Development of Lysosome-Targeted Fluorescent Probes for Cys by Regulating the Boron-dipyrromethene (BODIPY) Molecular Structure

Article abstract of DOI:10.1002/chem.201902301

Our previous discovery suggested that substituents on the 1,7 positions delicately modulate the sensing ability of the meso-arylmercapto boron-dipyrromethene (BODIPY) to biothiols. In this work, the impact of delicate modulations on the sensing ability is investigated. Therefore, 1,7-dimethyl, 3,5-diaryl substituted BODIPY is designed and developed and its conformationally restricted species with a meso-arylmercapto moiety (DM-BDP-SAr and DM-BDP-R-SAr) as selective fluorescent probes for Cys. Moreover, the lysosome-target probes (Lyso-S and Lyso-D) based on DM-BDP-SAr carrying one or two morpholinoethoxy moieties were developed. They were able to detect Cys selectively in vitro with low detection limits. Both Lyso-S and Lyso-D localized nicely in lysosomes in living HeLa cells and exhibited red fluorescence for Cys. Moreover, a novel fluorescence quenching mechanism was proposed from the calculations by density functional theory (DFT). The probes may go through intersystem crossing (from singlet excited state to triplet excited state) to result in fluorescence quenching.

Full text of DOI:10.1002/chem.201902301

A Journal of
Accepted Article
Title: Development of lysosome-targeted fluorescent probes for Cys by
regulating BODIPY molecular structure
Authors: Jinhua Gao, Yuanfang Tao, Jian Zhang, Nannan Wang, Xin
Ji, Jinling He, Yubing Si, and Weili Zhao
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To be cited as: Chem. Eur. J. 10.1002/chem.201902301
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Development of lysosome-targeted fluorescent probes for Cys by
regulating BODIPY molecular structure
Jinhua Gao,[a] Yuanfang Tao,[a] Jian Zhang,*[a] Nannan Wang,[a] Xin Ji,[a] Jinling He,[a] Yubing Si,*[c] and
Weili Zhao*[ab]
Abstract: Our early discovery suggested that substituents on 1,7
positions delicately modulate the sensing ability of the meso-
arylmercapto BODIPY to biothiols. In this work, we continue to
investigate the impact of delicate modulations on the sensing ability.
Therefore, we designed and developed 1,7-dimethyl, 3,5-diaryl
substituted BODIPY and its conformationally restricted species with
meso-arylmercapto moiety (DM-BDP-SAr and DM-BDP-R-SAr) as
selective fluorescent probes for Cys. Moreover, the lysosome-target
probes (Lyso-S and Lyso-D) based on DM-BDP-SAr carrying one
or two morpholinoethoxy moieties were developed. They were able
to detect Cys selectively in vitro with low detection limits. Both Lyso-
S and Lyso-D localized nicely in lysosomes in living HeLa cells and
exhibited red fluorescence for Cys. Moreover, a novel fluorescence
quenching mechanism was proposed from the calculations by
density functional theory (DFT). The probes may go through
intersystem crossing (from singlet excited state to triplet excited
state) to result in fluorescence quenching.
selective determination of biothiols is of great importance for
various investigations in biochemistry as w ell as assistance in
diagnosis of the related diseases in clinic.
A large number of innovative fluorescent probes have been
discovered to distinguish and measure biothiols.[12-31] Some
impressive w orks have succeeded in differentiating GSH from
Cys and Hcy.[19-27] How ever, it is still a challenge to construct
probes for discriminating Cys from Hcy due to the similar pKa
and steric congestion of Cys and Hcy, w hich also frequently
suffers from draw backs such as unsatisfied selectivity, long
response time, and undesired spectra overlap.[22,32 -40] We are
devoted to designing and developing new fluorescent probes
capable of effective differentiation of Cys, Hcy, GSH, and
favourably w ith lysosome targeting capability.
Our group has considerable interest in the design and synthesis
of BODIPY-based dyes as fluorescent probes for various
biothiols species.[41-49] The early w orks relying on the meso
substitution strategy of BODIPY w ere exhibited in Scheme 1.
With no substitution at 1,7 positions of BODIPY skeleton (BDP-
SAr), differentiate Cys/Hcy from GSH in dual emission channel
mode w as realized (Scheme 1a).[43] On the other hand, w hen
1,7-dimethyl groups present on BODIPY skeleton (TM-BDP-
SAr), w e found that differentiation in dual channel mode w as
changed to Cys from Hcy/GSH (Scheme 1b).[47] In addition, w e
also reported 1,7-diaryl substituted BODIPY and its
conformationally restricted species (DPh-BDP-R-SAr) for highly
selective detection of Cys and Hcy rather than bulky GSH
(Scheme 1c).[48] The steric congestion of the 1,7-diphenyl groups
completely blocked nucleophilic attack from bulky GSH. These
results implied the important functions of the steric congestion at
the 1,7 positions of BODIPY for the selective biothiols
recognition.
Introduction
As important biomolecules, biothiols such as cysteine (Cys),
homocysteine (Hcy) and glutathione (GSH) play vital roles in a
w ide range of physiological and pathological processes arising
from their biological redox chemistry.[1,2] Cys and Hcy are
essential biological molecules required for detoxifying function,
immunological competence, as w ell as grow th and delay of
ageing of cells and tissues in living systems.[3] Meanw hile, GSH
is the most abundant intracellular non-protein thiol (1–10 mM)
and a biomarker of oxidative stress.[4,5] It has been revealed that
GSH plays a critical role in controlling oxidative stress in order to
maintain the redox homeostasis for cell grow th and function.[6]
Aberrant levels of biothiols in humans may contribute to various
diseases. In addition, biothiols are closely associated w ith
proteolysis in the lysosome w hich reduces disulphide bonds.[7,8]
It is believed that GSH is involved in the stabilization of
lysosome’s membranes, w hereas Cys is an effective stimulator
[a]
J. Gao, Y. Tao, N. Wang, X. Ji, Dr. J. He, Dr. J. Zhang*, Prof. W.
Zhao*
Key Laboratory for Special Functional Materials of Ministry of
Education, School of Materials Science and Engineering,
Henan University
Kaifeng 475004, P. R. China.
