PH-dependent fluorescent probe that can be tuned for cysteine or homocysteine

Article abstract of DOI:10.1021/acs.orglett.6b03357

The very close structural similarities between cysteine and homocysteine present a great challenge to achieve their selective detection using regular fluorescent probes, limiting the biological and pathological studies of these two amino thiols. A coumarin-based fluorescent probe was designed featuring pH-promoted distinct turn-on followed by ratiometric fluorescence responses for Cys and turn-on fluorescence response for Hcy through two different reaction paths. These specific responses demonstrate the activity differences between Cys and Hcy qualitatively for the first time. The probe could also be used for Cys and Hcy imaging in living cells.

Full text of DOI:10.1021/acs.orglett.6b03357

Letter
pubs.acs.org/OrgLett
pH-Dependent Fluorescent Probe That Can Be Tuned for Cysteine or
Homocysteine
Yongkang Yue,†,⊥ Fangjun Huo,‡,⊥ Xiaoqi Li,† Ying Wen,† Tao Yi,*,§ James Salamanca,∥
Jorge O. Escobedo,∥ Robert M. Strongin,*,∥ and Caixia Yin*,†
†Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Key
Laboratory of Materials for Energy Conversion and Storage of Shanxi Province and ‡Research Institute of Applied Chemistry, Shanxi
University, Taiyuan 030006, China
§Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai
200433, China
∥Department of Chemistry, Portland State University, Portland, Oregon 97201, United States
S
* Supporting Information
ABSTRACT: The very close structural similarities between cysteine and
homocysteine present a great challenge to achieve their selective detection
using regular fluorescent probes, limiting the biological and pathological
studies of these two amino thiols. A coumarin-based fluorescent probe was
designed featuring pH-promoted distinct turn-on followed by ratiometric
fluorescence responses for Cys and turn-on fluorescence response for Hcy
through two different reaction paths. These specific responses demonstrate
the activity differences between Cys and Hcy qualitatively for the first time.
The probe could also be used for Cys and Hcy imaging in living cells.
For GSH, its tripeptide structure hinders the attack by the
ysteine (Cys), homocysteine (Hcy), and glutathione
C(GSH) are closely related to the pathology of many amino group. Other probes have been developed in recent
years that were based on this design strategy and have made
additional progress in biothiol detection.6d,7 Thus, the focus
should be to further develop fluorescent probes that address
discriminative detection between Cys and Hcy.8,7d Concen-
diseases. The deficiency of Cys was reported to cause slowed
growth, edema, liver damage, skin lesions, and weakness.1 The
normal intracellular concentration of Cys is 30−200 μM.2 For
Hcy, the normal concentration in serum is approximately 5−12
μM. Excess Hcy is associated with diseases including
Alzheimer’s, neural tube defects, and mental disorders.3 GSH
intracellular concentrations are 1−10 mM.4 Because of the
similar structures and chemical properties among Cys, Hcy, and
GSH, most commercially available probes for thiols do not
enable their discrimination, thus impeding further studies in
their roles on pathogenesis and physiological activities.5 There
have been few probes reported to date with distinct detection
characteristics for these three biothiols.6 In 2011, the Strongin
group utilized the acrylate moiety to synthesize a benzothiazole
derivative for discriminative detection of exogenous Cys and
Hcy in human plasma through a nucleophilic addition-
cyclization process.6a In a later work, a micelle-catalyzed
detection procedure specific for GSH was developed on the
basis of the same strategy.6b Subsequently, the Yang group
reported a BODIPY-based fluorescent probe for specific
detection of GSH over Cys and Hcy through a nucleophilic
chloride displacement by the thiol group.6c For Cys and Hcy,
the thiol-substituted products further undergo an intra-
molecular substitution of the thiol group by the amino group.
trated on this point, the distinction of the pKa values (pKa Cys
=
8.0; pKa Hcy = 8.87; pKa GSH = 9.20), which may reflect the
nucleophilic addition activity of thiol moieties depend on the
pH environment of the three biothiols and afford the
opportunity to detect them separately.9 Additionally, the
different steric-hindrance effects of the three biothiols can
also be used for discriminant detection.10 For the reaction site,
the acryloyl group displayed excellent sensitivity and selectivity
for Cys,11 especially the work for discriminant detection of Cys
and Hcy as reported by the Strongin group.6a Based on this
schematic, we modified the coumarin moiety to synthesize
probe 1 for fluorescence detection of thiols (Scheme 1). The
detection mechanisms of probe 1 toward Cys and Hcy were the
thiol-induced nucleophilic addition−cyclization process and
sensitive pH-promoted nucleophilic addition in aqueous
solution, respectively. 1H NMR titration experiments and
mass (MS) data confirmed these processes.
