Construction of a Near-Infrared Fluorescent Turn-On Probe for Selenol and Its Bioimaging Application in Living Animals

Article abstract of DOI:10.1002/chem.201502226

As selenocysteine (Sec) carries out the majority of the functions of the various Se-containing species in vivo, it is of high importance to develop reliable and rapid assays with biocompatibility to detect Sec. Herein, an NIR fluorescent turn-on probe for highly selective detection of selenol was designed and synthesized. The probe exhibits large turn-on signal upon treatment with selenocysteine (R-SeH), and it was further demonstrated that the new NIR fluorescent probe can be employed to image selenol in living animals. Simply red: The first near-infrared (NIR) fluorescent turn-on probe for highly selective detection of selenol was designed and synthesized (see figure). The probe exhibits a large turn-on signal upon treatment with selenocysteine (R-SeH), and it has been further demonstrated that the new NIR fluorescent probe can be employed to image selenol in living animals.

Full text of DOI:10.1002/chem.201502226

DOI: 10.1002/chem.201502226
Communication
&
Imaging Agents
Construction of a Near-Infrared Fluorescent Turn-On Probe for
Selenol and Its Bioimaging Application in Living Animals
Hua Chen,[b] Baoli Dong,[a] Yonghe Tang,[a] and Weiying Lin*[a, b]
lution.[5] However, designing highly selective probes for Sec
Abstract: As selenocysteine (Sec) carries out the majority
without suffering from interference of biological thiols is chal-
of the functions of the various Se-containing species in
lenging since thiols are usually present in high concentration
vivo, it is of high importance to develop reliable and rapid
(millimolar levels) in cells and have similar chemical properties
assays with biocompatibility to detect Sec. Herein, an NIR
as Sec. Some fluorescent probes for Sec have been achieved
fluorescent turn-on probe for highly selective detection of
by taking advantage of the mechanism of nucleophilic aromat-
selenol was designed and synthesized. The probe exhibits
ic substitution.[6,7] However, these reported fluorescent probes
large turn-on signal upon treatment with selenocysteine
for Sec are located in the visible region and are only suitable
(R-SeH), and it was further demonstrated that the new NIR
for imaging studies in living cells. It is essential to develop fluo-
fluorescent probe can be employed to image selenol in
rescent Sec probes with near-infrared emission for living
living animals.
animal imaging applications. Near-infrared (NIR) light (650–
900 nm) is advantageous in biological imaging due to mini-
mum photodamage to biological samples, deep tissue pene-
Selenium (Se) is a biologically essential element for cellular
redox balance, immune responses, cancer prevention, and in-
flammation protection.[1] Many different metabolites of Se,
such as hydrogen selenide, selenocysteine (Sec), selenite, sele-
nophosphate, selenodiglutathione, and charged Sec-tRNA, are
synthesized in animals in the course of converting inorganic Se
to organic forms and vice versa.[2] Insufficient or excessive
intake of Se has been associated with a number of diseases.[3]
Sec is a cysteine (Cys) analogue with a selenium-containing se-
lenol group in place of the sulfur containing thiol group in
Cys.[4] In mammalian tissues, selenocysteine (Sec), genetically
encoded as the 21st amino acid, appears to be the predomi-
nant chemical form of selenium, and is specifically incorporat-
ed into selenoproteins (SePs).[4] As Sec carries out the majority
of the function of the various Se-containing species in vivo, it
is of high demand to develop reliable and rapid assays with
biocompatibility to determine Sec.
tration, and minimum interference from background autofluor-
escence by biomolecules in living systems.[8,9] To the best of
our knowledge, no NIR fluorescent Sec probes have been re-
ported in the literature to date, although they are highly desir-
able for biological imaging of selenocysteine in living animals.
Recently, we introduced a unique family of NIR dyes (HD NIR
dyes), which are superior to the traditional 7-hydroxycoumarin
and fluorescein with absorption and emission in the NIR
region while retaining an optically tunable hydroxyl group.[10]
Based on this finding, we reasoned that their novel amino ana-
logue HD-NH2 could also exhibit an optically tunable function.
It is known that the strongly electron-withdrawing 2,4-dinitro-
benzenesulfonyl group has been used for the protection of an
amino group.[11] The resulting sulfonamide can be readily
cleaved by a nucleophilicity group, such as thiolate anions
(Figure 1). Several probes with sulfonamide linkage have been
reported to selectively detect thiolphenols at neutral pH.[12]
This mechanism underlines the importance of the anion form
of a nucleophilic group, which is the essentially reactive form
for the reaction. The pKa value of selenocysteine is around 5.8,
whereas that of aliphatic thiols is about 8.5. In a neutral reac-
tion medium (for example, pH 7.4), the high degree of dissoci-
ation of selenocysteine results in the predominant generation
of the corresponding selenolate, which can effectively react
with 2,4-dinitrobenzenesulfonamide.[7] However, under the
same neutral conditions, the less reactive neutral form of ali-
phatic thiols predominates, thus, the cleavage of the sulfona-
mide is very slow.