E-mail: zhangjian861208@126.com; zhaow@henu.edu.cn
Prof. W. Zhao*
School of Pharmacy, Institutes of Integrative Medicine,
Fudan University,
Shanghai, 201203, P. R. China.
Prof. Y. Si*
[b]
[c]
Henan Key Laboratory of Nanocomposites and Applications,
Institute of Nanostructured Functional Materials, Huanghe Science
and Technology College, Zhengzhou 450006, P. R. China
Scheme 1. Reaction mechanisms of BODIPY-based probes with
biothiolsin our group’spreviousstudies.
Supporting information for this article is given via a link at the end of
the document.
of albumin degradation in liver lysosomes.[9-11] Therefore,
1
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The required pyrrole skeleton (1a-3a) can be constructed using
Knorr pyrrole synthesis starting from 6-methoxy tetralone or from
3-acetyl benzene
derivative
w ith para
methoxy
or
morpholinoethoxy substitutent. Hydrolysis and decarboxylation
under alkaline conditions afforded the desired pyrroles (1b-3b).
Treatment of the pyrrole w ith triphosgene in the presence of
diisopropylethylamine and addition another portion of pyrrole
provided the dipyrrylmethanone (1c-4c). After reacting w ith
POCl3 to meso chloro dipyrromethene and complexation w ith
boron trifluoride etherate in the presence of base, the desired
BODIPY
dyes
(1d-4d)
w ere
obtained.
Using
4-
methoxythiophenol to replace the chloro group w ith the
assistance of mild base resulted in the desired probes ( DM-
BDP-SAr, DM-BDP-R-SAr, Lyso-S, and Lyso-D).
Spectroscopic properties of DM-BDP-SAr and DM-BDP-R-
SAr and the responses to biothiols
DM-BDP-SAr possessed absorption maximum at 591 nm, w hich
was shorter than that of DM-BDP-R-SAr (670 nm) in
acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.4) and low
fluorescence at 37 °C w as noticed (Figure 1). In the presence of
Cys, drastic changes in the absorption (w ith absorption at 470
nm) and fluorescence (enhanced emission at 562 nm) w ere
observed in 15 min for DM-BDP-SAr (Figures 1a and 1b). The
absorption spectra varied in a ratiometric response w ith an
isosbestic point at 506 nm, w hich could be adopted for the
quantitative estimation the concentration of Cys. In stark
contrast, w hen Hcy w as reacted w ith DM-BDP-SAr, only very
minor changes w ere found both in the absorption and emission
spectra (Figure S1). Moreover, GSH w as also investigated and
very slow reaction rate w as identified (Figure S1). Keep in mind
that our initial design purpose w as trying to develop dual
emission channel mode probe(s) in the green-red region, herein
unexpected selective detection of Cys w as achieved. More
astonishingly, the Stokes’ shift of 100 nm w as
Scheme 2. Chemical structuresof the probesdesigned.
We w ere curious to know w hether 1,7-dimethyl substitution
which enabled biothiols detection and differentiation of Cys from
Hcy/GSH in dual emission channel in blue-green region could be
extended to more favorable green-red region, and w hether
structure conformation restriction w ill affect the recognition.
Moreover, w e w ere also interested in developing lysosome-
targeted version. Hence, w e designed and synthesized 1,7-
dimethyl, 3,5-diaryl substituted BODIPY and its conformationally
restricted species carrying a p-methoxyphenylmercapto moiety
at meso position (DM-BDP-SAr and DM-BDP-R-SAr, Scheme
2a). Moreover, the lysosome targeted probes (Lyso-S and
Lyso-D) w ere designed by attaching
a single or dual
morpholinoethoxy group onto the phenyl moiety (Scheme 2b).
The Hela cells w ere popularly used for cell fluorescence imaging
due to the rapidly grow ing and reproduction, so w e chose HeLa
cells to identify the performence of Lyso-S and Lyso-D.
Results and Discussion
Syntheses of probes
The designed probes w ere prepared using the synthetic route as
show n in Scheme 3.
Figure 1. Time-dependent absorption and fluorescence spectra of
probes (10 μM) in the presence of 10 equiv. of Cys in acetonitrile/PBS
buffer (1 : 1, v/v, 10 mM, pH 7.4) at 37 °C. (a) Absorption spectra and (b)
fluorescence spectra (λex = 465 nm) of DM-BDP-SAr treated with Cys; (c)
Absorption spectra and (d) fluorescence spectra (λex = 525 nm) of DM-
BDP-R-SAr after addition of Cys. The excitation and emission slit widths
are set at 1 nm.
Scheme 3. Synthesisof probes.
2
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reached, w hich w as very unusual for BODIPY dyes w herein
small Stokes’ shift w as characteristic.[14,50,51] These unexpected
and unusual properties w ere obviously induced by the aryl
moieties w hich may squeeze the 1,7-dimethyl positions to block
the attack of the meso position by Hcy/GSH. As a reasonable
assumption, conformationally restricted DM-BDP-R-SAr should
be more difficult to the attack of thiol. Therefore, w hen DM-BDP-
R-SAr w as used for the detection of Cys, surprisingly over 90
min w as required to reach complete response (Figure 1c and
1d). DM-BDP-R-SAr w as also reluctant to react w ith Hcy or
GSH (Figure S2). Much diminished reacting rate of DM-BDP-R-
SAr compared to DM-BDP-SAr supported our assumption that
the more steric repulsion of the fused ring enhanced the steric
congestion of the 1,7-dimethyl moieties, w hich can also be
confirmed by the reduced reaction rate of conformationally
restricted probe to Cys and Hcy in our group’s previous studies
(Scheme 1c).[48]
dominated the decay w hich led to the low fluorescence of the
probe.[55] The modified mechanism based on DFT calculation
w as show n in Scheme 4b.
