Highly selective fluorescent OFF-ON thiol probes based on dyads of BODIPY and potent intramolecular electron sink 2,4-dinitrobenzenesulfonyl subunits

Article abstract of DOI:10.1039/c0ob00910e

Two highly selective OFF-ON green emitting fluorescent thiol probes (1 and 2) with intense absorption in the visible spectrum (molar extinction coefficient ε is up to 73800 M^-1 cm^-1 at 509 nm) based on dyads of BODIPY (as electron do

Full text of DOI:10.1039/c0ob00910e

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PAPER
Highly selective fluorescent OFF–ON thiol probes based on dyads of
BODIPY and potent intramolecular electron sink 2,4-dinitrobenzenesulfonyl
subunits†
Huimin Guo,*a Yingying Jing,a Xiaolin Yuan,b Shaomin Ji,a Jianzhang Zhao,*a Xiaohuan Lib and Yanyan Kanc
Received 20th October 2010, Accepted 15th December 2010
DOI: 10.1039/c0ob00910e
Two highly selective OFF–ON green emitting fluorescent thiol probes (1 and 2) with intense absorption
in the visible spectrum (molar extinction coefficient e is up to 73 800 M-1 cm-1 at 509 nm) based on
dyads of BODIPY (as electron donor of the photo-induced electron transfer, i.e.PET) and
2,4-dinitrobenzenesulfonyl (DNBS) (as electron acceptor of the PET process) were devised. The single
crystal structures of the two probes were determined. The distance between the electron donor
(BODIPY fluorophore) and the electron acceptor (DNBS) of probe 2 is larger than that of probe 1, as a
result the contrast ratio (or the PET efficiency) of probe 2 is smaller than that of probe 1. However,
fluorescence OFF–ON switching effects were observed for both probe 1 and probe 2 in the presence of
cysteine (the emission enhancement is 300-fold for probe 1 and 54-fold for probe 2). The fluorescence
OFF–ON sensing mechanism is rationalized by DFT/TDDFT calculations. We demonstrated with
DFT calculations that DNBS is ca. 0.76 eV more potent to accept electrons than the maleimide moiety.
The probes were used for fluorescent imaging of cellular thiols.
donating. Thus ratiometric as well as fluorescent OFF–ON signal
transduction was observed in the presence of cysteine for these
ICT-based probes.5a,9
Introduction
Thiols such as glutathione and cysteine play important roles
in living organisms.1–4 Thus, fluorescent molecular probes for
selective detection of thiols are of great interest. A wide variety
of fluorophores and sensing mechanisms have been used for
design of fluorescent thiol molecular probes.5 For fluorescent
thiol probes, usually the fluorophores bear electrophilic groups
(e.g., iodoacetamides and benzyl halides), to which thiols may
be covalently attached via electrophilic substitution. Alternatively
fluorophores attached with a maleimide unit are also used as thiol
probes.6–8 Fluorophores containing a formyl (–CHO) group are
also used for the construction of thiol probes, which are based on
tuning the intramolecular charge transfer (ICT) effect. In this kind
of probe, the –CHO group, as the electron acceptor of the ICT,
can be transformed into a thiazolidine structure, which is electron-
A photo-induced electron transfer (PET) thiol probe with
BODIPY as the fluorophore/electron donor and maleimide as the
electron acceptor is particularly interesting (probe 3, Scheme 1).7
BODIPY is ideal for assembling molecular probes due to its
characteristic photophysics, such as high fluorescence quantum
yield (even in aqueous media), pH-independent emission, visi-
ble excitation/emission wavelength and excellent photostability,
etc.10–14 On the other hand, maleimide is well-established as an
electron acceptor for the construction of PET thiol probes.5 It was
supposed that the C C double bond of maleimide serves as an
electron acceptor to quench the emission of the fluorophore, thus
the probe is non-fluorescent. 1,4-Michael addition with thiols will
saturate the C C double bond of the maleimide unit, thus the
PET quenching effect is prohibited and the fluorescence of the
fluorophore is switched on.5,7 However, this explanation of the
sensing mechanism is based on speculation, and to the best of our
knowledge, the sensing mechanism has not been rationalized from
a theoretical perspective.