Since the fluorescent indicators for calcium ion were report-
ed by Tsien in the early 1980s,[5a] fluorescent probes have been
recognized as the efficient molecular tools that can help moni-
tor and visualize trace amounts of samples in live cells or tis-
sues because of their high sensitivity and spatiotemporal reso-
[a] B. Dong, Y. Tang, Prof. W. Lin
Institute of Fluorescent Probes for Biological Imaging
School of Chemistry and Chemical Engineering
School of Biological Science of Jinan
Jinan, Shandong 250022 (P.R. China)
E-mail: weiyinglin2013@163.com
In this work, we developed HD-Sec as the first NIR fluores-
cent turn-on selenocysteine probe using HD-NH2 as the near-
infrared platform and 2,4-dinitrobenzenesulfonyl group as the
reactive site for selenocysteine (Figure 1). We thus anticipated
that the probe HD-Sec may be essentially non-fluorescent.
However, upon reaction with selenocysteine to release the 2,4-
[b] H. Chen, Prof. W. Lin
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering, Hunan University
Changsha, Hunan 410082 (P. R. China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502226.
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around 612 and 663 nm (Figure S1a). The fluorescence spec-
trum of HD-NH2 exhibits strong fluorescence with a maximum
emission peak at around 720 nm. As expected, the reference
compound HD-NHCOCH3, which bears no electron-donating
group, displays weak fluorescence (Figure 1b).
The molar extinction coefficients of the new compounds
HD-NH2 and HD-NHCOCH3 in CH3CH2OH are 99580 and
41420mÀ1 cmÀ1, respectively. Importantly, the dye HD-NH2 has
a fluorescence quantum yield of 0.53 in CH3CH2OH, which is
relatively large for NIR dyes. Furthermore, the fluorescence
quantum yield of the compound HD-NHCOCH3 is only 0.02,
much less than that of the dye HD-NH2. Thus, these data sug-
gest that the optical properties of the new dye HD-NH2 can be
regulated by structural modifications on the key amine group.
With the probe HD-Sec in hand, we first evaluated the capa-
bility of the probe to detect selenocysteine (R-SeH) in PBS
buffer (pH 7.4). As Sec is not stable, we generated it in situ by
mixing equal molar of dithiothreitol (DTT) and selenocysteine
((Sec)2).[14] As designed, the free probe is almost non-fluores-
cent in PBS (Figure 3a). However, addition of selenocysteine
(R-SeH) induces a dramatic change in the fluorescence spectra.
A significant fluorescence turn-on response at 712 nm (up to
65-fold) was observed (Figure 3b). Consistently, a marked red-
shift from 596 to 690 nm was noted in the absorption spectra
upon treatment of the probe with equal molar of DTT and
(Sec)2 (Figure S2 in the Supporting Information). Notably, the
emission wavelength of the probe is in the NIR region, which
is favourable for fluorescent imaging studies in living animals.
Furthermore, the fluorescence intensities at 712 nm have an
excellent linear relationship with the concentrations of equal
molar of DTT and selenocysteine ((Sec)2; Figure S3 in the Sup-
porting Information). The probe HD-Sec responded rapidly to
selenocysteine (Figure S4a in the Supporting Information). The
reaction was almost complete
Figure 1. The design of the near-infrared fluorescent turn-on probe HD-Sec
for selenocysteine.
dinitrobenzenesulfonate unit, the near-infrared fluorescence
should be recovered.
Probe HD-Sec was readily synthesized in four steps
(Figure 2). The staring material 1 was prepared according to
a reported procedure[13] and HD-NH2 was synthesized accord-
ing to the similar procedure for HD dyes.[10] Then, the HD-NH2
dye was treated with 2,4-dinitrobenzenesulfonyl chloride
under the basic conditions to give the target compound HD-
Sec. Reference compound HD-NHCOCH3 was synthesized by
acetylation of HD-NH2 (Scheme S1 in the Supporting Informa-
tion). All new compounds were characterized by NMR spec-
troscopy and mass spectrometry.
To further support our design concept of the NIR fluorescent
turn-on probes HD-Sec, we proceeded to investigate their opti-
cal properties. The absorption and emission profiles of com-
pounds HD-NH2 and HD-NHCOCH3 in CH3CH2OH solvent is
shown in Figure S1 in the Supporting Information. The HD-NH2
dye displays a maximum absorption peak at around 698 nm,
while HD-NHCOCH3 has two blue-shift absorption bands at
within 10 min, and the pseudo-
first-order rate constant was cal-
culated to be k=0.1644 minÀ1
(Figure S4b). The pH effect stud-
ies suggest that the probe HD-
Sec is suitable for detecting sele-
nocysteine (R-SeH) at physiologi-
cal pH (Figure S5 in the Support-
ing Information).