To find evidences for the mechanism of Scheme 4b, HRMS
studies w ere performed to monitor the reaction of DM-BDP-
SAr/DM-BDP-R-SAr w ith Cys. In the presence of Cys, DM-
BDP-SAr generated a species w ith characteristic peak at m/z =
552.1943 corresponding to the DM-BDP-N-Cys [M+H]+ (Figure
S4), w hile DM-BDP-R-SAr produced a characteristic peak at
m/z = 604.2247 corresponding to the DM-BDP-R-N-Cys [M+H]+
(Figure S5). An evidence for the intersystem crossing is the
increased rate of generation of 1O2. Enhancement of spin-orbit
interaction is a popular w ay to convert BODIPY fluorophore into
photosensitizer.[56] When 1,3-diphenylisobenzofuran (DPBF, a
know n efficient quencher of singlet oxygen) w as used to trap
singlet oxygen produced of DM-BDP-SAr under illumination of
light >500 nm using ZnPc as standard ( of 0.56 in DMF)
(Figure S6), the singlet oxygen quantum yield () w as
calculated to be 0.31. In comparison, a BODIPY dye w ith meso
chloro substitutent (BDP-Cl) only provided  of 0.06. Therefore,
the high population of T1 in DM-BDP-R-SAr could be reasonably
attributed to cause the fluorescence quenching.
So far, w e have established supporting evidences for the
plausible mechanism of both DM-BDP-SAr and DM-BDP-R-SAr
for the selective response to Cys w ith DM-BDP-SAr to be a
preferred one. We therefore further pursued to apply the current
system into a lysosome-targeted version. Such efforts w ere
accessed through the attachment of morpholinoethoxy group(s)
onto the para-position of one or tw o phenyl moieties (Scheme
2b). The designed lysosome targeting probes w ere prepared
similarly as DM-BDP-SAr as indicated in Scheme 3. The
responsive behaviors and targeting abilities of Lyso-S and
Lyso-D w ere investigated afterw ards.
Mechanistic Investigations
Based on the spectroscopic changes in comparison w ith general
rational observed,[43-48] the Cys selective responses of both
probes w ere attributed to the Cys-induced SNAr substitution-
rearrangement reaction to generate meso-amino-BODIPYs. The
low fluorescence of DM-BDP-SAr or DM-BDP-R-SAr w as
initially thought to be induced by photoinduced electron transfer
(PET) as demonstrated in previous publications (Scheme 4a).[52-
54]
How ever, when density functional theory (DFT) calculation
were performed on DM-BDP-SAr and DM-BDP-N-Cys (Figure
S3), it w as astonishingly found that electron densities of both
HOMO and LUMO orbitals mostly localized on the acceptor
(BODIPY moiety) and the spin-orbit coupling (SOC) values are
1.28 cm-1 the S1→T1 and 6.37 cm-1 for S2→T1 of DM-BDP-SAr,
18.3 and 12.5 times larger than those data of DM-BDP-N-Cys
(the SOC value of S1→T1 and S2→T1 for DM-BDP-N-Cys are
-1
0.07 cm and 0.51 cm-1 respectively). Therefore, it w as possible
that intersystem crossing (leading to the high population of T1)
Scheme 4 Proposed sensing mechanism of DM-BDP-R-SAr and DM-BDP-SAr for Cys.
3
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for Lyso-S and Lyso-D w ere further examined w ith various
amino acids as w ell as various sulfur-containing inorganic anion
species.
With the excitation of 465 nm, the fluorescence monitored at 566
nm indicated that no obvious fluorescence enhancement for
Lyso-S could be triggered by the treatment of various amino
acids, as w ell as various sulfur-containing inorganic anion
species (Figure 2c). To further demonstrate the selective
recognition of Lyso-S to Cys, the competition experiments w ere
also carried out by addition of Cys to the solution of Lyso-S in
the presence of other analytes. As show n in Figure 2d, the co-
existence of other analytes did not have obvious impact on
Lyso-S to detection of Cys. Interfering investigation and
competition experiments on Lyso-D also demonstrated selective
responses to Cys (Figure S10).
Considering that ROS/RNS species may potentially oxidize
thioethers,[57-62] the interference of ROS/RNS to the detection of
Cys w as also evaluated and the results w ere collected in Figure
S11. It can be clearly seen that the detection of Cys w ith our
probes (Lyso-S and Lyso-D) w as not affected by ROS/RNS
species.
Figure 2. Spectroscopic data measured for Lyso-S (10 μM) in
acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.4, λex = 465 nm) at 37 °C.