aState Key Laboratory of Fine Chemicals, School of Chemical Engineering,
Dalian University of Technology, Dalian 116024, P. R. China. E-mail:
zhaojzh@dlut.edu.cn, guohm@dlut.edu.cn
bCenter Laboratory, Affiliated Zhongshan Hospital of Dalian University,
Dalian 116001, P. R. China
However, the PET effect of probe 3 is narrowly effective; i.e. only
the ortho-isomer (3) shows negligible background emission (U =
0.002, Scheme 1),7 whereas the m- and p-isomers give significant
background emission (e.g. U = 0.37 for probe 4 and U = 0.54
for probe 5, Scheme 1), due to the poor PET effect of 4 and 5.7
Thus 4 and 5 cannot be used as effective thiol probes. The effective
PET of probe 3 was attributed to the shorter distance between
cDepartment of Immunology, Harbin Medical University, 194 Xuefu Road,
Harbin 150081, P. R. China
† Electronic supplementary information (ESI) available: General experi-
mental methods, 1H and 13C NMR spectra of the compounds, theoretical
calculations details (z-matrix), theoretical rationalization of the PET
effect of the probes. CCDC reference numbers 787588–787590. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c0ob00910e
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Scheme 1 Synthesis of the BODIPY-based thiol probes 1 and 2. The known thiol probes 3, 4 and 5 are presented for comparison (note 4 and 5 cannot
be used as probes due to significant background emission, i.e. the non-efficient PET effect).7
the BODIPY core and the maleimide unit, which ensures efficient
PET. The electron donor/acceptor distances in 4 and 5 are much
larger than that of probe 3. Thus the PET effect is not efficient
in probe 4 and 5. These results indicate that maleimide is not a
strong electron acceptor relative to BODIPY for the assembly of
PET fluorescence OFF–ON molecular probes. This postulation is
supported by another Ru(II) bispyridine complex/maleimide thiol
probe which also shows background emission (U = 0.036, note
the parent Ru(II) complex shows U of 0.06, thus the maximum
emission enhancement of the Ru(II)-based thiol probe in this case
will be only ca. 2-fold).8 These results indicate that maleimide is
not a universal electron acceptor for assembly of OFF–ON PET
probes.
To tackle this challenge, we set out to search for more potent
electron acceptors than maleimide to assemble PET fluorescent
probes, especially with electron-deficient fluorophores as photo-
excited electron donors, such as BODIPY, thus creating more
redundancy for design of the molecular probes, e.g., the PET
quenching will be effective even with the p-substitution (e.g. the
profile of probe 2 and dyad 5, Scheme 1), not restricted to o-
substitution (profile of probe 1 and probe 3, Scheme 1).
In order to tackle the aforementioned limitation of the current
thiol probes, herein we devised highly selective PET fluorescence
OFF–ON thiol probes 1 and 2 with BODIPY as the fluorophore
(photo-induced electron donor) and DNBS as the electron accep-
tor (Scheme 1). Probe 1 is devised with the o-substitution and
probe 2 is with the p-substitution profile. We demonstrated that
both probe 1 and 2 show the fluorescence OFF–ON switching
effect in the presence of biologically related thiols such as cysteine,
although probe 1 shows a much higher contrast ratio (300) than
probe 2 (54). Compared to the previously reported thiol probes
3 and 5, our results demonstrate that DNBS is a more potent
electron acceptor than maleimide to assemble PET fluorescent
OFF–ON probes with especially electron-deficient fluorophores
such as BODIPY, naphthalimide, etc.19 The fluorescence OFF–ON
sensing mechanism of the probes was rationalized by theoretical
calculations. The DFT/TDDFT calculations indicate the dark S1
excited state for probes 1 and 2 but radiative S1 excited state for
the fluorophores, and DNBS is ca. 0.76 eV more potent to accept
electrons than the maleimide moiety. The probes were used for
fluorescent imaging of cellular thiols.
Recently 2,4-dinitrobenzenesulfonyl (DNBS) was used for the
design of selective thiol probes and reactive oxygen species
(ROS).15–17 We demonstrated that by attachment of DNBS to
a fluorophore, the DNBS induces the dark state (S1) of the
photophysics of the fluorophore,9 thus the emission is switched
off. Cleavage of the DNBS will re-establish the radiative S1 state of
the fluorophore; as a result the emission is switched on.9 Recently
we extended this strategy to a phosphorescent OFF–ON thiol
probe with a Ru(II) polypyridine complex as the luminophore.18
However, these sensors usually give weak absorption in the visible
region and low luminescence quantum yields.
Experimental
Synthesis of BODIPY 1
2-Hydroxybenzaldehyde (0.75 g, 6.0 mmol) and 2,4-
dimethylpyrrole (1.26 g, 13.2 mmol) were dissolved in CH2Cl2
(200 mL). Then several drops of trifluoroacetic acid were added,
the mixture was stirred at room temperature for 10 h. After,
a solution of tetrachlorobenzoquinone (1.35 g, 6.0 mmol) in
CH2Cl2 (120 mL) was added. The mixture was stirred for 4 h.
After the addition of triethylamine (18 mL, 0.13 mol), BF3·Et2O
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(18 mL, 0.15 mol) was added dropwise, which was cooled in an
ice–water bath. The mixture was stirred for 2 h. The solvent was
removed under reduced pressure and the residue was dissolved
with CH2Cl2 (100 mL). Then the organic phase was washed
with water (2 ¥ 100 mL). The organic portion was collected and
dried over anhydrous Na2SO4, then the solvent was removed
under reduced pressure. The crude product was purified with
column chromatography (silica gel, ethyl acetate/petroleum ether,
1 : 20, v/v). Deep red solid powder was obtained. Yield: 0.71 g,
(s, 1H), 2.55 (s, 6H), 1.44 (s, 6H); 13C NMR (100 MHz, CDCl3):
d 156.5, 155.5, 143.4, 141.9, 132.0, 129.6, 127.5, 121.4, 116.3,
14.7; HRMS (TOF-MS-ESI) calcd for ([C19H19BF2N2O - H]-),
339.1480; Found, 339.1494.
