Development of ratiometric near-infrared fluorescent probes using analyte-specific cleavage of carbamate

Article abstract of DOI:10.1039/c3ob40932e

We report a facile method to design NIR ratiometric fluorescent probes in terms of analyte-induced carbamate cleavage. Two examples, CyNB and CyNN ₃, exhibit a significant analyte-triggered response with ratiometric fluorescence change in the NIR range and dual-emission ratiometry in living cells. is

Full text of DOI:10.1039/c3ob40932e

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Development of ratiometric near-infrared fluorescent
probes using analyte-specific cleavage of carbamate†
Cite this: DOI: 10.1039/c3ob40932e
Received 3rd May 2013,
Accepted 28th May 2013
Dongjian Zhu,a,b Guoping Li,a,b Lin Xue*a and Hua Jiang*a
DOI: 10.1039/c3ob40932e
www.rsc.org/obc
We report a facile method to design NIR ratiometric fluorescent nitrogen atom due to the internal charge transfer (ICT). When
probes in terms of analyte-induced carbamate cleavage. Two such an ICT process was affected by a certain sensing event,
examples, CyNB and CyNN3, exhibit a significant analyte-triggered sharp changes in absorption/fluorescence spectra were
response with ratiometric fluorescence change in the NIR range observed.6 With these facts in mind, we envision that modify-
and dual-emission ratiometry in living cells.
ing the amine substituent of tricarbocyanine with an electron-
withdrawing carbamate group would efficiently lower the elec-
tron density of the amine and could, in principle, give out an
obvious bathochromic shift of fluorescence emission com-
pared to that of normal amine-substituted tricarbocyanines.
The concept will provide a rationale for molecular design of a
generic model to construct NIR ratiometric probes by taking
advantage of a chemospecific cleavage of the caged carbamate.
Therefore, in this paper, we report the synthesis and photo-
physical properties of several carbamate-substituted tricarbo-
cyanines and further evaluation of the performances of two
NIR ratiometric probes utilizing this generic model
(Scheme 1).
Fluorescence imaging is a very powerful method for monitor-
ing biomolecules in living systems because of its high sensi-
tivity and selectivity, facility and fast response.1 Recently,
utilizing ratiometric fluorescent probes to analyze biological
species has been drawing intensive attention. This type of
probe, importing a built-in correction via the ratio of the emis-
sion intensity at two different wavelengths, can provide quanti-
tative information on analytes.2 Hence, a number of ratio-
metric fluorescence probes have been developed based on
small molecules, biological molecules, conjugated polyelectro-
lytes or nanoparticles, etc.3 However, most of them have
absorption and emission in the ultraviolet or visible range,
and are not adaptable to in vivo imaging and quantification.
Near-infrared (NIR) dyes provide a feasible opportunity for
in vivo imaging due to many distinct advantages, including
deeper tissue penetration, minimum photodamage to biologi-
cal samples, and minimum interference from background
autofluorescence in the living systems.4 Therefore, develop-
ment of ratiometric fluorescent probes based on NIR dyes will
contribute useful molecular tools for quantitative measure-
ments in vivo.
We initially synthesized benzylamine-substituted tricarbo-
cyanine (CyN) and modified the meso-nitrogen atom with a car-
boxybenzyl group (CyNC) (Scheme 1). The photophysical
properties of both CyN and CyNC are shown in Fig. 1 and
Among NIR dyes, amine-substituted tricarbocyanine has
been proved to be an excellent candidate for designing NIR
probes through modifying the amino group with well-defined
receptors.5 The photophysical properties of this type of
cyanine highly depend on the electron density of the meso-
aBeijing National Laboratory for Molecular Sciences, CAS Key Laboratory of
Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing,
P.R. China. E-mail: hjiang@iccas.ac.cn, zinclin@iccas.ac.cn; Tel: (+86) 10-82612075
bUniversity of Chinese Academy of Sciences, Beijing, P.R. China
†Electronic supplementary information (ESI) available: Synthetic procedures
and characterization of compounds CyCl, CyN, CyNC, CyNB and CyNN3, and
additional spectroscopic data. See DOI: 10.1039/c3ob40932e
Scheme 1 Design strategy for NIR ratiometric fluorescent probes.