(a) Time-dependent absorption spectra changes upon treatment with 10
equiv. of Cys; (b) Time-dependent fluorescence intensity changes and
spectra changes (inset) upon treatment with 10 equiv. of Cys (λex = 465
nm); (c) Fluorescence intensities monitored at 566 nm upon treatment
with 100 μM of individual analyte for 15 min. (0) Blank, (1) Cys, (2) Hcy,
(3) GSH, (4) Ala, (5) Arg, (6) Asp, (7) Gla, (8) Gln, (9) Glu, (10) Gly, (11)
His, (12) Ile, (13) Leu, (14) Lys, (15) Met, (16) Phe, (17) Pro, (18) Ser,
(19) Thr, (20) Try, (21) NaSH, (22) HSO3−, (23) SO32−, (24) S2O32−, (25)
S2O42−, (26) S2O52−; (d) Fluorescence intensities (black bars) upon
treatment with individual analyte (100 μM) for 15 min in comparison with
those (red bars) after addition of Cys (100 μM). (0) Blank, (1) Hcy, (2)
GSH, (3) Ala, (4) Arg, (5) Asp, (6) Gla, (7) Gln, (8) Glu, (9) Gly, (10) His,
(11) Ile, (12) Leu, (13) Lys, (14) Met, (15) Phe, (16) Pro, (17) Ser, (18)
Thr, (19) Try, (20) NaSH, (21) HSO3−, (22) SO32−, (23) S2O32−, (24)
S2O42−, (25) S2O52−. The excitation and emission slit widths are set at 1
nm and Error bars are relative standard deviations(RSD), n=3.
Time-dependent spectral changes of Lyso-S and Lyso-D
with Cys
We first examined the time-dependent responses on absorption
and emission behaviours of Lyso-S and Lyso-D to Cys,
respectively. The absorption maximum of Lyso-S w as 592 nm
and Lyso-S exhibited fairly low fluorescence at 624 nm under
excitation w ith light of 465 nm. Upon addition of Cys in
acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.4) at 37 °C, the
absorption at 592 nm decreased abruptly, accompanied by an
increase in the absorption at 469 nm displaying significant
hypsochromic shift (123 nm, Figure 2a). Meanw hile, a new
fluorescence band centered at 566 nm illustrated dramatic
increment of fluorescence up to 113-fold over the intrinsic
fluorescence of Lyso-S in 15 min (Figure 2b). For Lyso-D,
similar trend w as also observed as show n in Figure S7a and
S7b. These results indicated that both Lyso-S and Lyso-D
exhibit fast responses to Cys, w hich w ould be favorable for the
detection of Cys in the living systems.
Figure 3. Fluorescence spectral changes of (a) Lyso-S and (c) Lyso-D
upon addition of increased concentrations of Cys (10 μM, λex = 465 nm).
Each spectrum was recorded 15 min after Cys addition in
acetonitrile/PBS buffer (1 : 1, v/v, 10 mM, pH 7.4) at 37 °C; A linear
relationship of fluorescence intensity changes at 566 nm of (b) Lyso-S
and (d) Lyso-D against Cys concentration, which can be linearly fitted by
the equation Y = 4.331 X  0.445, with R2 = 0.990 and Y = 4.696 X 
0.197, with R2 = 0.989 respectively. The excitation and emission slit
widths are set at 1 nm and Error bars are relative standard deviations
(RSD), n=3.
The sensitivities of probes toward various concentrations of
Cys
To check w hether Lyso-S and Lyso-D fluorescence response
changes over Cys concentration, the Cys titration experiments of
these tw o probes w ere measured (Figure 3). Figures 3a and 3c
show s the changes in the emission of Lyso-S and Lyso-D in the
presence of increasing concentration of Cys. Further studies
suggested a concentration-dependent response manner w ith the
linear response of Lyso-S tow ard Cys ranging from 0 μM to 15
Selective responses of Lyso-Sand Lyso-D to Cys
To evaluate the selectivity of Lyso-S/Lyso-D, Hcy and GSH
were measured for the fluorescence changes upon reacting w ith
the probes (Figure 2c, Figure S8 and Figure S9). It w as found
that neither Lyso-S, nor Lyso-D show ed significant
spectroscopic changes w ith Hcy/GSH. The selectivity patterns
4
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μM and the detection limit w as estimated as 46 nM (Figure 3b),
while Lyso-D show ed a linear response tow ard Cys ranging
from 0 μM to 20 μM and the detection limit w as estimated as 76
nM (Figure 3d).
The effect of pH
The effect of the pH value on the fluorescence behaviors of
Lyso-S and Lyso-D tow ard Cys w ere carried out as w ell and the
data w ere collected in Figure S12. Both the probes of Lyso-S
and Lyso-D w ere fairly stable in pH range of 4.0–8.0 w ith very
low fluorescence emission. In the presence of Cys, noticeable
fluorescence enhancement at pH range from 4.8 to 8.0 w as
observed. The increased reactivity tow ards Cys w as observed
along w ith increased pH value for both probes. These
observations correlated w ell w ith the previous reports ascribed
to the larger concentration of thiolate species in higher pH
environment.[12-31] Lysosomes are spherical-shaped, catabolic
organelles w ith an acidic interior (pH 4.0–6.0).[63] Though
diminished reactivity tow ards Cys w as found, both Lyso-S and
Lyso-D could function properly under lysosomal pH environment.
Figure 4. Confocal fluorescence and bright-field images of living HeLa
cells. (a) fluorescence and (b) bright field images of HeLa cells after
incubated with Lyso-S (10 μM) for 30 min; (c) fluorescence and (d) bright
field images of HeLa cells pretreated with 5 mM of NEM for 30 min and
then incubated with Lyso-S (10 μM) for 30 min. All fluorescence was
taken in red channel at 580−620nm inidentical conditions.