Synthesis of probe 2
BODIPY 2 (100.0 mg, 0.3 mmol) was dissolved in dry CH2Cl2
(10 mL). Then dry triethylamine (0.1 mL) was added to the
solution, and the mixture was stirred for 5 min. A solution of 2,4-
dinitrobenzenesulfonyl chloride (235.0 mg, 0.9 mmol) in CH2Cl2
was added ◦dropwise at 0 ◦C. The reaction mixture was stirred for
10 h at 50 C. The solvent was removed under reduced pressure
and the crude product was subjected to column chromatography
(silica gel, DCM/Petroleum ether, 1 : 3, v/v). Probe 2 was obtained
as an orange-red solid (102.0 mg, 59.7%). M.p. 209 ◦C. lmax
35.0%. M.p. 211 ◦C. lmax (MeOH/H2O = 4 : 1): 500 nm, emax
:
1
75 200 M-1 cm-1. H NMR (400 MHz, CDCl3) d (ppm) = 7.35
(t, 1H, J = 7.2 Hz), 7.01–7.11 (m, 3H), 5.99 (s, 2H), 5.24 (s, 1H),
2.54 (s, 6H), 1.50 (s, 6H); 13C NMR (100 MHz, CDCl3): d 156.6,
152.61, 143.6, 135.6, 131.6, 129.4, 121.8, 121.8, 121.2, 116.8, 14.8,
13.9; HRMS (TOF-MS-ESI) calcd for ([C19H19BF2N2O - H]-),
339.1480; Found, 339.1482.
1
(MeOH/H2O = 4 : 1): 501 nm, emax: 84 300 M-1 cm-1. H NMR
(400 MHz, CDCl3) d (ppm) = 8.67 (d, 1H, J = 4.0 Hz), 8.46–9.49
(dd, 1H, J = 4.0 Hz, J = 8.0 Hz), 8.16 (d, 1H, J = 8.0 Hz), 7.37
(d, 2H, J = 8.0 Hz), 7.31 (d, 2H, J = 8.0 Hz), 5.99 (s, 2H), 2.53 (s,
6H), 1.32 (s, 6H); 13C NMR (100 MHz, CDCl3): d 156.5, 151.3,
149.4, 149.3, 142.7, 139.2, 135.5, 134.0, 133.5, 131.3, 130.5, 126.4,
123.2, 121.9, 120.6, 14.8, 14.7. HRMS (TOF-MS-ES) calcd for
([C25H21BF2N4O7S + Na]+), 593.1090; Found, 593.1066.
Synthesis of probe 1
BODIPY 1 (100.0 mg, 0.3 mmol) was dissolved in 10 mL dry
CH2Cl2. Then, adding 0.1 mL dry triethylamine to the solution,
the mixture was stirred for 5 min. After that the solution of 2,4-
dinitrobenzenesulfonyl chloride (235.0 mg, 0.9 mmol) in CH2Cl2
◦
was added dropwise to the above mixture at 0 C. The reaction
mixture was stirred for 10 h at room temperature. The solvent
was removed under vacuum and the crude product was subjected
to column chromatography (silica gel, DCM/Petroleum ether,
1 : 5, v/v). Probe 1 was obtained as a deep red solid (26.0 mg,
X-Ray single crystal determination
Single-crystal X-ray diffraction data were obtained on a Bruker
SMART APEX CCD diffractometer using graphite monochro-
16.0%). M.p. 214 ◦C. lmax (MeOH/H2O = 4 : 1): 509 nm, emax
:
˚
mated MoKa (l = 0.71073 A) radiation with the SMART and
73 800 M-1 cm-1. 1H NMR (400 MHz, CDCl3) d (ppm) = 8.60 (br,
1H), 8.13–8.16 (dd, 1H, J = 4.0 Hz, J = 8.0 Hz), 7.70 (d, 1H, J =
8.0 Hz), 7.58–7.65 (m, 2H), 7.48 (t, 1H, J = 8.0 Hz), 7.29–7.31 (m,
1H), 5.96 (s, 2H), 2.41 (s, 6H), 1.48 (s, 6H); 13C NMR (100 MHz,
CDCl3): d 157.1, 150.8, 147.4, 143.4, 134.7, 133.9, 131.7, 131.2,
128.7, 127.6, 125.9, 125.4, 122.0, 121.3, 14.6, 14.3; HRMS (TOF-
MS-ES) calcd for ([C25H21BF2N4O7S + Na]+), 593.1090; Found,
593.1103.
SAINT programs. The data were collected using the j/w scan
mode and corrected for Lorentz and polarization effects, during
data reduction with SHELXTL 97 software, the absorption
effect was corrected for as well. Crystallographic data for the
structural analyses have been deposited with the Cambridge
Crystallographic Data Center (CCDC). CCDC reference numbers
for BODIPY 1, probe 1 and probe 2, are CCDC 787588, 787590
and 787589, respectively.†
Synthesis of BODIPY 2
Fluorescent imaging of the cellular thiols
4-Hydroxybenzaldehyde (0.37 g, 3.0 mmol) and 2,4-
dimethylpyrrole (0.63 g, 6.6 mmol) were dissolved in THF
(90 mL). Then several drops of trifluoroacetic acid were added,
and the mixture was stirred at room temperature for 10 h. A
solution of 2,3-dichloro-5,6-dicyano-p-benzoquinone (0.68 g,
3.0 mmol) in THF (120 mL) was added. The mixture was stirred
for 4 h. After the addition of triethylamine (9 mL, 0.08 mol),
BF3·OEt2 (9 mL, 0.08 mol) was added dropwise to the mixture,
which was cooled in an ice–water bath. The mixture was stirred
overnight, and the solvent was removed under reduced pressure.
The residue was dissolved with CH2Cl2 (100 mL) and the solution
was washed with 5% aqueous NaHCO3 (100 mL) followed by
water (2 ¥ 100 mL). The organic layers were dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure.
The crude product was purified with column chromatography
(silica gel, CH2Cl2), 0.54 g red solid powder was obtained, yield:
The NCI-H446 cells were incubated with PBS (phosphate buffered
saline) at 37 C in DMSO–PBS solution (1 : 200, v/v, pH 7) for
◦
1 h. Then the cells were washed with PBS buffer three times, and
the probe (20 mm, 0.4 : 200 DMSO/PBS, v/v, pH 7) was added
and incubated at 37 ◦C for 10 min. After that, the fluorescence
imaging was performed after washing the cells with PBS buffer
three times. The luminescence imaging of the cells was obtained
using a Nikon ECLIPSE-Ti confocal laser scanning microscope.
The green emission was observed with 488 nm laser excitation.
The offset value is ‘70’, and the pinhole is ‘medium’.
Computational details
The ground state structures of the sensors were optimized using
density functional theory (DFT) with B3LYP functional and 6-
31G(d) basis set (considered as gas phase). The excited state
related calculations were carried out with the time dependent DFT
(TDDFT), based on the optimized structure of the ground state.
There are no imaginary frequencies in the frequency analysis of all
53.0%. M.p. 203 ◦C. lmax (MeOH/H2O = 4 : 1): 498 nm, emax
:
1
81 100 M-1 cm-1. H NMR (400 MHz, CDCl3) d (ppm) = 7.11
(d, 2H, J = 8.0 Hz), 6.93 (d, 2H, J = 8.0 Hz), 5.98 (s, 2H), 5.13
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calculated structures. All these calculations were performed with
Gaussian 09W.20
Results and discussion
Design and synthesis of the BODIPY-based thiol sensors
We envisioned that fluorescent OFF–ON PET thiol probes could
be designed with DNBS as an electron acceptor and BODIPY as
an electron donor/fluorophore.9,18 Thus BODIPY/DNBS probes
1 and 2 were devised and expected to show a fluorescence OFF–
ON effect in the presence of thiols (Scheme 1). To compare
the electron-withdrawing capability of DNBS and maleimide,
probe 2 was designed as para-substituted (Scheme 1), thus the
PET efficiency of 2 and 5 can be directly compared by their
background emission. A weaker electron acceptor cannot perturb
the photophysics of BODIPY; the emission of BODIPY will not
be efficiently suppressed. If probe 2 can give weaker background
emission than 5, then DNBS is a more potent electron acceptor
than maleimide.
The syntheses of probes 1 and 2 are straight forward (Scheme 1).
Firstly, phenolic BODIPY 1 and 2 were prepared.14 Then reaction
with DNBS-Cl gives 1 in 16.0% overall yield. Probe 2 was prepared
with a similar approach (31.6% overall yield).
Single-crystal structures of probes 1 and 2
The single crystals of BODIPY 1, and probes 1 and 2 were obtained
by slow evaporation of solutions of the compounds in CH2Cl2.
The single crystal structures are presented in Fig. 1. In BODIPY
1, the 2-hydroxyphenyl attached at C7 takes a perpendicular
geometry against the BODIPY core (90.1◦). This is due to the steric
hindrance imposed by the methyl groups at C4 and C9 positions
of the dipyrromethane core. For probe 1 (Fig. 1b), the phenyl ring
at the C7-position of the BODIPY core takes a dihedral angle of
72.4◦ toward the BODIPY core. The BODIPY fluorophore takes
a co-planar geometry, which is essential for strong fluorescence.
The nitro groups (–NO2) on the phenyl ring take non-coplanar
geometry toward the sulfonylphenyl ring to which the nitro groups
are attached. The o-nitro takes a 36.2◦ dihedral angle and the p-
nitro takes a 21.5◦ dihedral angle. The distortion is probably due to
the steric hindrance between the nitro groups and the phenyl ring.
Furthermore, the sulfonylphenyl ring takes a parallel geometry to
the BODIPY core and the distance between the two subunits of
˚
probe 1 is 3.68 A. Thus p–p stacking is possible for the molecules
packed in the single crystal.19
The single crystal of probe 2 was also determined (Fig. 1c). No
p–p stacking was found. The distortion between the phenyl ring
and the BODIPY core is 75.4◦. Distortions of 80.7◦ and 12.8◦
were found for the o- and p- nitro groups, respectively.
These single crystal structures unambiguously verified the
molecular structures of the thiol probes. To the best of our
knowledge, this is the first time that the single crystal structures
of BODIPY-based fluorescent molecular thiol probes were deter-
mined.
Fig. 1 ORTEP drawings of the compounds. (a) BODIPY 1, (b) probe 1
and (c) probe 2, with thermal ellipsoids shown at the 50% probability level.
All H atoms are omitted for clarity.
after addition of cysteine are presented in Fig. 2a. Both precursor
BODIPY 1 and probe 1 show similar absorption at 509 nm
(Fig. 2a). The intense absorption is due to the S0→S1 transition
of the BODIPY fluorophore, whereas the minor absorption at
350 nm may be due to the S0→S2 transition. The molar extinction
coefficient (e) at 509 nm is up to 73 800 M-1 cm-1. This intense
absorption of visible light is beneficial for fluorescence bioimaging
Selective detection of thiols with probe 1
Firstly probe 1 was studied for selective detection of thiols such as
cysteine. The UV-vis absorption spectra of the probe 1 before and
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Fig. 3 Response of probe 1 to different analytes and pH. (a) Relative
fluorescence intensity of 10 mM probe at 514 nm (lex = 450 nm) before and
after incubation in the presence of 2 mM analytes, incubated for 30 min at
37 ◦C. Neutral pH, in MeOH/water (4/1, v/v) solution. (b) The response
Fig. 2 (a) UV-vis absorption of BODIPY 1 and probe 1 before and after
addition of L-cysteine. (b) Fluorescence emission spectra (lex = 450 nm) of
probe 1 before and after addition of L-cysteine. The fluorescence photos
of the probe 1 alone and the mixture of probe 1/cycsteine (incubated for
30 min) are also presented as the inset in (b). In MeOH/water (4/1, v/v)
solution. At neutral pH, c (probe) = 1.0 ¥ 10-5 mol L-1, c (L-cysteine) =
2.0 ¥ 10-3 mol L-1. 20 ◦C.
of the emission of probe 1 and BODIPY 1 to the variation of pH. lex
=
503 nm. lem = 514 nm. c = 1.0 ¥ 10-5 mol L-1. 20 ◦C.
sensing kinetics with glutathione are much slower than that with
cysteine (see the ESI†).
∑
The responses of probe 1 to ROS, such as O2 and OH∑
-
because without excitation in the UV region, the background
fluorescence of the biological samples can be greatly suppressed.
The similar UV-vis absorption of probe 1 and its precursor
BODIPY 1 indicated that the Frank-Condon excited state of the
BODIPY core is not perturbed by the introduction of DNBS,
thus suggesting a supramolecular photochemical feature for the
interaction between DNBS and BODIPY, i.e. there is no significant
electronic communication between the BODIPY and the DNBS
subunit at the ground state (S0 state). With the addition of cysteine,
the absorption band does not show significant variation (Fig. 2a).
Probe 1 is non-fluorescent in aqueous buffer (U = 0.002)
(Fig. 2b). In the presence of cysteine, however, the emission at
516 nm was intensified by 300-fold (U = 0.470). Thus a fluorescent
OFF–ON switching effect was achieved with probe 1. The
detection limit to cysteine reaches 4.0 ¥ 10-7 mol L-1. The sensing
mechanism is most probably PET. Our later DFT/TDDFT
calculations proved this speculation. Probe 1 is transformed
into BODIPY 1 with cleavage of DNBS by thiols, thus the
green emission will be switched on.9,15–18 The proposed sensing
mechanism was proved by mass spectrometry of the reaction
mixtures (see the ESI†).
were also studied.15b We found that probe 1 gives a response
to OH∑ but not to O2 ∑, which is different from a previously
-
reported DNBS/fluorescein-based molecular probe for detection
of superoxides.15b
Furthermore, the cleavage product of probe 1, i.e. BODIPY 1,
can be ionized and the fluorescence of BODIPY 1 can be probably
quenched. Thus the pH-dependencies of the emission of probe
1 and BODIPY 1 were also investigated (Fig. 3b). The results
show that probe 1 is stable in the physiological pH range and
the emission of BODIPY 1 is constant (the intensive emission is
persistent) in the physiological pH range. To our knowledge, probe
1 is the only example of a thiol probe with BODIPY as an electron
donor (i.e. d-PET) that shows fluorescent OFF–ON switching with
green emission, except the previously reported probe 3.7
Lower contrast ratio of OFF–ON probe 2: effect of the distance
between electron donor/acceptor on the PET efficiency
The distance between the electron donor/acceptor in probe 2
is larger than that of probe 1. This distance is tremendously
important for the contrast ratio (or the PET efficiency) of the
PET fluorescent molecular sensors. This is demonstrated by the
Other biologically related analytes were also tested against
probe 1. Glutathione induces positive responses (Fig. 3a). The
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reported probes 3 and 5 (Scheme 1).7 Probe 3 shows very weak
background emission (U = 0.002, PET is efficient) but 5 shows
undesired intense fluorescence (U = 0.54), due to the poor PET
efficiency, which is believed to be caused by the larger distance
between the electron donor and the electron acceptor.7
Table 1 Photophysical properties of the BODIPY thiol probes and the
BODIPY precursors
lAbs (nm)/ea
lex/nm
l
em/nm
Ub
BODIPY 1
Probe 1
BODIPY 2
Probe 2
Probe 1+ L-cys
Probe 2+ L-cys
500/7.52 ¥ 104
509/7.38 ¥ 104
498/8.11 ¥ 104
501/8.43 ¥ 104
503/7.44 ¥ 104
499/8.72 ¥ 104
503
509
501
502
504
501
514
514
512
516
516
512
0.598
0.002
0.344
0.012
0.470
0.283
Probe 2 shows similar absorption at 501 nm (see the ESI†).
Different from 5 (U = 0.54), probe 2 is very weakly fluorescent (U =
0.012) but shows drastically enhanced emission in the presence of
thiols such as cysteine. The detection limit to cysteine is 4.9 ¥
10-6 mol L-1, slightly lower than that of probe 1. The cleavage
product of probe 2 by thiols shows the intense BODIPY emission
at ca. 512 nm (U = 0.283). Probe 2 is transformed into BODIPY 2
with cleavage of DNBS by thiols, thus the intrinsic photophysics
of BODIPY are re-established and the green emission will be
switched on.9,15–18 The emission of probe 2 in the presence of thiols
was intensified by 54-fold (Fig. 4a). This value is much smaller
than that of probe 1, which indicates that the PET efficiency of
probe 2 is lower than that of probe 1. The selectivity of probe
2 toward amino acids and biological thiols was also investigated
(Fig. 4b). It was found probe 2 is highly selective toward cysteine.
We found that the reaction of probe 2 with cysteine is faster than
that with glutathione (see the ESI†). Probe 2 gives faster sensing
kinetics than probe 1, possibly due to the small steric hindrance
of probe 2.
a Measurements were performed in solvent MeOH/water (4/1, v/v)
and extinction coefficients e (M-1 cm-1) are also shown. b Luminescence
quantum yields were measured with BODIPY (Scheme 1) as the reference
(U = 0.48 in acetonitrile).10b
The main photophysical parameters of BODIPY-based probes
1 and 2 and the precursors are summarized in Table 1. We can
see that probe 1 gives a much higher contrast ratio than probe 2.
We propose that this is due to the smaller distance between the
electron donor and the electron acceptor. Compared to previously
reported BODIPY-based thiol probes, such as 5 (U = 0.54), probe
2 is non-fluorescent.
Thus we propose that DNBS is a more potent electron acceptor
than maleimide to construct PET OFF–ON fluorescent probes.
DFT/TDDFT calculations: elucidation of the fluorescence
OFF–ON sensing mechanism and the electron accepting ability of
DNBS
Recently theoretical calculations have been used for the study
of the photophysics of fluorophores and fluorescent molecular
probes.21–24 Previously we used theoretical calculations based on
density functional theory (DFT) and the time-dependent DFT
(TDDFT) to study the d-PET effect of fluorescent boronic acid
sensors,25,26 as well as fluorescent OFF–ON thiol sensors.9,18
The strategy we used in the rationalization of the fluorescence
properties of the molecular sensors is to study the properties of
the lowest-lying singlet excited state (S1), which is responsible for
the fluorescence emission (Kasha’s rule).27 The S1 state will be
a dark state if the oscillator strength of the S0→S1 transition is
close to zero (forbidden transition). In this case the probe is non-
fluorescent. Large oscillator strength indicates an allowed S0→S1
transition and the molecular probe is probably fluorescent.
The geometry of probe 1 was optimized with DFT methods.
The geometry of the probe is similar to the single crystal structure
(Fig. 1). For example, the optimized dihedral angle between the
phenyl ring at the 7-position and the BODIPY core is 75.3◦, which
is very close to the 72.4◦ of the single crystal. The distortion of the
o-nitro group toward the phenyl ring is 43.1◦, close to the 36.2◦ of
the single crystal.
Next, we studied the singlet excited states of the probes with the
TDDFT calculations (Table 2). The excitation energy, oscillator
strength (f ) and the electronic composition of the singlet excited
states are presented in Table 2. For probe 1, we found that the
S1 state is a dark state.24 Since transitions with complete charge
transfer are forbidden, S1 is a dark state, supported by the oscillator
strength of S0→S1 (f = 0.0004); this means that probe 1 is non-
fluorescent, which is in agreement with the experimental results
(Fig. 2b). For the thiol-cleaved product, i.e. BODIPY 1, however,
Fig. 4 (a) Fluorescence emission spectra (lex = 450 nm) of probe 2 before
and after addition of L-cysteine. (b) Relative fluorescence intensity of
10 mM probe 2 at 514 nm (lex = 450 nm) before and after incubation
in the presence of 2 mM analytes for 30 min. Neutral pH, in MeOH/water
(4/1, v/v) solution. c (probe) = 1.0 ¥ 10-5 mol dm-3, c(L-cysteine) = 2.0 ¥
10-3 mol dm-3. 20 ◦C.