Received: November 10, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b03357
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Encouraged by our previous work12 and the emergence of
the weak responses of probe 1 toward Hcy in the above system,
we further measured the time-dependent UV−vis and
fluorescence spectra in Hepes buffer/DMSO (1:1, v/v) with
a slightly higher pH value (pH = 7.8). As expected, the
responses of probe 1 toward both Cys and Hcy were
accelerated (Figure 2). As a result, the turn-on fluorescence
Scheme 1. Design of Probe 1 and the Divisional Detection of
Cys and Hcy
The spectroscopic properties of probe 1 toward biothiols
were measured through time-dependent UV−vis and fluo-
rescence spectrometry in Hepes buffer/DMSO (1:1, v/v, pH
7.4) solution. As shown in Figure 1a, probe 1 itself displayed
Figure 2. (a, b) Time-dependent fluorescence emission spectra of 1
(30 μM) in the presence of 10 equiv of Cys in Hepes buffer/DMSO
(1:1, v/v, pH 7.8) at 25 °C. (c) Time-dependent fluorescence
emission spectra of 1 (30 μM) in the presence of 10 equiv of Hcy in
Hepes buffer/DMSO (1:1, v/v, pH 7.8) at 25 °C. (d) Fluorescence
spectra of 1 (30 μM) upon addition of 10 equiv of Cys, Hcy, GSH,
Ala, Asn, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, Val in Hepes buffer/DMSO (1:1, v/v, pH 7.8) at 25 °C.
(λex = 447 nm, slit: 5 nm/5 nm).
responses of probe 1 to Cys at 499 nm peaked within 12 min,
then decreased, accompanied by the increase of a new signal
centered at 554 nm over extended periods of time (Figure
2a,b). Corresponding to the fluorescence changes, the UV−vis
responses of probe 1 to Cys displayed an apparent blue shift
within the first 30 min and then reversed with red shift changes
in the subsequent 90 min (Figure S6). Neither Hcy nor GSH
induced the second fluorescence signal in the present detection
system. Interestingly for GSH, pH 7.8 induced a slight
responses compared to pH 7.4, which further proved the pH-
regulated nucleophilic addition activities of the three thiols. The
specific pH-promoted changes of probe 1 toward Cys may be
caused by the stepwise detection processes presented in
Scheme 1. The pH modulated turn-on and subsequent
ratiometric fluorescence responses of probe 1 with Cys, Hcy,
and GSH provided chances for distinct detection of the three
similar biothiols. To verify the threshold of the pH-regulated
two-stage responses, we further studied the detection systems
at pH 7.6 and 8.0. The result showed that the differences in the
fluorescence sensing of probe 1 toward Cys, Hcy, and GSH
occur when the pH value of the system exceeds 7.6 and that
there is a proportional relation between fluorescence change
and increasing pH value (Figures S5 and S7). These specific
fluorescence responses induced by minor pH changes reflected
the activity differences among Cys, Hcy, and GSH qualitatively.
The probe not only showed a pH-promoted sensitive
response to the biothiols but also had an excellent selectivity
toward Cys, Hcy, and GSH over various canonical amino acids.
As shown in Figure 1c, only Cys and Hcy could induce the
Figure 1. (a) Time-dependent fluorescence emission spectra of 1 (30
μM) in the presence of 10 equiv of Cys in Hepes buffer/DMSO (1:1,
v/v, pH 7.4) at 25 °C. (b) Corresponding time-dependent UV−vis
spectra of 1 (30 μM) in the presence of 10 equiv of Cys in Hepes
buffer/DMSO (1:1, v/v, pH 7.4) at 25 °C. (c) Fluorescence emission
spectra of 1 (30 μM) upon addition of 10 equiv of Cys, Hcy, GSH,
Ala, Asn, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser,
Thr, Trp, Tyr, and Val in Hepes buffer/DMSO (1:1, v/v, pH 7.4) at
25 °C. (d) Time-dependent fluorescence emission intensity changes of
1 toward 10 equiv of biothiols at 499 nm (λex = 447 nm, slit: 5 nm/5
nm).
dim fluorescence emission at 559 nm. However, the addition of
Cys into the probe 1 containing buffer induced a significant
blueshifted fluorescence emission at 499 nm with the excitation
at 447 nm and plateaued with 44-fold enhancement in 30 min.