Bioimaging applications of Lyso-S and Lyso-D
To further assess the biological application w ith our probes,
toxicity studies performed on HeLa cells via MTT for both Lyso-
S and Lyso-D (Figure S13). The results suggested that both
probes lack of toxicity up to 20 μM. Then, confocal laser
scanning microscopic fluorescence imaging w as used to
examine the capability of Lyso-S and Lyso-D to detect Cys in
living cells (Figures 4 and S14). Bright red fluorescence could be
visualized in HeLa cells upon addition of Lyso-S and Lyso-D
respectively after incubated for 30 min. By contrast, w hen HeLa
cells w ere pre-treated w ith 5 mM of NEM, a w ell-know n
scavenger of biothiols,[64] for 30 min and subsequently incubated
w ith Lyso-S and Lyso-D, only w eak red fluorescence w as
noticed. The result exhibits that Lyso-S and Lyso-D could
sense intracellular Cys from red emission channels, w hich are
favourable to bioimaging applications due to the reduced
background of absorption, fluorescence, and light scattering in
long w avelength region.[65-67]
Next, the colocalization experiments w ere performed by co-
staining Lyso-S w ith various commercially available organelle-
tracing probes such as Lyso-Tracker Green DND-26, Mito-
Tracker Green, as w ell as Hoechst (a nucleus-specific dye) in
HeLa cells to figure out the location of the probe. As show n in
Figure 5, good overlap of the red emission from Lyso-S w ith the
green emission from Lyso-Tracker Green DND-26 w as observed
from the merged image show n in the yellow color, the Pearson's
colocalization coefficient calculated from the intensity correlation
plots w as 0.79. The intensity profiles of the linear regions of
Figure 5. Confocal laser scanning microscopic images for intracellular
localization of Lyso-S in HeLa cells. Cells were treated with 10 μM Lyso-
S for 30 min and co-stained with 100 nM Lyso-Tracker DND-26, 200 nM
Mito-Tracker Green, or 5 μg/mL Hoechst respectively. Red channel at
580−620 nm. Green channel at 490−520 nm for Lyso-Tracker Green
DND-26 and Mito-Tracker Green, 440−480 nm for Hoechst.
interest (ROI) across HeLa cells stained w ith Lyso-S and Lyso-
Tracker Green DND-26 vary in close synchrony. Comparatively,
no good overlap w as observed betw een Lyso-S and Mito-
Tracker Green or Hoechst. These results confirmed the majority
of Lyso-S localized w ithin lysosomes of the cells and suggested
the potential of Lyso-S as a probe for the detection of Cys in
lysosome of living HeLa cells. Similarly, the colocalization
experiments of Lyso-D co-stained w ith Lyso-Tracker Green
DND-26, Mito-Tracker Green, and Hoechst demonstrated that
Lyso-D could also efficiently localized in the lysosomes
(Pearson's colocalization coefficient w as 0.78) to selective
detection Cys in living HeLa cells (Figure S15). Therefore, both
Lyso-S and Lyso-D in the lysosomes to selective detect Cys in
living HeLa cells could be achieved. It is also revealed that one
morpholino group is sufficient to provide the lysosome targeting.
5
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v/v) to yield white powder (0.27 g, 50%). 1H-NMR (400 MHz, CDCl3): δ
8.00 (s, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.81 (d, J = 2.3 Hz 1 H), 6.72 (dd, J
= 8.3 ,2.5 Hz, 1H), 6.51 (s, 1H), 3.81 (s, 3H), 2.93 (t, J = 7.6 Hz, 2H),
2.66 (t, J = 7.6 Hz, 2H), 2.08 (s, 3H).
Additional morpholine moiety did not further enhance the
targeting capability.
Conclusions
Bis(7-methoxy-3-methyl-4,5-dihydro-1H-benzo[g]indol-2-yl)methano-
ne (1c). A solution of triphosgene (0.90 g, 3.1 mmol) in THF (30 mL) was
added to a stirred solution of compound 1b (1.0 g, 4.7 mmol) and
ethyldiisopropylamine (0.70 mL, 4.7 mmol) in THF (10 mL) over 20 min at
0 °C under nitrogen. After stirred for 2 h at 0 °C, the reaction mixture was
treated with saturated NaCl solution and the resulting solution was
extracted with CH2Cl 2. The extract was washed with H2O and brine, dried
with anhydrous Na2SO4, filtered and evaporated. The purification of the
crude product was carried out by silica gel column chromatography
(CH2Cl2/ethyl acetate, 6 : 1, v/v) to yield yellow solid (0.42 g, 40%). 1H-
NMR (400 MHz, d6-DMSO): δ 11.46 (s, 2H), 7.78 (d, J = 8.5 Hz, 2H),
6.84 (d, J = 2.3 Hz 2 H), 6.78 (dd, J = 8.5 ,2.5 Hz, 2H), 3.75 (s, 6H), 2.86
(t, J = 7.5 Hz, 4H), 2.58 (t, J = 7.4 Hz, 4H), 2.04 (s, 6H).
In an attempt to design new long w avelength fluorescent probes
to detect biothiol species in dual emission mode through meso
replacement, w e attached aromatic moiety at 3 and 5 positions
of BODIPY dye w ith 1,7-dimethyl to control the replacement and
obtained DM-BDP-SAr and DM-BDP-R-SAr. The expected dual
emission detection w as not reached and unexpected selective
detection to Cys over Hcy/GSH w as found w ith enormous 100
nm Stokes’ shift. DM-BDP-SAr turned out to be a preferred
probe for Cys w ith faster reaction rate. We further modified the
probe w ith lysosomal targeting morpholine moiety and
developed Lyso-S and Lyso-D as selective probes for Cys
detection in lysosome. Good linear responses ranged from 0 μM
to 15 μM w ith the detection limit of 46 nM for Lyso-S, and 020
μM (w ith a detection limit of 76 nM) for Lyso-D w ere reached.
As desired, both Lyso-S and Lyso-D could localize in
lysosomes nicely. Moreover, a novel fluorescence quenching
mechanism w as proposed based on density functional theory
(DFT) calculation. The probes show ed significant intersystem
crossing in the excited state (from singlet to triplet excited
states) w hich induced fluorescence quenching.