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Table 2 Selected electronic excitation energies (eV) and oscillator strengths (f ), configurations of the low-lying excited states of the probe 1 and the
precursor BODIPY 1, calculated by TDDFT//B3LYP/6-31G(d), based on the optimized ground state geometries
TDDFT//B3LYP/6-31G(d)
Compounds
Electronic transitionsa
Excitation energy
f b
Compositionc
CId
Probe 1
S0→S1
S0→S4
1.84 eV (674 nm)
2.96 eV (419 nm)
0.0004
0.3694
H→L
0.7065
0.4850
0.2393
0.2246
0.5689
0.1268
0.1212
H→L+2
H→ L+1
H–1→L
H→L
BODIPY 1
S0→S1
3.02 eV (411 nm)
0.4704
H–1→L
H–2→L
a Only selected excited states were considered. The numbers in parentheses are the excitation energy in wavelength. b Oscillator strength. c H stands for
HOMO and L stands for LUMO. Only the main configurations are presented. d Coefficient of the wavefunction for each excitation. The CI coefficients
are in absolute values.
the oscillator strength of the S0→S1 transition is 0.4704. This
result indicates that S0→S1 transition is quantum mechanically
allowed, as well as the S1→S0 transition. Thus, BODIPY 1 is
probably fluorescent. This theoretical postulation is fully proved
by experimental results (Fig. 2b).
In order to validate the aforementioned rationalization of the
fluorescence OFF–ON mechanism of thiol probe 1, the reported
thiol probe 3 was also studied with the same approach and similar
results was obtained, i.e. the BODIPY core serves as an electron
donor and the maleimide subunit acts as the electron acceptor to
constitute the dark S1 state (see the ESI†).
1. The HOMO of probe 2 is localized on BODIPY but the LUMO
and LUMO+1 are located on DNBS, i.e. DNBS is an electron sink.
However, the LUMO+2 of probe 2 is localized on BODIPY. The
singlet excited states of probe 2 were studied with TDDFT
calculations (see the ESI†). The HOMO→LUMO transition is
involved in S1. S1 is a dark state, supported by the oscillator
strength of S0→S1 (f = 0.0000), i.e. probe 2 is non-fluorescent.8
This prediction was proved by experimental results. The main
component of the allowed S0→S5 transition (f = 0.4708) of 2 is
the BODIPY localized HOMO→LUMO+2. S5 can be populated
by direct photo-excitation, then via internal conversion the S1 state
is populated. However, S1→S0 is non-radiative. Therefore, the lack
of emission of probe 2 is rationalized by the dark state S1, which is
induced by DNBS. More sophisticated computation was reported
to rationalize the PET sensing mechanism;24b however, we propose
that the DFT/TDDFT-based calculation is sufficient to predict
the correct order of the dark excited state and the emissive excited
state (Fig. 6).
The frontier molecular orbitals of probe 1 and the thiol cleaved
product BODIPY 1 are presented in Fig. 5.
With the same p-substituted profile, probe 5 (Scheme 1) shows
undesired significant background emission (U = 0.54, thus it can-
not be used as a fluorescent thiol probe), but probe 2, with the same
p-substituted scaffold, shows negligible background emission, thus
probe 2 can potentially be used as a fluorescent thiol probe. Our
results infer that DNBS is a more potent electron acceptor than
maleimide because the p-substitution ensures effective PET for
probe 2, but for BODIPY–maleimide thiol probe 3–5 (Scheme 1),
the PET is only effective with the o-substitution.7 We set out to
explain the different PET efficiency, or contrast ratio of probes 2
and 5 with theoretical calculations.
The optimization of 2 shows that the phenyl linker between
BODIPY and DNBS takes a perpendicular geometry against
BODIPY, thus there is no p-conjugation between BODIPY and
DNBS moieties (the ESI†). This is corroborated by the similar UV-
vis absorption of probe 1 and BODIPY (Fig. 2a). The energies
of the HOMO are -5.44 eV and -5.33 eV for probes 2 and
5, respectively (Fig. 6). TDDFT calculations indicate that the
electron will be excited from H to L+2 or L+1 for probe 2 and 5,
respectively. Both L+2 and L+1 are originated from the BODIPY
and have similar energies of -2.45 eV and -2.34 eV, respectively.
The electron will relax to the unoccupied MOs with lower energy,
such as L+1 and L for probes 2 and 5, respectively. For 5, the L
(-2.97 eV) is localized on maleimide. For probe 2, however, both
Fig. 5 The frontier molecular orbitals (MOs) of (a) probe 1 and (b)
BODIPY 1 (i.e. the cleaved product of 1 by thiols). The energy levels of the
MOs are shown (eV). Calculations are based on ground state geometry by
DFT at the B3LYP/6-31G(d)/level using Gaussian 09.
This theoretical approach is applied to probe 2 and the precursor
2 (see the ESI†). Similar results were obtained compared to probe
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Fig. 6 Energy levels of the frontier molecular orbitals of (a) probe 2 and (b) probe 5. The locations of the molecular orbitals are marked. DFT calculation
is based on the ground state geometry at the B3LYP/6-31G(d) level using Gaussian 09. (c) Sensing mechanism of the probes 1 and 2 from an electronic
excited state perspective. G stands for ground state, ET stands for electron transfer excited state (dark state) and LE stands for locally excited state
(emissive state). Cleavage of the DNBS moiety will remove the dark state (ET) from the photophysics of the probe and thus emission will switch on.
L+1 and L are located on DNBS. Interestingly, the energy of the L
of probe 2 is -3.73 eV, which is ca. 0.76 eV lower than maleimide
in 5. Thus, we propose DNBS is a much more potent electron
acceptor than maleimide. Therefore, the background emission
of probe 2 is much weaker than 5. This is supported by the
fluorescence quantum yields, i.e. U = 0.54 for 5,7 vs. U = 0.012 for
probe 2. Electrochemical measurements (cyclic voltammograms,
i.e. CV, see the ESI†) corroborate these theoretical predictions.
CV measurement of probe 2 indicate a reduction at more positive
voltage (-890 mV, i.e. -3.53 eV) which is assigned to the DNBS
moiety, whereas BODIPY shows the reduction at a more negative
value (-1500 mV, i.e. 2.92 eV). Therefore, the DNBS disturbs the
electronic structure of the BODIPY fluorophore. The ET dark
excited state is beneath the bright state.24b
The driving force for PET can be evaluated with the free
energy changes (DG◦) derived from the Rehm-Weller equation
(eqn (1)).28–30 Where E◦(D+/D) is the oxidation potential of
the electron donor, E◦(A/A-) is the reduction potential of the
electron acceptor, herein the values are approximated as the energy
difference of HOMO and LUMO (calculated with DFT). DE0,0 is
the zero-zero transition energy (approximated as the crossing point
Fig. 7 Fluorescence images of NCI-H446 cells. (a) Fluorescence im-
age of cell; (d) fluorescence image of cells incubated with probe 1
(20 mM) for 10 min; (g) fluorescence images of cells pre-treated with
of the excitation and the emission spectra of the sensors). wp was
not considered in the calculation of the DG◦ values. DG◦ of probes
2 and 5 was approximated as -0.76 eV and -0.11 eV, respectively.
The much larger DG◦ of probe 2 than 5 indicates that DNBS is a
more potent electron acceptor than maleimide. This finding will be
helpful for the design of fluorescent molecular probes with more
distinct OFF–ON switching abilities.
N-methylmaleimide (0.5 mM) for 1 h and then incubated with probe 1
(20 mM) for 10 min; (b, e, h) are the corresponding bright field images of
(a, d, g); (c), (f) and (i) are the overlays of the respective fluorescent and
bright images. 37 ◦C.
N-methylmaleimide to pre-treat cells to remove the intracellular
thiols, then the cells were incubated with probe 1, and no green
fluorescence was observed (Fig. 7g).9,18
DG◦ = E◦ (D+/D) - E◦ (A/A-) - DE0,0 + wp
(1)
Probe 2 was also tested for fluorescent imaging of the cellular
thiols. Green fluorescence emission was observed (see the ESI†).
Interestingly, the green fluorescence emission is persistent even
with pre-treatment of the cells with N-methylmaleimide.9,18 This
result indicates that, different from probe 1, probe 2 can be cleaved
by an unknown intracellular species, probably enzymes. Thus the
N-methylmaleimide cannot inhibit the cleavage of DNBS from
In vivo fluorescent imaging with probe 1 and 2
Fluorescent imaging of intracellular thiols with probe 1 was
carried out (Fig. 7). The NCI-H446 cells were incubated with
probe 1 and intense green emission was observed (Fig. 7d). In
order to prove that probe 1 is specific to intracellular thiols, we used
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probe 2. Our result demonstrated that the regioisomers probe 1
and probe 2 show different intracellular specificities.
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Conclusions
In conclusion, we have synthesized two new fluorescent OFF–
ON green-emitting thiol probes 1 and 2 based on BODIPY/2,4-
dinitrobenzenesulfonyl (DNBS) dyads. Probe 1 has the o-
substituted phenyl scaffold and probe 2 has the p-substitution
profile, thus the electron donor/electron acceptor distance of
probe 2 is larger than that of probe 1. This different electron
donor/acceptor distance may impose a significant effect on the
contrast ratio (PET efficiency) of the probes. Both probes show
intense absorption at approximately 500 nm. Probe 1 is non-
fluorescent. Cleavage of DNBS with thiols re-establishes the
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was observed. A similar result was observed for probe 2 but
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The sensing mechanism of the thiol probes was rationalized by
DFT/TDDFT calculations, which indicate the dark S1 excited
state (oscillator strength f = 0.0004 for the S0→S1 transition) for
probe 1 but the emissive S1 state for the cleaved products (i.e.
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0.4704 for the S0→S1 transition of BODIPY 1). The probes 1
and 2 were used for fluorescent imaging of intracellular thiols.
Probe 1 is specific for intracellular thiols (the green emission
will be inhibited by N-methylmaleimide). For probe 2, however,
the green emission is persistent even with pretreatment of the
cells with N-methylmaleimide. Our results show that DNBS
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molecular probes, especially with electron-deficient fluorophores as
the photo-excited electron donor, such as BODIPY. Furthermore,
rationalization of the sensing mechanism of the molecular probes
with DFT/TDDFT calculations will be useful for the design of
fluorescent molecular sensors with predetermined photophysical
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Acknowledgements
We thank the NSFC (20972024 and 21073028), the Fundamental
Research Funds for the Central Universities (DUT10ZD212),
Ministry of Education (SRFDP-200801410004 and NCET-08-
0077), the Royal Society (UK) and NSFC (China-UK Cost-Share
Science Networks, 21011130154), State Key Laboratory of Fine
Chemicals (KF0802), State Key Laboratory of Chemo/Biosensing
and Chemometrics (2008009), the Education Department of
Liaoning Province (2009T015) and Dalian University of Tech-
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