A cysteamine-selective two-photon fluorescent probe for ratiometric bioimaging

Article abstract of DOI:10.1039/c4cc09416f

We report a two-photon fluorescent probe for ratiometric imaging of cysteamine in situ. This probe can detect the levels of endogenous cysteamine with statistical significance in live cells and brain hippocampal tissues, revealing that cysteamine is localized mainly in the perikaria of the pyramidal neurons and the granule cells.

Full text of DOI:10.1039/c4cc09416f

ChemComm
View Article Online
View Journal
COMMUNICATION
A cysteamine-selective two-photon fluorescent
probe for ratiometric bioimaging†
Cite this:DOI: 10.1039/c4cc09416f
Avik R. Sarkar,‡a Cheol Ho Heo,‡a Eunjin Kim,b Hyo Won Lee,a Hardev Singh,a
Jeong Jin Kim,a Hyuk Kang,a Chulhun Kang*b and Hwan Myung Kim*a
Received 25th November 2014,
Accepted 17th December 2014
DOI: 10.1039/c4cc09416f
www.rsc.org/chemcomm
We report a two-photon fluorescent probe for ratiometric imaging of in the pKa values and the bulkiness of the target thiol. However,
cysteamine in situ. This probe can detect the levels of endogenous the selectivity of these thiols toward CS has never been reported.
cysteamine with statistical significance in live cells and brain hippo- Moreover, in application to tissues or animals, short excitation
campal tissues, revealing that cysteamine is localized mainly in the wavelengths ranging from UV to visible light used in these
perikaria of the pyramidal neurons and the granule cells.
studies may be limited by cellular autofluorescence, artifactual
generation of reactive oxygen species (ROS), and shallow penetra-
Cysteamine (CS), a simple and reactive aminothiol originating tion depths.
from the degradation of coenzyme A (CoA) to taurine, is distri-
Two-photon microscopy (TPM) has become an important
buted in all living organisms.1–6 CS plays key roles in various tool for biomedical applications. TPM uses two near-infrared
physiological processes such as amino acid transport,2 protein photons with femtosecond pulses as the excitation source, provid-
synthesis,3 reduction of disulfides,4 protection against oxidative ing various advantages including inherent sectioning capability,
damage,5 and improvement of embryo development rates.6 low photodamage, longer observation time, and greater tissue
In medical application, CS has been approved by FDA for the penetration depth.11–14 In combination with TPM, emission ratio-
treatment of cystinosis, a progressive disorder of multiple organs metric probes utilizing a single excitation source would be an ideal
caused by abnormal intracellular accumulation of cystine.7 Also, approach to quantitative detection of biological species in living
CS treatment has been suggested to be clinically effective in specimens, because the dual-intensity ratio between the reacted
neurodegenerative disorders.8 Despite the clinical uses of CS in and unreacted populations of the probe can eliminate the experi-
human diseases, its intracellular level and underlying therapeutic mental artifacts such as local probe concentration and incident
mechanism are barely known. The precise measurement of CS at laser power.13 Hence, there is a strong need to develop an emission
the cell, tissue, and organism levels would be crucial to further the ratiometric probe for CS that can be applicable to TPM.
understanding of the roles of CS in biology and medicine.
However, to the best of our knowledge, the detection method of for selectively and precisely monitoring CS over other biothiols
CS in situ has never been exploited. in live cells and tissues. The probe was designed by a combi-
Herein, we report a ratiometric TP fluorescent probe (SCS1)
To detect thiols in cells, a number of fluorescent probes have nation of three main components, an acrylate unit as the thiol
been developed which contain various functional groups includ- reactive site, 6-(benzo[d]thiazol-20-yl)-2-(N,N-dimethylamino)
ing disulfide groups as selective thiol detection moieties.9 These naphthalene (BTDAN) as a two-photon reporter, and an oligo-
efforts, however, have been hampered by their poor selectivity meric ethylene glycol unit as a solubility enhancing group.
toward a specific thiol. Recently, probes bearing acrylates as the Acrylates are well-known reaction sites for Cys and Hcy which
thiol-reactive moieties have been actively introduced.10 Attempts undergo a condensation reaction followed by intramolecular
to control their selectivity toward cysteine (Cys), homocysteine cyclization to produce 5-oxoperhydro-1,4-thiazepine derivatives.10h
(Hcy), or glutathione (GSH) have been based upon the differences To design a CS-selective probe, a flexible long chain of ethylene
glycol was employed to interact strongly with the fluorophore part
a Department of Chemistry and Department of Energy Systems Research,
Ajou University, Suwon 443-749, Korea. E-mail: kimhm@ajou.ac.kr;
Fax: +82-31-219-1615
in an aqueous medium, so that the interaction would render the
thiol group in the less sterically hindered CS more reactive than
those of Cys/Hcy in the reaction with the acrylates. In addition, the
acrylate moiety is attached at the electron donating amine of
BTDAN via a self-immolative spacer, [4-((carbamoyloxy)methyl)-
phenolate] (Scheme 1).14a Therefore, a thiol-mediated reaction
b East–West Medical Science, Kyung Hee University, Yongin 446-701, Korea.
E-mail: kangch@khu.ac.kr
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc09416f
‡ These authors equally contributed to this work.
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun.

View Article Online
Communication
ChemComm
Scheme 1 Structures of SCS1 and 1, and the proposed mechanism of the
reaction of SCS1 with cysteamine.
would trigger the elimination of the carbamate linkage and
liberate the strong electron donor, thereby shifting the emission
maximum to the red region.
The synthetic procedure of SCS1 is described in the ESI†
(Scheme S1). The solubility values of SCS1 and 1 in phosphate
buffered saline (PBS, 10 mM, pH = 7.4) as determined by
fluorescence methodology15 were 2 and 6 mM, respectively
(Fig. S1, ESI†). The solubility values of SCS1 in fresh culture
media for live cells and tissues were approximately 6 mM (Fig. S1,
ESI†), which are higher than measured in the PBS. In PBS solution,
SCS1 exhibited an absorption maximum (labs) at 335 nm
(e = 31 719 Mꢀ1 cmꢀ1) and a fluorescence maximum (lfl) at
460 nm (F = 0.75) whereas the corresponding values for 1, the
reaction product of SCS1 according to Scheme 1, were 376 nm
(e = 26 551 Mꢀ1 cmꢀ1), and 540 nm (F = 0.34), respectively
(Table 1). The larger Stokes shift observed in 1 compared to
SCS1 can be attributed to the greater stabilization of the charge-
transfer excited state in the former due to the presence of a
stronger electron-donating group.16
Fig. 1 (a) One-photon fluorescence response with time for the reaction
of SCS1 (1 mM) with cysteamine (CS, 1 mM) in PBS buffer (10 mM, pH 7.4).
(b) Plot of kobs vs. concentration of CS. (c) Fluorescence intensity ratio of
SCS1 toward CS, thiol derivatives, H2O2, and Na2S. Bars represent the ratio
(F540/F460) of the emission intensities before, 30 min and 1 h after addition
of the CS, thiol derivatives, H2O2, and Na2S. Data were acquired in PBS
buffer (10 mM, pH 7.4). The excitation wavelength was 370 nm.
The detection limit of CS with SCS1 was found to be 0.81 mM
(Fig. S4, ESI†).
In the presence of 1 mM CS, SCS1 showed a 47-fold incre-
ment of the intensity ratios (Fyellow/Fblue) at 410–460 (Fblue) and
530–580 nm (Fyellow) (Fig. 1a, Table 1). Moreover, the change in
the Fyellow/Fblue remained unperturbed in the presence of other thiol
derivatives including Cys, Hcy, GSH, cystine, 2-mercaptoethanol
(2-ME), taurine, and cystamine (an oxidized form of CS), and
H2O2 and H2S (Fig. 1c). Similar results were obtained with
various amino acids and metal ions (Fig. S5, ESI†). Further,
SCS1 showed a strong response upon addition of CS even in the
presence of excess amounts of other thiols (Fig. S6, ESI†). The
high selectivity for CS over Cys, Hcy, and GSH is particularly
interesting because other acrylate derivatives do not have such
selectivity.10
To explain this phenomenon, we computationally investi-
gated the structure of SCS1 by molecular dynamics (MD) and
subsequently by density functional theory (DFT).18 We first
searched for stable conformations of SCS1 by MD, in which
stable conformers including the most stable one had an ethylene
glycol chain folded around the fluorophore, while the least stable
one (DE E 60 kJ molꢀ1) had a linear chain. The structures of the
most stable conformer and two more representative folded ones
were further optimized with DFT at the B3LYP/6-31G level. The
polarization continuum model (PCM) with water as a solvent was
used to account for the solvent effect.18b The results suggested
that SCS1 always adopts folded conformations (Fig. 2 and
Fig. S7, and Table S1, ESI†). In addition, it has been established
The reaction between SCS1 and CS produced 1 as the only
product as monitored by emission spectra (Fig. 1a) and HPLC
analysis (Fig. S2, ESI†). The emission spectra of a 1 mM solution
of SCS1 treated with 1 mM CS in PBS buffer increased gradually at
540 nm with a simultaneous decrease at 460 nm and with an
intense isoemmisive point at 505 nm (Fig. 1a). This process followed
pseudo-first-order kinetics with kobs = (1.7 ꢁ 0.1) ꢂ 10ꢀ3 sꢀ1 (Fig. S3,
ESI†). Moreover, the plot of kobs vs. various concentrations
(0.90–5.0 mM) of CS was a straight line passing through the
origin (Fig. 1b) indicating that the reaction is overall second-
order with k2 = 4.1 ꢁ 0.1 Mꢀ1
s
ꢀ1. This value was more than
175-times larger than those for disulfide-derived probes.17
Table 1 Photophysical data for SCS1 and 1a
(1)
max
fl
max
c
e
(2) f
Cpd
l
(10ꢀ4e)b
l
Fd
Rmax/Rmin
l
max
Fdg
SCS1
1
335 (3.17)
376 (2.66)
460
540
0.75
0.34
47
740
750
27
82
a
b
All data were measured in PBS buffer (10 mM, pH 7.4). lmax of the
one-photon absorption spectra in nm. The numbers in parentheses are
c
molar extinction coefficients in Mꢀ1 cmꢀ1
.
lmax of the one-photon
d
e
emission spectra in nm. Fluorescence quantum yield, ꢁ10%. Emis- that the fluorophore part and/or peripheral group can affect the
binding affinity.19 Therefore, the selectivity for CS may be attri-
buted to the folded-like structure of SCS1, which may increase the
sion ratio (F530–580/F410–460) conversion factor, Rmax/Rmin, measured by
one-photon processes before and 1 h after addition of 1 mM cysteamine.
f
The peak two-photon cross section in 10ꢀ50 cm4 s per photon (GM),
g
ꢁ15%. Two-photon action cross section.
electric and/or steric repulsion for the carboxylate-containing Cys,
Chem. Commun.
This journal is ©The Royal Society of Chemistry 2015

View Article Online
ChemComm
Communication
Fig. 2 Optimized geometries of SCS1: (a) the linear conformer and (b) the
most stable conformer. The structure of (a) was optimized only by Amber*
force field because it converged to a folded structure at the DFT level, and
the folded structure (b) was optimized by B3LYP/6-31G with a polarization
continuum model for the solvent effect.
Hcy and GSH compared to more neutral and smaller CS. The
selectivity over 2-mercaptoethanol might be due to the slow rate
of the cyclization step, as reported in ref. 10c. Further, SCS1 was
pH-insensitive over a biologically relevant pH range (Fig. S8,
ESI†). These results implied that SCS1 can serve as a ratiometric
fluorescent probe for CS without interference by pH or from
other biologically relevant analytes.
The TP action cross section values (Fdmax, where d is the TP
absorption cross section) of SCS1 and 1 were determined in PBS
buffer at pH 7.4. The values of SCS1 and 1 were 28 and 85 GM
(1 GM = 10ꢀ50 cm4 per photon) at 740 and 750 nm, respectively
(Table 1 and Fig. S9, ESI†). The larger value for 1 compared to
SCS1 can be attributed to the enhanced internal charge transfer
character.16 These Fdmax values were comparable to those of
existing TP probes and offered a bright TPM image of the living
samples. Indeed, upon 750 nm excitation, bright TPM images
of SCS1-labeled cells and tissues were obtained (Fig. 3 and 4).
Further, this probe displayed high photostability as monitored
by collecting TPEF for 1 h in the cells (Fig. S10, ESI†) and
negligible cytotoxicity under the imaging conditions as revealed
by cell viability tests using MTS assay (Fig. S11, ESI†).
Fig. 3 Pseudocolored ratiometric TPM images (Fyellow/Fblue) of U87 cells
incubated with 2 mM (a) SCS1 and (f) 1. U87 cells were pretreated with
(b) N-ethylmaleimide (NEM, 100 mM) for 15 min, (c) cysteamine (CS, 100 mM) for
30 min, (d) mercaptoethylguanidine (MEG, 25 mM) for 2 h, and (e) cysteamine
(100 mM) for 30 min and MEG (25 mM) for 2 h before labeling with SCS1.
(g) Average Fyellow/Fblue intensity ratios in the TPM images. Images were
acquired using 750 nm excitation and emission windows of 410–460 nm
(blue) and 530–580 nm (yellow). Cells shown are representative images
from replicate experiments (n = 5). Scale bars: 24 mm.
Next we tested whether SCS1 could detect intracellular CS in
live cells. A series of TPM experiments were conducted with U87
cells, a human astrocytoma cell. Using 750 nm excitation, the TPM
images of SCS1-labeled cells at 410–460 (Fblue) and 530–580 nm
(Fyellow) were simultaneously obtained and their ratios (Fyellow/Fblue
)
were analyzed (Fig. 3 and Fig. S12, ESI†). The average ratios
(Fyellow/Fblue) of U87 cells labeled with SCS1 and 1 were
0.53 ꢁ 0.04 and 1.13 ꢁ 0.05, respectively (Fig. 3a and f). Then
we treated the cells with N-ethylmaleimide (NEM), a well-known
thiol-blocker,20 for the depletion of intracellular thiol species.
We used 100 mM NEM because the damaged-cells were signifi-
cantly observed when greater amounts of NEM were applied.
Upon treatment of NEM, the ratio decreased to 0.44 ꢁ 0.04
(Fig. 3b). Further, the ratio increased from 0.53 to 0.75 ꢁ 0.04 by
addition of 100 mM CS into the media (Fig. 3a and c), revealing that
SCS1 can measure the intracellular level of CS with statistical signi-
ficance. Then the cells were treated with mercaptoethylguanidine
Fig. 4 Pseudocolored ratiometric TPM images (Fyellow/Fblue) of a rat hippo-
campal slice stained with 20 mM SCS1 for 1 h. (a) 35 TPM images along the
z-direction at the depths of approximately 90–180 mm were accumulated
with 10ꢂ magnification. (b–d) Enlarged images of the white box in (a) taken
with 40ꢂ magnification, (b) CA1, (c) CA3, and (d) DG regions. Images were
acquired using 750 nm excitation and emission windows of 410–460 nm
(blue) and 530–580 nm (yellow). Images shown are representative from
replicate experiments (n = 5). Scale bars: (a) 420 and (d) 48 mm.
(MEG), a potent inhibitor of cysteamine dioxygenase responsible for When the cells were treated with MEG, the ratio increased to
CS degradation,21 to induce accumulation of intracellular CS. 0.70 ꢁ 0.04 and even further to 0.93 ꢁ 0.05 with co-treatment of
This journal is ©The Royal Society of Chemistry 2015
Chem. Commun.

View Article Online
Communication
ChemComm
5 P. Guerin, S. El Mouatassim and Y. Menezo, Hum. Reprod. Update,
2001, 7, 175–189.
100 mM CS and MEG (Fig. 3d and e). These data indicate that
SCS1 is a sensitive and selective CS probe, with a signal strong
enough to allow the measurement of even endogenously pro-
duced CS in live cells.
6 D. G. de Matos, D. Nogueira, R. Cortvrindt, C. Herrera, T. Adriaenssens,
R. S. Pasqualini and J. Smitz, Mol. Reprod. Dev., 2003, 64, 214–218.
7 (a) A. Brodin-Sartorius, M. J. Tete, P. Niaudet, C. Antignac, G. Guest,
C. Ottolenghi, M. Charbit, D. Moyse, C. Legendre, P. Lesavre, P. Cochat
and A. Servais, Kidney Int., 2012, 81, 179–189; (b) W. A. Gahl, J. Z. Balog
and R. Kleta, Ann. Intern. Med., 2007, 147, 242–250.
8 (a) C. Gibrat and F. Cicchetti, Prog. Neuropsychopharmacol. Biol.
Psychiatry, 2011, 35, 380–389; (b) M. Bousquet, C. Gibrat, M. Ouellet,
C. Rouillard, F. Calon and F. Cicchetti, J. Neurochem., 2010, 114,
1651–1658.
We even further attempted CS measurement with SCS1 in a
living tissue, a freshly prepared rat hippocampus, a part of the
brain that plays an important role in learning and memory. As
displayed in Fig. 4a, CS is surprisingly abundant in the pyramidal
neuron layers and the granule cells in the CA1–CA3 regions and
the DG, respectively. Moreover, the higher-magnification images
(Fig. 4b–d) of these regions clearly showed the levels of CS in the
individual cells, in which CS mainly existed in the perikaryon of
the neurons. Indeed, this image is the first image showing the CS
distribution in brain tissue, which would certainly be highly
valuable for further understanding of the therapeutic mechanism
of CS in neurodegenerative diseases. These results clearly confirm
that SCS1 is suitable for monitoring CS deep inside living tissues,
possibly serving as a research tool for cysteamine-related therapy.
To close, we have exploited for the first time a CS-selective
and ratiometric TP fluorescent probe, SCS1. This probe shows a
marked blue-to-yellow emission color change in response to CS
in physiological buffer, high selectivity for CS over other thiols
and biologically relevant species, and pH insensitivity in the
biologically relevant pH ranges. This probe can precisely detect
CS at the subcellular level in live cells and living brain tissues
using TPM with minimum interference from cytotoxicity and
photobleaching artifacts. In addition, its application to hippo-
campal tissues revealed that CS is abundant in the cell bodies of
pyramidal cells and granule cells in CAs and DG, respectively.
These findings demonstrate that SCS1 could play an indispensable
role in understanding the biological and medicinal roles of CS in
living systems.
9 (a) M. H. Lee, Z. Yang, C. W. Lim, Y. H. Lee, S. Dongbang, C. Kang
and J. S. Kim, Chem. Rev., 2013, 113, 5071–5109; (b) X. Chen,
Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120–2135.
10 (a) Q. Zhang, D. Yu, S. Ding and G. Feng, Chem. Commun., 2014, 50,
14002–14005; (b) Y. Zhang, J.-H. Wang, W. Zheng, T. Chen, Q.-X.
Tong and D. Li, J. Mater. Chem. B, 2014, 2, 4159–4166; (c) X. Xiong,
F. Song, G. Chen, W. Sun, J. Wang, P. Gao, Y. Zhang, B. Qiao, W. Li,
S. Sun, J. Fan and X. Peng, Chem. – Eur. J., 2013, 19, 6538–6545;
(d) H. Zhang, P. Wang, Y. Yang and H. Sun, Chem. Commun., 2012,
48, 10672–10674; (e) H. Wang, G. Zhou, H. Gai and X. Chen, Chem.
Commun., 2012, 48, 8341–8343; ( f ) Z. Guo, S. Nam, S. Park and
J. Yoon, Chem. Sci., 2012, 3, 2760–2765; (g) H. S. Jung, T. Pradhan,
J. H. Han, K. J. Heo, J. H. Lee, C. Kang and J. S. Kim, Biomaterials,
2012, 33, 8495–8502; (h) X. Yang, Y. Guo and R. M. Strongin, Angew.
Chem., Int. Ed., 2011, 50, 10690–10693.