Compound 1d. Compound 1c (0.30 g, 0.66 mmol) was dissolved in 1,2-
dichloroethane (10 mL). Phosphorus oxychloride (0.30 mL, 2.6 mmol)
was added at 0 °C under nitrogen, and the reaction mixture was allowed
to come to room temperature and stirred for 12 h, then cooled in an ice
bath. Triethylamine (0.60 mL, 4.0 mmol) was added, and the reaction
was stirred at 0 °C for 5 min. Boron trifluoride etherate (0.70 mL, 5.3
mmol) was added dropwise while maintaining the temperature at 0 °C.
The reaction mixture was allowed to come to room temperature and
stirred for an additional 30 min. The resulting solution was poured out in
diethyl ether (300 mL) and extracted with water. After drying over Na2SO4,
filtration, and evaporation, the crude product was purified by silica gel
column chromatography (CH2Cl2/petroleum ether, 1 : 1, v/v) to yield
mazarine solid (0.12 g, 35%). 1H-NMR (400 MHz, CDCl3): δ 8.69 (d, J =
8.9 Hz, 2H, 3,5-ArH), 6.95 (dd, J = 8.9, 2.5 Hz, 2H, 3,5-ArH), 6.80 (s, 2 H,
3,5-ArH), 3.87 (s, 6H, 3,5-Ar-OCH3), 2.88 (t, J = 6.2 Hz, 4H, -CH2CH2-),
2.64 (t, J = 6.1 Hz, 4H, -CH2CH2-), 2.47 (s, 6H, 1,7-CH3); 13C-NMR (100
MHz, CDCl3): δ 160.57; 149.83; 143.09; 135.10; 132.28; 131.69; 131.21;
130.30; 121.21; 114.16; 112.27.
Experimental Section
Ethyl 2-(hydroxyimino)-3-oxobutanoate, 1a-1c, 2a-2c, 3a-3c and 4c
were prepared according thereported methods.[68-72]
Ethyl
7-methoxy-3-methyl-4,5-dihydro-1H-benzo[g]indole-2-
carboxylate (1a). In a 1000 mL flask, 6-methoxytetralone (19.4 g, 110
mmol) and sodium acetate (54.0 g, 660 mmol) were dissolved in
propionic acid (300 mL). When the mixture was heated to 150 °C, a
solution of compound 1a (17.5 g, 110 mmol) in 200 mL propionic acid
and zinc dust (71.0 g, 1.10mol) were slowly added to the stirred mixture
at 150 °C. After the addition, the mixture was stirred at 150 °C for 2 h and
cooled to about 70 °C. It was then poured into ice/water mixture and was
allowed to stand overnight. The precipitate was filtered and washed with
water until the pH value of filtrate was 7.0. The residue was then
recrystallized from ethanol several times to give compound 1a (4.6 g,
15%) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 9.20 (s, 1H), 7.32 (d,
J = 8.3 Hz, 1H), 6.87–6.71 (m, 2 H), 4.35 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H),
2.92 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.30 (s, 3H), 1.38 (t, J =
7.1 Hz, 3H).
DM-BDP-R-SAr. Compound 1d (52 mg, 0.10 mmol) was dissolved in
CH2Cl2 (50 mL), p-methoxythiophenol (70 mg, 0.50 mmol) and NEt3 (51
mg, 0.50 mmol) were added. The reaction mixture was purged with
nitrogen and stirred at room temperature for 2 h. The crude mixture was
poured out in diethyl ether, washed with aqueous Na2CO3, and the
organic layer was evaporated to dryness. DM-BDP-R-SAr was obtained
as a brown solid (55 mg, 88%) after filtration over a silica gel column
chromatography (CH2Cl2/petroleum ether, 2 : 1, v/v). M. P.  300 °C ; 1H-
NMR (400 MHz, CDCl3): δ 8.71 (d, J = 9.0 Hz 2H, 3,5-ArH), 7.19 (d, J =
8.8 Hz, 2H, 8-ArH), 6.94 (dd, J = 8.9, 2.6 Hz, 2H, 3,5-ArH), 6.83 (d, J =
8.9 Hz, 2H, 8-ArH), 6.79 (d, J = 2.5 Hz, 2H, 3,5-ArH), 3.87 (s, 6H, 3,5-Ar-
OCH3), 3.76 (s, 3H, 8-Ar-OCH3), 2.86 (t, J = 6.9 Hz, 4H, -CH2CH2-), 2.58
(t, J = 6.5 Hz, 4H, -CH2CH2-), 2.43 (s, 6H, 1,7-CH3); 13C-NMR (100 MHz,
CDCl3): δ 160.61; 158.01; 150.51; 143.33; 137.13; 136.28; 131.75;
130.57; 129.85; 128.43; 127.66; 121.38; 115.20; 114.12; 112.28; 55.32;
7-Methoxy-3-methyl-4,5-dihydro-1H-benzo[g]indole (1b). NaOH (0.40
g, 10 mmol) and compound 1a (0.70 g, 2.5 mmol) were added to
ethylene glycol (40 mL) and heated to 160 °C under nitrogen. After
stirring at 160 °C for 2 h, the reaction mixture was treated with saturated
NaCl solution and the resulting solution was extracted with CH2Cl2. The
extract was washed with H2O and brine, dried with anhydrous Na2SO4,
filtered and evaporated. The purification of the crude product was carried
out by silica gel column chromatography (CH2Cl2/petroleum ether, 1 : 1,
30.84; 29.73;
20.57; 14.62. HRMS-MALDI: Calculated for
[C36H33BF2N2O3S]+: 621.2278;found621.2303.
6
This article is protected by copyright. All rights reserved.

10.1002/chem.201902301
Chemistry - A European Journal
FULL PAPER
Bis(5-(4-methoxyphenyl)-3-methyl-1H-pyrrol-2-yl)methanone
(2c).
CH3); 13C-NMR (100 MHz, CDCl3): δ 160.70; 159.78; 155.92; 155.71;
142.56; 142.45; 136.59; 131.32; 131.05; 125.02; 124.80; 122.83; 114.25;
113.69; 66.89; 65.71; 57.54; 55.27; 54.05;17.17.
