 2017, 46,
2076ꢀ2090. (c) X. Jiao, Y. Li, J. Niu, X. Xie, X. Wang, B.
Tang, Anal. Chem., 2018, 90, 533ꢀ555. (d) W. Sun, S. Guo, C.
Hu, J. Fan, X. Peng, Chem. Rev., 2016, 116, 7768ꢀ7817. (e)
W. Xu, Z. Zeng, J.ꢀH. Jiang, Y.ꢀT. Chang, L. Yuan, Angew.
Chem. Int. Ed, 2016, 55, 13658ꢀ13699.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 6
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 7 of 7
View Article Online
DOI: 10.1039/C8TB01152D
Journal Name
ARTICLE
10 M. Li, P. Cui, K. Li, J. Feng, M. Zou, X. Yu. Chine. Chem.
Lett., doi.org/10.1016/j.cclet.2017.11.011.
11 (a) H. Zhu, J. Fan, Q. Xu, J. Wang, P. Gao, X. Peng, Chem.
Commun., 2012, 48, 11766–11768. (b) K. Surendran, S. P.
Vitiello, D. A. Pearce, Pediatr. Nephrol., 2014, 29, 2253–
2261.
12 U. T. Phan, B. arunachalam, P. Cresswell, J. Biol. Chem.
,
2000, 275, 25907–25914.
13 D. Kand, T. Saha, M. Lahiri, P. Talukdar, Org. Biomol.
Chem., 2015, 13, 8163–8168.
14 J. Fan, Z. Han, Y. Kang, X. Peng, Sci. Rep., 2016, 6, 19562.
15 (a) B. Dong, X. Song, X. Kong, C. Wang, Y. Tang, Y. Liu
and W. Lin, Adv. Mater., 2016, 28, 8755–8759; (b) L. Yuan,
W. Lin, Y. Yang, H. Chen, J. Am. Chem. Soc., 2012, 134,
1200ꢀ1211. (c) Y. Wei, D. Cheng, T. Ren, Y. Li, Z. Zeng, L.
Yuan, Anal. Chem., 2016, 88, 1842ꢀ1849.
16 (a) X. Xie, M. Li, F. Tang, Y. Li, L. Zhang, X. Jiao, X. Wang,
B. Tang. Anal. Chem., 2017, 89, 3015−3020. (b)H. Maeda, H.
Matsuno, M. Ushida, K. Katayama, K. Saeki, N. Itoh. Angew.
Chem. Int. Ed., 2005, 44, 2922 –2925. (c) Y. Pan, T. –B. Ren,
D, Cheng, Z. –B. Zeng, L. Yuan, X. –B. Zhang. Chem. Asian.
J., 2016, 11, 3575ꢀ3582. (d) R. PhilosofꢀOppenheimer, I.
Pecht, M. Fridkin, Int. J. Pept. Protein Res., 1995, 45,
116ꢀ121. (e) W. Lin,; L. Long, W. Tan, Chem. Commun.
2010, 46, 1503ꢀ1505.
,
17 L. Yuan, W. Lin, H. Chen, Biomaterials
, 2013, 34,
9566ꢀ9571.
18 (a) K. Liu, H. Shang, X. Kong, M. Ren, J. –Y. Wang, Y. Liu,
W. Lin. Biomaterials, 2016, 100, 162ꢀ171. (b) K. Liu, H.
Shang, X. Kong, W. Lin, J. Mater. Chem. B, 2017, 5,
3836ꢀ3841.
19 B. C. A. Parker, W. T. Rees, Analyst, 1960, 85, 587ꢀ600.
20 B. P. Joshi, J. Park, W. I. Lee and K. Lee, Talanta., 2009, 78,
903ꢀ909.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins