Highly selective red- and green-emitting two-photon fluorescent probes for cysteine detection and their bio-imaging in living cells

Article abstract of DOI:10.1039/c2cc00093h

Two highly selective two-photon fluorescent probes for cysteine over homocysteine, N-acetyl-l-cysteine, dithiothreitol, glutathione and other amino acids, and their fluorescent imaging in living cells have been shown.

Full text of DOI:10.1039/c2cc00093h

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COMMUNICATION
Highly selective red- and green-emitting two-photon fluorescent probes
for cysteine detection and their bio-imaging in living cellsw
a
a
Zhiguang Yang,z Ning Zhao,z Yuming Sun,b Fang Miao,a Yong Liu,a Xin Liu,a
Yuanhong Zhang,a Wentao Ai,a Guofen Song,a Xiaoyuan Shen,c Xiaoqiang Yu,*a
Jingzhi Sun*c and Wai-Yeung Wong*d
Received 5th January 2012, Accepted 14th February 2012
DOI: 10.1039/c2cc00093h
Two highly selective two-photon fluorescent probes for cysteine
over homocysteine, N-acetyl-L-cysteine, dithiothreitol, glu-
tathione and other amino acids, and their fluorescent imaging
in living cells have been shown.
in the red region corresponds to the transparency window of a
biological specimen, enabling optimal penetration of light into
various biological media.8 Comparatively, the fluorescence in
the blue-green spectral region can be strongly attenuated by a
biological specimen.9 More recently, Zhao et al. reported a
red-emitting Cys probe based on styryl-BODIPY,10 and its
fluorescent intensity induced by Cys is about three times as
strong as that by Hcy, but the influence of NAC has not been
studied. Furthermore, it is important to stain simultaneously
one or more targets using two or more probes with different
light-emitting range during the cell imaging,8,9 thus it is also
essential to offer two, or even more, highly selective sensors
for Cys with distinct fluorescence. For example, commercial
nucleic acid fluorescent dyes, such as 40,6-diamino-2-phenyl-
indole (DAPI) and Hoechst, show blue emission, whereas
acridine homodimer and hexidium iodide are green- and
red-emitting dyes, respectively.
In a biological system, a nuance sometimes leads to a significantly
different function. For example, cysteine (Cys), homocysteine
(Hcy), and N-acetyl-L-cysteine (NAC), three analogous amino
acids with the only difference in a methylene or an acetyl group
at their residues, have been shown to play different yet important
roles in cellular processes.1 Some diseases such as decreased
hematopoiesis, leucocyte loss and psoriasis are associated with
the deficiency of Cys;2 while excess of Hcy was shown to be a risk
factor for cardiovascular and Alzheimer’s diseases.3 At the same
time, a number of studies indicate that NAC, a precursor of
glutathione (GSH) in cells and a thiol antioxidant, has the
potential to prevent cancer and other mutation-related diseases.4
Consequently, it is a fundamental and vital work for researchers in
biochemistry and biomedicine to separately probe Cys, Hcy and
NAC. However, the structural similarity of Cys, Hcy and NAC
makes it challenging to discriminate anyone from the others.
Recently, some probes that can distinguish between Cys and
Hcy have been reported,5 but interference from NAC has not
been referred to. A report by Zhang et al. discriminated the total
amount of Cys and Hcy from NAC, but not between Cys and
Hcy.6a More recently, a probe by Strongin et al. discriminated
Cys, Hcy and NAC from each other.6b
Investigations have demonstrated that two-photon micro-
scopy (TPM) has unique merits in imaging specimen alive11
and thus a variety of fluorescent probes with large two-photon
absorption cross section (d),12 including those for thiols,13
have been explored. So far, three two-photon excitation
fluorescence (TPEF) probes for detecting the total amount
of Cys and Hcy have been reported,6a,14 and only one probe
reported by Zhang et al. has shown the TPEF image of cells in
TPM,15 with the d of the mixture with Hcy of 11 GM (1 GM
= 10ꢀ50 cm4 s photonꢀ1). To the best of our knowledge,
TPEF probes, especially with red-emitting properties, that can
distinguish Cys from Hcy and NAC are not yet available.
In our previous works, we have synthesized various TPEF
probes for DNA,9,16 RNA17 and mitochondria18 using pyridyl
and carbazole units and carried out their TPEF imaging in
alive and fixed cells. Here, we present two new TPEF probes
AM1y and CA1y with different emitting wavelengths
(Scheme 1). CA1 is constructed by linking a pyridyl and an
aldehyde moiety specifically to the 3,6-positions of a 9-ethyl-
carbazole core. At the same time, due to the strong electron-rich
properties of triphenylamine, many TPEF molecules containing
triphenylamine are red-emitting.8 Thus, AM1 was designed and
synthesized by linking a pyridyl ring to a triphenylamine core
containing two aldehydes. From Scheme 1, both AM1 and CA1
afford A–p–A electronic structure. Upon the reaction with Cys,
In fluorescent bio-imaging, the red-emitting probes have
gained tremendous attention,7 because the wavelength range
a Center of Bio & Micro/Nano Functional Materials, State Key Lab
of Crystal Materials, Shandong University, Jinan 250100,
P. R. China. E-mail: yuxq@sdu.edu.cn; Tel: +8653188366418
b School of Information Science and Engineering, Shandong
University, P. R. China
c Institute of Biomedical Macromolecules, Department of Polymer
Science and Engineering, MoE Key Laboratory of Macromolecule
Synthesis and Functionalization, Zhejiang University, P. R. China.
E-mail: sunjz@zju.edu.cn
d Institute of Molecular Functional Materials and Department of
Chemistry and Institute of Advanced Materials, Hong Kong Baptist
University, Hong Kong, P. R. China. E-mail: rwywong@hkbu.edu.hk
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc00093h
z Yang and Zhao made equal contributions to this work.
c
3442 Chem. Commun., 2012, 48, 3442–3444
This journal is The Royal Society of Chemistry 2012

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Fig. 2 (a) TPEF of AM1 and its mixtures. (b) TPEF of CA1 and its
mixtures. Inset: two-photon absorption spectra of AM1 + Cys (a) or
CA1 + Cys (b), error limit: 20%.9 lex: 800 nm. All buffers and other
experimental conditions are identical to those in Fig. 1.
Scheme 1 Reaction of AM1 and CA1 with Cys.
two thiazolidines19 have A–p–D motifs. Thus, they possess high
d values and exhibit notable TPEF enhancement in comparison
with AM1 and CA1. Moreover, the thiazolidines from AM1 and
UV light (lex = 365 nm) are given in Fig. S23 (ESIw). The
SPEF results of CA1 are depicted in Fig. 1d, and SPEF intensity
of CA1 + Cys is 2.5 times as strong as that of CA1 + NAC.
The photos of CA1 are also given (Fig. S24, ESIw).
1
CA1 were characterized by ESI-MS and H NMR (see ESIw).
In acetonitrile–Tris/HCl buffer solution,5b upon addition of
Cys to AM1, the colorless solution becomes a bright yellow
one, showing a ‘‘naked-eye’’ probe for Cys (Fig. 1a, inset).
Meanwhile, a new absorption band between 450 nm and
550 nm indicates the interaction between AM1 and Cys (Fig. 1a).
Clearly, the addition of other bioanalytes cannot result in the
absorption and color change of AM1 (Fig. S20a and S21,
ESIw). In methanol–Tris/HCl buffer solution,15 upon addition
of Cys to CA1, the color and absorption also show obvious
changes and the addition of NAC, GSH and His can also bring
about some absorption changes (Fig. 1b and Fig. S20b, ESIw). The
addition of other bioanalytes does not induce any change in color
(Fig. S22, ESIw) and absorption. The free AM1 is colorless and
weakly fluorescent (Fig. 1c), while the one-photon excitation
fluorescence (SPEF) of AM1 + Cys is notably enhanced with a
red-shift of 84 nm (from 493 to 577 nm), accompanied with a
fluorescent color change from dark to deep red (Fig. 1c, inset).
Upon addition of NAC, the SPEF intensity of AM1 also increases
slightly, and the intensity of AM1 + Cys is 6.3 times as strong as
that of AM1 + NAC. Hcy did not alter the fluorescence of AM1.
The photos of AM1 and AM1s in different bioanalytes under
As revealed from the absorption measurements, the linear
absorption wavelengths of AM1 + Cys and CA1 + Cys are in
the range of 350–500 nm, hence their two-photon absorption
can be excited by a Ti:sapphire laser source in the range of
720–930 nm.8 Notably, the intensity of AM1 + Cys is far higher
than that of AM1. NAC slightly enhances the TPEF intensity
of AM1, but Hcy does not (Fig. 2a). The TPEF intensity of
AM1 + Cys at 630 nm is 11, 67 and 60 times as strong as that of
AM1 + NAC, AM1 + Hcy and AM1, respectively. The corres-
ponding ratios of CA1 + Cys at 551 nm are 2.64, 77 and 59,
respectively (Fig. 2b). From Fig. 2, inset, the d of AM1 + Cys is
1700 GM at 920 nm and that of CA1 + Cys is 90 GM at 810 nm,
whereas AM1 and CA1 are not TPEF-active, indicating that both
are promising TPEF turn-on sensors for Cys.
To study the interaction between AM1 and Cys, three
titrations on absorption, SPEF and TPEF have been carried
out (Fig. S25, ESIw). When the molar ratio between Cys and
AM1 is less than 30, the photophysical properties of the
mixture do not change. At 30–60, absorbance, SPEF and
TPEF intensities increase fleetly. At larger than 60, these
optical properties remain stable.
Before using AM1 and CA1 to image living cells, we
confirmed again their selectivity. As shown in Fig. 3Ia and
IIa, the SPEF and TPEF responses of AM1 to Cys are
markedly higher than to other bioanalytes except for NAC