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Fig. 1 (a) Absorption and (b) emission spectra of 1 µM CyN (black line) and
1 µM CyNC (red line) in a mixture of H2O–DMSO (9 : 1, v/v, 10 mM HEPES,
100 mM NaCl, pH = 7.4) λex = 671 nm.
Fig. 2 (a) Fluorescence response of 2 μM CyNB to 300 μM H2O2. Spectra were
acquired every 5 min after H2O2 was added. (b) Fluorescence responses of 2 μM
CyNB to various ROS and RNS (300 μM). Bars represent emission intensity ratios
F
747 nm/F808 nm at 0, 15, 30, 45, 60, 90, and 120 min after addition of each ROS
or RNS. Data were acquired in a mixture of H2O–DMSO (9 : 1, v/v, 10 mM HEPES,
100 mM NaCl, pH = 7.4), λex = 676 nm.
Table 1 Photophysical data of CyN and CyNCa
Dye
λabs/nm
εmax b/×105 M−1 cm−1
λem/nm
Φc
CyN
CyNC
645
785
0.42
1.71
747
808
0.062
0.044
evaluation of our design concept through two analyte-respon-
sive NIR ratiometric probes CyNB and CyNN3.
a 10 mM HEPES, 100 mM NaCl, 10% (v/v) DMSO, pH = 7.4. b Molar
extinction coefficients are in the maximum of the highest peak.
c Cardiogreen was used as the standard for quantum yield
measurements (Φ = 0.13 in DMSO).7
The probes can be easily obtained through the reaction
between CyN and the corresponding carbonochloridate (see
the ESI†). As shown in Fig. 2, we assessed the photophysical
properties of CyNB in a mixture of H2O–DMSO (9 : 1, v/v,
10 mM HEPES, 100 mM NaCl, pH = 7.4). CyNB exhibits a
Table 1. We found that benzylamine-substituted tricarbo- maximal absorbance at 785 nm (ε = 1.49 × 105 M−1 cm−1) and
cyanine, CyN, exhibits a major absorption maximum at 645 nm a characteristic emission maximum at 808 nm (Φ = 0.036),
and a characteristic NIR fluorescence emission maximum at which are almost the same as those of CyNC. Treatment of
747 nm (Φ = 0.062) very similar to other amine-substituted tri- CyNB with H2O2 in a time period of 2 h resulted in remarkable
carbocyanine analogues.5 As expected, the electron-withdraw- blue shifts of 140 nm in absorption and 61 nm in fluorescence
ing carboxybenzyl group in CyNC induces remarkable spectra (Fig. 2a and Fig. S1a†). These spectral changes clearly
bathochromic shifts in both absorption of 140 nm and emis- demonstrate that the self-immolative process was triggered by
sion of 61 nm, which is consistent with the ICT process H2O2 and subsequently liberated the NIR dye, CyN with a
involved in the meso-nitrogen atom of CyNC. Hence, these fea- characteristic of much shorter wavelengths in both absorption
tures afford an excellent opportunity to design a generic model and emission spectra (Table 1). The ratio of emission inten-
or scaffold for single-excitation, dual-emission NIR ratiometric sities (F747 nm/F808 nm) varied from 0.15 to 3.4 in the reaction
probes through specific chemoselective triggers.
with H2O2, indicating that CyNB is an excellent NIR dual-emis-
To confirm the feasibility of our concept, we subsequently sion ratiometric fluorescent probe for H2O2 (R/R0 up to 22).
tried to introduce diverse caging groups into the cleavable Kinetic measurement of CyNB (1 μM) with H2O2 (1 mM) under
carbamate group. It is well known that H2O2 and H2S are the pseudo-first-order conditions gave an observed rate con-
small-molecule signal transducers and play vital roles in stant of kobs = 1.26 × 10−3 s−1 (Fig. S3a†). At low concentration
physiological and pathological processes in living organisms.8 of H2O2 (30 μM), CyNB also exhibited distinct fluorescence
Developments of fluorescent probes for in vivo detection and ratiometric response, but with a slower reaction rate (Fig. S4†).
visualization of H2O2/H2S are still challenging research topics Further, the selectivity of CyNB for H2O2 was also examined in
of chemistry and biochemistry. Recent disclosures show that the presence of other biologically relevant reactive oxygen
reaction-based approaches are very efficient for designing species (ROS) and reactive nitrogen species (RNS), such as
H2O2/H2S probes by taking advantage of caging groups, such ONOO− (peroxynitrite), OCl−, TBHP (tert-butylhydroperoxide),
as boronic acid/ester or azide, which can be specifically ˙OH, ˙OtBu, 1O2, O2−, NO, NO2− and NO3− under the identical
reacted with H2O2 or H2S.9,10 On the other hand, on the basis conditions. Due to the high H2O2-specificity of boronate
of the fact that the p-hydroxybenzyl or aminobenzyl moiety is groups, the ratiometric emission response of CyNB to H2O2 is
able to self-immolate through an intramolecular 1,6-elimi- highly selective. However, other ROS/RNS exerted no obvious
nation,11 we reason that importing an analyte-responsive group fluorescence ratio response (Fig. 2b). These results suggest
to the carbamate of CyNC could make the caged CyNC respon- that the sensing scaffold of CyN/CyNB is practicable for NIR
sive to a particular molecule while not affecting the photo- ratiometric H2O2-selective probes in aqueous media.
physical properties of the fluorophore. If the cleavage process
We further test the feasibility of our concept using another
was triggered by a specific analyte so as to release the NIR dye NIR probe, CyNN3, containing an azide group which is highly
of CyN, remarkable ratiometric fluorescence signals would be specific to H2S.10 The spectroscopic properties of CyNN3 were
obtained (Scheme 1 and Table 1). Herein, we demonstrate the measured in a mixture of H2O–CH3CN (8 : 2, v/v, 40 mM
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Fig. 4 Fluorescent imaging of H2O2 in CyNB-labeled NIH 3T3 cells. (a) Bright-
field image; (b) cells were stained with 5 μM CyNB at 37 °C for 30 min; (c) CyNB-
labeled cells were treated with 100 μM H2O2 for 30 min at 25 °C. λex = 635 nm,
scale bar: 20 μm, ratio bar: 0–4.
Fig. 3 (a) Fluorescence response of 2 μM CyNN3 to 4 mM H2S. Spectra were
acquired every 5 min after H2S was added. (b) Fluorescence responses of 2 μM
CyNN3 to various relevant species for 1 h. Data shown: H2S, Cl−, Br−, I−, AcO−,
N3−, SCN−, HCO3−, HPO42−, H2O2, TBHP, O2−, NO, NO2−, NO3−, HSO3−, SO3
,
2−
S2O32−, S2O42−, S2O52−, Cys, GSH, and Hcy at 4 mM. Data were acquired in a
signals (Fig. S7†). Accordingly, the pseudocolor ratio image
(Em700–770 nm/Em780–800 nm) gave an average value of 0.87 0.12
(Fig. 4b) by using the FV10-ASW software (Olympus, Ver. 2.1c).
When the cells were treated with 100 μM H2O2 at 25 °C
for 30 min, significant enhancements in the ratio were
obtained with an average value of 1.63 0.13 (Fig. 4c). These
are consistent with the observations in aqueous buffer, indicat-
ing that CyNB can visualize H2O2 in living cells by dual-emis-
sion ratiometry under biological conditions. We then tested
the ability of CyNN3 to detect intracellular H2S in living cells
(Fig. 5 and Fig. S8†). Similarly, CyNN3 is also cell-permeable
and can stain the NIH 3T3 cells in the same optical windows.
The ratio imaging (Em700–770 nm/Em780–800 nm) gave an average
value of 1.20 0.14 (Fig. 5a). Treatment of CyNN3-loaded cells
with 1 mM H2S for 30 min at 37 °C led to a sharp increase in
the ratio to 2.05 0.28 (Fig. 5b). Moreover, in the presence of
ZnCl2 (an efficient eliminator of H2S),10b,g addition of H2S did
not obviously induce the increase of the fluorescence ratio
(1.16 0.13, Fig. 5c). The data establish that CyNN3 is capable
of imaging intracellular H2S in living cells.
In summary, we have developed a new tricarbocyanine-
based scaffold for constructing NIR ratiometric fluorescent
probes by specific analyte-induced cleavage of carbamate.
Accordingly, we prepared two NIR ratiometric probes: CyNB for
H2O2 and CyNN3 for H2S. By highlighting the significant ratio-
metric fluorescence signal output in aqueous solution and
dual-emission ratiometry in living cells of both probes, we
expect that this design concept can contribute an effective
method for development of NIR probes through a simple
modulation of the caging site on the carbamate moiety.
mixture of H2O–CH3CN (8 : 2, v/v, 40 mM HEPES, pH = 7.4), λex = 698 nm.
HEPES, pH = 7.4). Again, we found the tuning of the caging
site in CyNN3 did not apparently affect the photophysical pro-
perties of the fluorophore of CyNC. CyNN3 shows a maximal
emission at 810 nm (Φ = 0.033) in the absence of H2S upon
excitation at 698 nm. By treating with 4 mM H2S (Na2S was
used as a hydrogen sulfide source in all experiments),10b the
fluorescence spectrum shows a gradual decrease at 810 nm
with a concomitant increase at 744 nm (Fig. 3a). These spectro-
scopic changes are similar to those of CyNB. The ratio of emis-
sion intensities (F744 nm/F810 nm) varied from 0.16 to 2.9 in
response to H2S during 1 h, and ca. 18-fold emission ratio
change was thereby obtained, indicating that a self-immolative
process can also be triggered by H2S. Under the pseudo-first-
order conditions (2 μM CyNN3, 4 mM H2S), the observed rate
constant was measured to be 9.91 × 10−4 −1 (Fig. S3b†). When
s
CyNN3 was treated with a low concentration of H2S (0.4 mM), a
distinct fluorescence ratiometric response but with a slower
reaction rate was observed (Fig. S5†). The fluorescence
responses of CyNN3 to other biological species were also inves-
tigated in parallel under the identical conditions. As shown in
Fig. 3b, the ratiometric emission response of CyNN3 is highly
selective for H2S over other anions, ROS, RNS, reactive sulfide
species (RSS), and, in particular, different thiols. The results
indicate that CyNN3 is a good candidate to selectively detect
H2S in aqueous solution.
Moreover, in order to verify the practicability of our tricarbo-
cyanine scaffold for intracellular imaging, we also
measured the fluorescence spectra of CyNB, CyNN3, and CyN
at different pH values. Emission intensities and ratio calib-
rations remain almost constant in the range of pH 6–8 (Fig. S6†).
This evidence indicates that both CyNB and CyNN3 are pH-
independent in the physiological pH range and are suitable
for ratiometric detection of H2O2 and H2S in living cells,
respectively.
We next applied CyNB and CyNN3 to confocal fluorescence
ratiometric imaging of H2O2 and H2S in living cells, respecti-
vely. The fibroblast NIH 3T3 cells were incubated with 5 μM
CyNB in serum-free DMEM (0.5%, v/v, DMSO) for 30 min at
37 °C, and then washed with PBS three times before imaging.
Upon excitation at 635 nm, both emission channels of
Fig. 5 Fluorescent imaging of H2S in CyNN3-labeled NIH 3T3 cells. (a) Cells
were stained with 5 μM CyNN3 at 37 °C for 30 min and further incubated in
serum-free DMEM for 30 min; (b) CyNN3-labeled cells were treated with 1 mM
H2S for 30 min at 37 °C; (c) cells were pretreated with 2 mM ZnCl2 for 30 min,
then incubated with 5 μM CyNN3 for 30 min, and further treated with 1 mM
700–770 nm and 780–800 nm displayed evident fluorescence H2S for 30 min at 37 °C. λex = 635 nm, scale bar: 20 μm, ratio bar: 0–5.
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We thank the National Natural Science Foundation of
China (21102148, 21125205), National Basic Research Program
of China (2009CB930802, 2011CB935800), and the State Key
Laboratory of Fine Chemicals, Department of Chemical Engi-
neering, Dalian University of Technology for financial support.
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