The synthesis of compound 2c was analogous to the synthesis of
compound 1c starting from 2-(4’methoxyphenyl)-4-methyl pyrrole (2b).
1H-NMR (400 MHz, CDCl3): δ 9.05 (s, 2H), 7.58–7.43 (m, 4H), 6.94 (d, J
= 8.8 Hz, 4H), 6.37 (s, 2H), 3.84 (s, 6H), 2.31 (s, 6H).
Lyso-S. The synthesis of Lyso-S was analogous to the synthesis of DM-
BDP-R-SAr. M. P. 110–-113 °C ; 1H-NMR (400 MHz, CDCl3): δ 7.79 (dd,
J = 8.6, 4.1 Hz, 4H, 3,5-ArH), 7.21 (d, J = 8.7 Hz, 2H, 8-ArH), 6.91 (d, J =
8.7 Hz, 4H, 3,5-ArH), 6.86 (d, J = 8.7 Hz, 2H, 8-ArH), 6.36 (d, J = 2.7 Hz,
2H, Pyrrole-H), 4.15 (t, J = 5.5 Hz, 2H, -ArOCH2-), 3.82 (s, 3H, 3-Ar-
OCH3), 3.80–3.73 (m, 7H, 8-Ar-OCH3 and -O(CH2)2), 2.82 (t, J = 5.5 Hz,
2H, -CH2N-), 2.60 (s, 4H, -N(CH2)2), 2.51 (s, 6H, 1,7-CH3); 13C-NMR (100
MHz, CDCl3): δ 160.71; 159.73; 158.36; 156.45; 156.20; 143.72; 143.59;
136.40; 136.31; 135.69; 131.10; 128.12; 127.48; 125.35; 125.09; 123.41;
123.34; 115.35; 114.21; 113.66; 66.83; 65.65; 57.54; 55.38; 55.27; 54.02;
17.60; HRMS-MALDI: Calculated for [C37H38BF2N3O4SNa]+: 691.2596;
found 691.2573.
8-Chloro-1,7-dimethyl-3,5-di(p-methoxyphenyl)-4,4-difluoro-4-bora-
3a,4a-diaza-s-indacene (2d). The synthesis of compound 2d was
analogous to the synthesis of compound 1d. 1H-NMR (400 MHz, CDCl3):
δ 7.78 (d, J = 8.8 Hz, 4H, 3,5-ArH), 6.92 (d, J = 8.9 Hz, 4H, 3,5-ArH),
6.41 (s, 2H, Pyrrole-H), 3.83 (s, 6H, 3,5-Ar-OCH3), 2.56 (s, 6H, 1,7-CH3);
13C-NMR (100 MHz, CDCl3): δ 160.63, 155.81, 142.51, 136.58, 131.27,
131.04, 124.80, 122.83, 113.68, 55.31,17.26.
DM-BDP-SAr. The synthesis of DM -BDP-SAr was analogous to the
synthesis of DM-BDP-R-SAr. M. P. 186–188 °C ; 1H-NMR (400 MHz,
CDCl3): δ 7.79 (d, J = 8.8 Hz, 4H, 3,5-ArH), 7.21 (d, J = 8.8 Hz, 2H, 8-
ArH), 6.92 (d, J = 8.8 Hz, 4H, 3,5-ArH), 6.86 (d, J = 8.8 Hz, 2H, 8-ArH),
6.36 (s, 2H, Pyrrole-H), 3.83 (s, 6H, 3,5-Ar-OCH3), 3.77 (s, 3H, 8-Ar-
OCH3), 2.51 (s, 6H, 1,7-CH3); 13C-NMR (100 MHz, CDCl3): δ 160.63;
158.31; 156.36; 143.64; 136.32; 135.57; 131.10; 128.11; 127.45; 125.09;
123.36; 115.32; 113.66; 55.29; 29.73; 17.62. HRMS-MALDI: Calculated
for [C32H29BF2N2O3S]+: 569.2005; found 569.1991.
Bis(3-methyl-5-(4-(2-morpholinoethoxy)phenyl)-1H-pyrrol-2-yl)meth-
anone (4c). The synthesis of compound 4c was analogous to the
synthesis of compound 1c. Only the characterization data was given here.
1H-NMR (400 MHz, CDCl3): δ 9.96 (s, 2H), 7.45 (d, J = 8.5 Hz, 4H), 6.82
(d, J = 8.6 Hz, 4H), 6.30 (s, 2H), 4.02 (t, J = 5.5 Hz, 4H), 3.72–3.67 (m,
8H), 2.73 (t, J = 5.5 Hz, 4H), 2.53 (d, J = 3.9 Hz, 8H), 2.22 (s, 6H).
2-(4’-Morpholineethoxyphenyl)-4-methyl pyrrole (3b). The synthesis
of compound 3b was analogous to the synthesis of compound 1b. 1H-
NMR (400 MHz, CDCl3): δ 8.31 (s, 1H), 7.35 (d, J = 8.7 Hz, 2H), 6.88 (d,
J = 8.7 Hz, 2H), 6.56 (s, 1H), 6.26 (s, 1H), 4.11 (t, J = 5.7 Hz, 2H), 3.78–
3.72 (m, 4H), 2.81 (t, J = 5.7 Hz, 2H), 2.64–2.56(m, 4H), 2.16 (s, 3H).