and the TPEF intensity of AM1 + Cys is 11 times as strong as
that of AM1 + NAC. From Fig. 3Ib and IIb, NAC, GSH, His
and Asp can obviously depress the detection sensitivity of
CA1. At the same time, the competition experiments show that
addition of other bioanalytes did not result in the visible
variation of SPEF and TPEF intensities of AM1 + Cys and
CA1 + Cys (Fig. S26, ESIw). Based on the above discussion,
TPEF selectivity of AM1 to Cys should be the best combi-
nation. At the same time, in pH 4.0–7.5, a wide physiological
pH range, the AM1 was weakly fluorescent, but AM1 + Cys
exhibited high TPEF intensity (Fig. S27, ESIw). Especially,
after adding Cys, the TPEF intensity increased sharply within
3 min, suggesting a high reaction speed, which is suitable for
making full use of the intracellular AM1 molecules. The
detection limits of AM1 and CA1 for Cys (S/N = 3)5b
are about 293–408 mM (Fig. S28, ESIw). Moreover, the
TPEF from AM1 + Cys showed very good photostability,
and high fluorescence intensity could last for at least 60 min
Fig. 1 (a) Absorption of AM1 and its mixtures in acetonitrile–Tris/HCl
buffer solution (tris: 10 mM, KCl: 100 mM) with pH: 7.27 and v/v of 4 : 1.
(b) Those of CA1 and its mixtures in methanol–Tris/HCl buffer solution
(Tris: 10 mM, KCl: 100 mM) with pH: 7.00 and v/v of 4 : 1. (c) and (d)
Fluorescence spectra of AM1 (lex = 427 nm) and CA1 (lex = 410 nm)
under the same conditions as their absorption. Inset: the colors under
natural light (Fig. 1a and b) and UV light (Fig. 1c and d) (lex = 365 nm).
From left to right: AM1 or CA1, +Cys, +Hcy, +NAC. [AM1] =
[CA1] = 10 mM, [Cys] = [Hcy] = [NAC] = 600 mM.
c
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We propose that the red fluorescence in Fig. 4I and Fig. S30I
(ESIw) comes primarily from AM1 + Cys, and green fluorescence
in Fig. 4II and Fig. S30II (ESIw) from CA1 + Cys as well as
CA1 + NAC and CA1 + GSH. On the other hand, molecules
containing 2-aminoethanethiol moieties, such as endogenous
N-terminal Cys peptides/proteins, may also induce fluorescence
of AM1 and CA1, although such proteins are rare.20
In summary, we have reported two novel TPEF turn-on
probes for selective detection of Cys, and their fluorescent
images in living HeLa cells in TPM have been shown.
We thank NSFC (50673053, 50873086, 50573065 and
50990061), 2011JC006, SKLSSM201116), Research Grants
Council of HKSAR (HKBU202709, HKUST2/CRF/10 and
AoE/P-03/08) and help from Prof. Minyong Li in Department of
Medicinal Chemistry, School of Pharmacy, Shandong University.
Fig. 3 One- (I) and two-photon (II) fluorescence responses of AM1
(a) and CA1 (b) to various bioanalytes. lex: I: 488 nm, II(a): 800 nm,
II(b): 910 nm. All buffers and other experimental conditions are
identical to those in Fig. 1. Amino acids and biothiols are consistent
with Fig. S21 (ESIw).
Notes and references
y Both AM1 and CA1 are available free.
(Fig. S29, ESIw). Consequently, AM1 is a TPEF probe that
has great potential to excel to selectively respond to Cys if it is
permeable to living cells.
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The fluorescent imaging was firstly carried out by confocal
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Fig. 4 TPM images of AM1 or CA1. (a) Fluorescence imaging,
(b) DIC pictures, (c) overlay images of a and b. Detection wavelengths,
(I): 4565 nm. (II): 510–540 nm. Scale bar = 50 mm.
c
3444 Chem. Commun., 2012, 48, 3442–3444
This journal is The Royal Society of Chemistry 2012