11 F. Helmchen and W. Denk, Nat. Methods, 2005, 2, 932–940.
12 (a) H. M. Kim and B. R. Cho, Acc. Chem. Res., 2009, 42, 863–872;
(b) S. Yao and K. D. Belfield, Eur. J. Org. Chem., 2012, 3199–3217;
(c) F. Liu, T. Wu, J. Cao, S. Cui, Z. Yang, X. Qiang, S. Sun, F. Song,
J. Fan, J. Wang and X. Peng, Chem. – Eur. J., 2013, 19, 1548–1553;
(d) L. Li, C. W. Zhang, G. Y. Chen, B. Zhu, C. Chai, Q. H. Xu, E. K.
Tan, Q. Zhu, K. L. Lim and S. Q. Yao, Nat. Commun., 2014, 5, 3276;
(e) X. Dong, C. H. Heo, S. Chen, H. M. Kim and Z. Liu, Anal. Chem.,
2014, 86, 308–311; ( f ) L. Zhou, X. Zhang, Q. Wang, Y. Lv, G. Mao,
A. Luo, Y. Wu, Y. Wu, J. Zhang and W. Tan, J. Am. Chem. Soc., 2014,
136, 9838–9841.
13 (a) S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E. H. Joe, B. R. Cho and
H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915–9923; (b) H. J. Kim,
C. H. Heo and H. M. Kim, J. Am. Chem. Soc., 2013, 135, 17969–17977.
14 (a) H. W. Lee, C. H. Heo, D. Sen, H. O. Byun, I. H. Kwak, G. Yoon and
H. M. Kim, Anal. Chem., 2014, 86, 10001–10005; (b) L. Li, X. Shen,
Q. H. Xu and S. Q. Yao, Angew. Chem., Int. Ed., 2013, 52, 424–428;
(c) L. Li, J. Ge, H. Wu, Q. H. Xu and S. Q. Yao, J. Am. Chem. Soc., 2012,
134, 12157–12167.
15 H. M. Kim, H. J. Choo, S. Y. Jung, Y. G. Ko, W. H. Park, S. J. Jeon,
C. H. Kim, T. Joo and B. R. Cho, ChemBioChem, 2007, 8, 553–559.
16 H. M. Kim and B. R. Cho, Chem. Commun., 2009, 153–164.
17 C. S. Lim, G. Masanta, H. J. Kim, J. H. Han, H. M. Kim and B. R. Cho,
J. Am. Chem. Soc., 2011, 133, 11132–11135.
This work was supported by National Research Foundation (NRF)
grants funded by the Korean Government (2012R1A2A1A03670456)
and the Basic Science Research Program through the NRF
(2012R1A1A2006259).
Notes and references
18 (a) M. J. Frisch, et al., Gaussian 09, Gaussian, Inc., Wallingford, CT,
ˇ
1 T. Nitto and K. Onodera, J. Pharmacol. Sci., 2013, 123, 1–8.
2010; (b) S. Miertus, E. Scrocco and J. Tomasi, Chem. Phys., 1981, 55,
2 A. Jezegou, E. Llinares, C. Anne, S. Kieffer-Jaquinod, S. O’Regan,
117–129.
J. Aupetit, A. Chabli, C. Sagne, C. Debacker, B. Chadefaux-Vekemans, 19 L. Dode, L. Raeymaekers, L. Missiaen, B. Vilsen, J. P. Andersen and
A. Journet, B. Andre and B. Gasnier, Proc. Natl. Acad. Sci. U. S. A., 2012,
109, E3434–E3443.
F. Wuytack, in Calcium Signaling, ed. J. W. Putney, Jr., CRC Press,
Boca Raton, FL, 2nd edn, 2006.
3 M. J. Wilmer, L. A. Kluijtmans, T. J. van der Velden, P. H. Willems, 20 C. R. Yellaturu, M. Bhanoori, I. Neeli and G. N. Rao, J. Biol. Chem.,
P. G. Scheffer, R. Masereeuw, L. A. Monnens, L. P. van den Heuvel
and E. N. Levtchenko, Biochim. Biophys. Acta, 2011, 1812, 643–651.
4 F. Drago and C. Montoneri, Life Sci., 1997, 61, 21–28.
2002, 277, 40148–40155.
21 R. B. Richerson and D. M. Ziegler, Methods Enzymol., 1987, 143,
410–415.
Chem. Commun.
This journal is ©The Royal Society of Chemistry 2015