8-Chloro-1,7-dimethyl-3,5-di(p-morpholinoethoxypheny)l-4,4-difluo-
ro-4-bora-3a,4a-diaza-s-indacene (4d). The synthesis of compound 4d
was analogous to the synthesis of compound 1d. Only the
characterization data were given here. 1H-NMR (400 MHz, CDCl3): δ
7.75 (d, J = 8.7 Hz, 4H, 3,5-ArH), 6.90 (d, J = 8.7 Hz, 4H, 3,5-ArH), 6.40
(s, 2H, Pyrrole-H), 4.14 (t, J = 5.5 Hz, 4H, -ArOCH2-), 3.77–3.71 (m, 8H, -
O(CH2)2), 2.83 (t, J = 5.5 Hz, 4H, -CH2N-), 2.62 (d, J = 3.9 Hz, 8H, -
N(CH2)2), 2.56 (s, 6H, 1,7-CH3); 13C-NMR (100 MHz, CDCl3): δ 159.69;
155.76; 142.56; 136.70; 131.30; 131.04; 125.06; 122.81; 114.23; 66.73;
65.56; 57.49;53.97; 17.17.
(5-(4-Methoxyphenyl)-3-methyl-1H-pyrrol-2-yl)(3-methyl-5-(4-(2-mor-
pholinoethoxy)phenyl)-1H-pyrrol-2-yl)methanone (3c). A solution of
triphosgene (0.30 g, 1.0 mmol) in 1,2-dichloroethane (10 mL) was added
to
a stirred solution of compound 2b (561 mg, 3 mmol) and
ethyldiisopropylamine (0.50 mL, 3.0 mmol) in 1,2-dichloroethane (10 mL)
over 10 min at 0 °C under nitrogen. After stirred for 1 h at 0 °C , the
compound 3b (0.86 g, 3.0 mmol) was added. The reaction mixture was
stirred at 35 °C for 4 h. Then, the reaction mixture was treated with
saturated NaCl solution and the resulting solution was extracted with
CH2Cl2. The extract was washed with H2O and brine, dried with
anhydrous Na2SO4, filtered and evaporated. The purification of the crude
product was carried out by silica gel column chromatography
(CH2Cl2/ethyl acetate, 3 : 1, v/v) to yield yellow solid (0.53 g, 35%). 1H-
NMR (400 MHz, CDCl3): δ 7.84–7.70 (m, 4H), 6.91 (d, J = 8.5 Hz, 4H),
6.41 (d, J = 10.4 Hz, 2H), 4.39 (s, 2H), 3.95 (s, 4H), 3.83 (s, 3H), 3.16 (s,
2H), 2.99 (s, 4H), 2.57 (s, 6H).
Lyso-D. The synthesis of Lyso-D was analogous to the synthesis of DM-
BDP-R-SAr. Only the characterization data were given here. M. P. 92–
95 °C ; 1H-NMR (400 MHz, CDCl3): δ 7.77 (d, J = 8.8 Hz, 4H, 3,5-ArH),
7.20 (d, J = 8.8 Hz, 2H, 8-ArH), 6.90 (d, J = 8.8 Hz, 4H, 3,5-ArH), 6.86 (d,
J = 8.9 Hz, 2H, 8-ArH), 6.35 (s, 2H, Pyrrole-H), 4.14 (t, J = 5.6 Hz, 4H, -
ArOCH2-), 3.77 (s, 3H, 8-Ar-OCH3), 3.76–3.71 (m, 8H, -O(CH2)2), 2.81 (t,
J = 5.6 Hz, 4H, -CH2N-), 2.63–2.55 (m, 8H, -N(CH2)2), 2.51 (s, 6H, 1,7-
CH3); 13C-NMR (100 MHz, CDCl3): δ 159.78; 158.36; 156.28; 143.67;
136.35; 135.75; 131.10; 128.12; 127.46; 125.29; 123.37; 115.35; 114.20;
66.90; 65.73; 57.57; 55.38; 54.06; 17.58; HRMS-MALDI: Calculated for
[C42H47BF2N4O5SNa]+: 791.3232; found 791.3220.
8-Chloro-1,7-dimethyl-3-(p-methoxyphenyl)-5-(p-morpholinoethoxy-
phenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
(3d).
The
Acknowledgements
synthesis of compound 3d was analogous to the synthesis of compound
1d. 1H-NMR (400 MHz, CDCl3): δ 7.77 (dd, J = 8.8, 3.7 Hz, 4H, 3,5-ArH),
6.91 (d, J = 8.8 Hz, 4H, 3,5-ArH), 6.40 (s, 2H, Pyrrole-H), 4.12 (t, J = 5.6
Hz, 2H, -ArOCH2-), 3.81 (s, 3H, 3-Ar-OCH3), 3.77–3.71 (m, 4H, -O(CH2)2),
2.80 (t, J = 5.6 Hz, 2H, -CH2N-), 2.61–2.51 (m, 10H, -N(CH2)2 and 1,7-
This research w as supported by the National Natural Science
Foundation of China (No. 21372063, No. 21702046, No.
21703077) and Project Funded by China Postdoctoral Science
Foundation (2018M632757). The authors thank Dr. Weiw ei
7
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10.1002/chem.201902301
Chemistry - A European Journal
FULL PAPER
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10.1002/chem.201902301
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Layout 1:
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In this w ork, w e investigate the impact
of substituents on 1,7 positions of the
Jinhua Gao,[a] Yuanfang Tao,[a] Jian
Zhang,*[a] Nannan Wang,[a] Xin Ji,[a]
Jinling He,[a] Yubing Si,*[c] and Weili
Zhao*[ab]
meso-arylmercapto
BODIPY
to
biothiols. DM-BDP-SAr turned out to
be a preferred probe for Cys w ith
faster reaction rate. We further
modified the probe w ith lysosomal
targeting morpholine moiety and
developed Lyso-S and Lyso-D as
selective probes for Cys detection in
lysosome.
Page No. – Page No.
Development of lysosome-targeted
fluorescent probes for Cys by
regulating
structure
BODIPY
molecular
10
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