The corresponding UV−vis spectrum displayed 7 nm blue shift
within 60 min (Figure 1b). For Hcy addition, a quarter-fold
fluorescence emission enhancement at 499 nm was observed
compared with the Cys-probe system, and the UV−vis
spectrum was almost invariable (Figure S4). At the same
time, the responses of probe 1 toward Hcy in this system were
hysteretic (Figure 1d). However, GSH was virtually inert to
probe 1 in the detection environment (Figure S4). These
optical responses of probe 1 toward the three thiol-containing
amino acids might be caused by the nucleophilic addition
process displayed in Scheme 1, which resulted in the specific
detection of Cys in Hepes buffer/DMSO (1:1, v/v, pH 7.4).
B
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turn-on fluorescence responses at 499 nm within 60 min, and
other amino acids (which included GSH) did not interfere with
the detection process. Furthermore, in the Hepes buffer/
DMSO (1:1, v/v, pH 7.8) system, probe 1 displayed distinct
fluorescence responses toward Cys and Hcy without interfer-
ence of other amino acids (Figure 2d). Similarly, the detection
processes in the pH 7.6 and 8.0 systems maintained the same
results as above (Figure S8). These data showed that probe 1
could selectively detect Cys and Hcy through two emission
channels.
Figure 4. (a, d) Confocal fluorescence image of HepG2 cells
preincubated with 1 mM NEM for 30 min and further incubated
with HBSS (Hanks’ Balanced Salt Solution (with Ca2+, Mg2+)) of pH
7.4 in the presence of 10 μM nigericin and 5 μM probe 1 for 30 min.
(b, e) Using the control procedures, the cells were further incubated
with 100 μM Cys in HBSS of pH 7.4 in the presence of 10 μM
nigericin for 60 min. (c, f) Using the control procedures, the cells were
further incubated with 100 μM Hcy in HBSS of pH 7.4 in the presence
of 10 μM nigericin for 60 min. (g) Cross-sectional analysis along the
white line in the insets (single cell in white squares in (b) and (c),
respectively). λex = 458 nm; scale bar =30 μm. Green channel: 500
20 nm.
To further confirm the detection mechanisms of probe 1
1
toward Cys and Hcy, we carried out time-dependent H NMR
experiments upon addition of equivalent amounts of Cys and
Hcy to probe 1 in DMSO-d6 (Figure S1). As shown in Figure 3,
emission (a). However, the further exogenous Cys and Hcy
induced noticeable fluorescence emission (b, c), and interest-
ingly, the cross-sectional analysis of a single cell for Cys and
Hcy respectively displayed distinct intensity differences (g). At
the same time, the exogenous GSH displayed nonvariance with
the controlled trial (Figure S10). These results demonstrated
that probe 1 could detect Cys and Hcy specifically under
physiological conditions.
To further value the discriminative detection of Cys and Hcy
in living cells, we measured the fluorescence imaging
experiments with HepG2 cells in pH 7.8. As shown in Figure
5, cells precultured with 1 mM NEM and then 5 μM probe 1 in
HBSS buffer (containing 10 μM nigericin) displayed nearly
nonfluorescence emission in both the green and red channels
(a, d). Further incubation with Cys induced distinct
1
Figure 3. Time-dependent H NMR experiments of probe 1 toward
Cys and Hcy in DMSO-d6. Spectra for probe 1 + Cys, and probe 1 +
Hcy were obtained 30 min after addition.
the addition of Cys reduced the signals at 6.19, 6.44, and 6.57
ppm (j, k) that belong to the acryloyl group and disappeared
completely after 30 min. At the same time, the signals at 7.29
(h) and 7.80 ppm (g) reduced gradually and two new signals at
6.84 and 7.57 emerged which are consistent with the 1H NMR
data of compound 5. These signal changes demonstrated how
the nucleophilic addition−cyclization process occurred during
the detection process of probe 1 toward Cys selectivity.
Furthermore, the HR-MS data of the Cys-probe 1 system in
Figure S2 also supported the same aforementioned mechanism.
1
For Hcy, however, the original H NMR signals at 6.19, 6.44,
and 6.57 ppm (j, k) decreased while other signals downfield
remained the same (Figure 3), which indicated that the olefinic
bond of the acryloyl group in probe 1 was broken but the ester
group still remained intact. Moreover, in the ESI-MS data in
Figure S3 we found m/z = 574.75 for compound 6 (n = 2) [M
+ Na]+. These changes in the reaction processes may be caused
by the more stable 7-membered ring in the case of Cys
compared to the 8-membered ring for Hcy.
To evaluate the applicability of probe 1 in biological systems,
we measured the MTT assay with HepG2 cells, and the results
showed minimal cytotoxicity of probe 1 at a concentration of
50 μM (87.6% viability) (Figure S9). The cell-imaging
experiments were measured with HepG2 cells in the pH 7.4
system. As shown in Figure 4, cells preincubated with 1 mM
NEM and then 5 μM probe 1 in HBSS buffer (containing 10
μM nigericin, an H+/K+ ionophore to homogenize the intra-
and extracellular pH)13 displayed nearly nonfluorescence
Figure 5. (a, d, g) Confocal fluorescence image of HepG2 cells
preincubated with 1 mM NEM for 30 min and further incubated with
HBSS (Hanks’ Balanced Salt Solution (with Ca2+, Mg2+)) of pH 7.8 in
the presence of 10 μM nigericin and 5 μM probe 1 for 30 min. (b, e,
h) Using the control procedures, the cells were further incubated with
100 μM Cys in HBSS of pH 7.8 in the presence of 10 μM nigericin for
60 min. (c, f, i) Using the control procedures, the cells were further
incubated with 100 μM Hcy in HBSS of pH 7.8 in the presence of 10
μM nigericin for 60 min. (j, k) Cross-sectional analysis along the white
line in the insets (single cell in white squares in (b) and (c), (e) and
(f) respectively). λex = 458 nm; scale bar =30 μm. Green channel: 500
20 nm; Red channel: 600 25 nm.
C
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fluorescence emission in the two emission channels (b, e). As
for Hcy, these cells displayed strong fluorescence emission in
the green channel but very low emission in the red channel (c,
f). The cross-sectional analysis of a single cell in the green and
red channels for Cys and Hcy, respectively, displayed a large
signal ratio that demonstrated the utility of probe 1 for
discrimination detection of Cys and Hcy in living cells (j, k)
(Figure S11). Consistently, the exogenous GSH could not
induce the fluorescence responses in the pH 7.8 system (Figure
S12). These minor pH changes induced fluorescence responses
of probe 1 toward Cys and Hcy in living cells promoted a
deeper insight into the activities of the biothiols in biological
systems.
In conclusion, we designed a coumarin-based fluorescent
probe for discriminative detection of Cys and Hcy through two
emission channels. The reaction mechanism involves a
nucleophilic addition step followed by intramolecular cycliza-
tion and cleavage. Probe 1 was able to detect Cys through a
turn-on-ratiometric fluorescence response when the pH of the
reaction was set at 7.6, while Hcy would only undergo the
nucleophilic addition step with a turn-on fluorescence signal.
Increasing the reaction pH up to 8.0 modulated the turn-on
fluorescence at 499 nm of probe 1 toward Cys, Hcy, and GSH
with high selectivity. Further, imaging studies with HepG2 cells
showed that probe 1 can detect Cys and Hcy in live cells using
two emission channels. These results have promoted ongoing
related studies of fluorescent probes for thiols in subcellular
structures at either pH 8.0 in mitochondria or at pH 4.5 in
lysosomes. The pH-promoted detection mechanism provides a
new pathway for the design of thiol probes and may bring a
deeper insight into the biological activities of these amino
thiols.
2014401), Shanxi Province Outstanding Youth Fund (No.
2014021002), Shanxi University funds for study abroad 2014,
and the National Institutes of Health (No. R15EB016870).
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b03357.
Synthesis of the probe, structure characterizations,
additional UV−vis and fluorescence spectra, and addi-
tional fluorescence images (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
* E-mail: yitao@fudan.edu.cn
*E-mail: strongin@pdx.edu
*E-mail: yincx@sxu.edu.cn
ORCID
Caixia Yin: 0000-0001-5548-6333
Author Contributions
⊥Y.Y. and F.H. contributed equally.
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O.; Huynh, K. T.; Hakuna, L.; Strongin, R. M. Chem. Commun. 2014,
50, 8219−8222. (c) Barve, A.; Lowry, M.; Escobedo, J. O.;
Thainashmuthu, J.; Strongin, R. M. J. Fluoresc. 2016, 26, 731−737.
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Chem., Int. Ed. 2014, 53, 10916−10920. (c) Chen, Y.; Zhu, C.; Cen, J.;
Bai, Y.; He, W.; Guo, Z. Chem. Sci. 2015, 6, 3187−3194.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported by the National Natural Science
Foundation of China (Nos. 21472118 and 21672131), the
Program for the Top Young and Middle-aged Innovative
Talents of Higher Learning Institutions of Shanxi (No.
2013802), Talents Support Program of Shanxi Province (No.
D
DOI: 10.1021/acs.orglett.6b03357
Org. Lett. XXXX, XXX, XXX−XXX