To confirm the sensing pro-
cess, we decided to study the
product of HD-Sec reaction with
selenocysteine by both NMR
spectroscopy and mass spec-
trometry. Both the mass spec-
trometry and NMR analyses con-
firmed that the fluorescence
turn-on is indeed due to the se-
lenocysteine-mediated removal
of the 2,4-dinitrobenzenesulfo-
nate moiety (Figure S6 and Fig-
ure S7 in the Supporting Infor-
mation).
Figure 2. Synthesis of the probe HD-Sec. Conditions: a) NaH, DMF, 508C, 2 h; b) CF3COOH, CH2Cl2, room tempera-
ture, 6 h; c) NaH, DMF, 908C, 5 h; d) Cs2CO3, CH2Cl2, 2,4-dinitrobenzenesulfonyl chloride, room temperature, 1 h.
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Figure 4. Fluorescence probe HD-Sec (5 mm) in the presence of various ana-
lytes in PBS buffer (pH 7.4, containing 5% DMSO as a co-solvent). 1: blank;
25 mm for: 2: Arg, 3: Glu, 4: Val, 5: Ser, 6: Leu, 7: Lys, 8: K+, 9: Ca2+, 10: Na+,
11: Mg2+, 12: Zn2+, 13: DTT, 14: NADH, 15: glucose, 16: NaHS; 17: GSH
(10 mm), 18: Hcy (1 mm), 19: Cys (1 mm), 20: DTT (25 mm); and selenocys-
teine (25 mm). Excitation at 650 nm and emission at 712 nm. Data are ex-
pressed as mean ÆSD of three experiments.
Figure 3. a) Emission spectra (excited at 650 nm) of the novel probe HD-Sec
(5 mm) in the presence of increasing concentrations of selenocysteine at
room temperature in PBS (pH 7.4, containing 5% DMSO as a co-solvent),
generated by the treatment of equal molar (Sec)2 with DTT; b) fluorescence
intensity ratio (F/F0) changes at 712 nm of the probe (5 mm) with increasing
concentrations of selenocysteine at room temperature in PBS, generated by
the treatment of equal molar (Sec)2 with DTT.
To verify the specific response of probe HD-Sec toward sele-
nocysteine (R-SeH), we then examined the selectivity of the
probe HD-Sec toward other species (such as representative
amino acids, glucose, metal ions, reducing agents, nucleosides,
and small-molecule thiols) in buffer solution (25 mm phosphate
buffer, pH 7.4) and monitored the response by emission spec-
troscopy. As exhibited in Figure 4, no noticeable changes were
observed upon addition of amino acids (Arg, Glu, Val, Ser, Leu,
and Lys), metal ions (K+, Ca2+, Na+, Mg2+, and Zn2+), a reduc-
ing agent (DTT, NADH, and NaHS) or glucose. Notably, small-
molecule thiols such as glutathione (GSH) at 10 mm, Cys and
Hcy at 1 mm trigger only a minor fluorescence enhancement
(Figure 4). By contrast, upon treatment of equal molar of DTT
and selenocysteine ((Sec)2) with the probe, a large fluorescence
signal (65-fold fluorescence enhancement) was observed. Thus,
these data demonstrate that the probe has a high selectivity
for selenocysteine over other biological species tested includ-
ing GSH and cysteine at the biologically relevant concentra-
tions, suggesting that the new NIR probe is favourable for
imaging studies in biological systems.
Figure 5. a–c) MCF-7 cells co-incubated with HD-Sec (5 mm) for 30 min.
a) Brightfield image; b) emission from the red channel; and c) overlay of the
brightfield image and red channel. d–f) MCF-7 cells pre-incubated with
(Sec)2 (5 mm) for 6 h, and then co-incubated with HD-Sec (5 mm) for 30 min.
d) Brightfield image; e) emission from the red channel; f) overlay of the
brightfield image and red channel. Scale bars: 10 mm.
ine the possibility of the probe to monitor endogenously pro-
duced selenocysteine in living cells. Sodium selenite (Na2SeO3)
is a precursor of Sec biosynthesis, and supplement of cells
with sodium selenite could significantly increase the Sec level
in cells.[15] Sodium selenite (Na2SeO3) itself did not react with
the probe HD-Sec. After the cells were stimulated with sodium
selenite for 6 h, a marked fluorescence appeared (Figure 6e).
HD-Sec was also incubated with HeLa cells, and similar imag-
ing results were obtained (Figure S8 and Figure S9 in the Sup-
porting Information). Taken together, these results indicate
that HD-Sec is cell membrane permeable and capable of sens-
ing selenocysteine in living cells.
For the preliminary fluorescence imaging applications, the
probe HD-Sec was incubated with living MCF-7 cells pretreated
with or without (Sec)2 (Figure 5 and Figure 6).
As the physiological concentration of Sec is low in cells, the
probe HD-Sec cannot be directly used to image endogenous
selenocysteine. As shown in Figure 5b, the cells treated with
only the probe exhibit relatively weak fluorescence in the red
channel. By contrast, when the cells were pretreated with
(Sec)2 for 6 h and then incubated with HD-Sec, much more in-
tense fluorescence in the red channel was observed (Fig-
ure 5e). Prompted by the above desirable results of imaging
exogenous selenocysteine in living cells, we decided to exam-
The prominent features of HD-Sec include NIR absorption
and emission, rapid response, excellent selectivity, and good
cell membrane permeability. These desirable attributes encour-
aged us to further investigate the suitability of the probe for
detecting selenocysteine in the context of living animals.
Kunming mice were divided into three groups (Figure 7). One
group was given saline (100 mL) in the peritoneal cavity, fol-
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Figure 7. Representative fluorescent images (pseudo-colour) of selenocys-
teine in mice, 30 min after injection of HD-Sec: a) only the probe HD-Sec
was injected in the peritoneal cavity of the mouse; b) the mouse was given
an i.p. injection of (Sec)2 and followed by i.p. injection with the sensor HD-
Sec after 1 h; c) the mouse was given an i.p. injection of (Sec)2 and followed
by i.p. injection with the sensor HD-Sec after 6 h. The mice were imaged
using an IVIS Lumina XR (IS1241N6071) in vivo imaging system with an exci-
tation filter of 640 nm and an emission range of 695–770 nm; d) quantifica-
tion of fluorescence emission intensity from the abdominal area of the mice
of groups a–c: 0 (a), 1 (b), 2 (c). Data are expressed as mean ÆS.D. of three
experiments.
Figure 6. a–c) MCF-7 cells co-incubated with HD-Sec (5 mm) for 30 min.
a) Brightfield image; b) emission from the red channel; and c) overlay of the
brightfield image and red channel. d–f) MCF-7 cells pre-incubated with
Na2SeO3 (5 mm) for 6 h, and then co-incubated with HD-Sec (5 mm) for
30 min. d) Brightfield image; e) emission from the red channel; f) overlay of
the brightfield image and red channel. Scale bars: 10 mm.
lowed by intraperitoneal (i.p.) injection with HD-Sec (20 mm, in
20 mL DMSO) as the negative control group; The second group
was given an i.p. injection of (Sec)2 (20 mm in 100 mL saline)
and followed by i.p. injection with the probe HD-Sec (20 mm, in
20 mL DMSO) after 1 h; and the third group was given an i.p.
injection of (Sec)2 (20 mm in 100 mL saline) and followed by i.p.
injection with the probe HD-Sec (20 mm, in 20 mL DMSO) after
6 h. The mice were anesthetized, and the abdominal fur was
removed. After the probe HD-Sec was injected, the mice were
imaged using an IVIS Lumina XR (IS1241N6071) in vivo imaging
system. As shown in Figure 7b, the mice pretreated with (Sec)2
after 1 h and then treated with the probe HD-Sec exhibit
a much higher fluorescence readout (pseudocolor) than the
mice treated with only the probe (Figure 7a), but less than the
mice pretreated with (Sec)2 after 6 h and then treated with the
probe (Figure 7c). The fluorescent intensity from the abdomi-
nal area of the mice was quantified, and the data indicate that
the mice injected with treated (Sec)2 after 6 h and then treated
with the probe have approximately 5.6-fold higher fluores-
cence intensity than the mice injected with only the probe
(Figure 7d). Thus, these results demonstrate that the new NIR
probe HD-Sec can image selenocysteine in the living animals.
To the best of our knowledge, this represents the imaging of
selenol in living animals for the first time.
(Changsha, China). All animal procedures for this study were
approved by the Animal Ethical Experimentation Committee of
Central South University according to the requirements of the
National Act on the use of experimental animals (China).
Acknowledgements
Funding was partially provided by NSFC (21172063, 21472067)
and the Startup Fund of University of Jinan.
Keywords: fluorescence sensing
·
fluorescent probes
·
imaging agents · NIR-fluorescent fluorophores · selenocysteine
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Received: June 7, 2015
Published online on July 14, 2015
Chem. Eur. J. 2015, 21, 11696 – 11700
www.chemeurj.org
11700
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