Tuning the intramolecular charge transfer of alkynylpyrenes: Effect on photophysical properties and its application in design of OFF-ON fluorescent thiol probes

Article abstract of DOI:10.1021/jo900588e

(Figure Presented) Green and yellow-emitting 1,6- and 1,8- bis(phenylethynyl) pyrenes (dyes 7, 8, 9, and 10) with different intramolecular charge transfer (ICT) feature were synthesized and the effect of ICT on the photophysical properties of these deriva

Full text of DOI:10.1021/jo900588e

Tuning the Intramolecular Charge Transfer of Alkynylpyrenes: Effect
on Photophysical Properties and Its Application in Design of OFF-ON
Fluorescent Thiol Probes
Shaomin Ji,† Jun Yang,‡ Qing Yang,‡ Shasha Liu,§ Maodu Chen,§ and Jianzhang Zhao*,†
State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Department of Bioscience and
Biotechnology, School of Biological and EnVironmental Science and Technology, and School of Physics and
Optoelectronic Technology, Dalian UniVersity of Technology, 158 Zhongshan Road, Dalian 116012, P.R. China
zhaojzh@dlut.edu.cn
ReceiVed March 18, 2009
Green and yellow-emitting 1,6- and 1,8-bis(phenylethynyl) pyrenes (dyes 7, 8, 9, and 10) with different
intramolecular charge transfer (ICT) feature were synthesized and the effect of ICT on the photophysical
properties of these derivatives were studied by UV-vis absorption spectra, fluorescence emission spectra,
and DFT/TDDFT calculations. For the dyes with electron-pushing group (e.g., -dimethylamino, dye 8 and
dye 10), structureless and solvent polarity-sensitive fluorescence emission spectra were observed. Conversely,
dye with electron-withdrawing group (e.g., -CN, dye 7) shows structured and solvent polarity-independent
emission spectra. OFF-ON fluorescent thiol probes 11 and 12 with 2,4-dinitrobenzenesulfonyl protected
ethynylpyrene fluorophore were designed based on DFT/TDDFT calculations, which predicts dark state (S1)
for these thiol probes (e.g., oscillator strength f ) 0.0086 for S1rS0 transition of the probe 11). This dark
state is induced by the ICT effect with ethynylated pyrene fluorophore as electron donor and 2,4-
dinitrobenzenesulfonyl unit as electron acceptor. Cleavage of the 2,4-dinitrobenzenesulfonyl unit by thiol releases
the free fluorophore, for which the lowest-lying excited state S1 is no longer a dark state, but an emissive state
(f ) 0.9776 for S1rS0 transition). These theoretical predictions on the photophysical properties of the molecular
probes were fully proved by experimental results. Our results demonstrated that the fluorescence OFF-ON
switching of this kind of thiol probe is due to the termination of the ICT effect (which quenches the emission,
by a dark S1 state) by cleavage of the 2,4-dinitrobenzenesulfonyl unit (as acceptor of ICT effect) with thiols,
not the re-establishment of the D-π-A feature of the fluorophore. These investigation on the pyrene derived
green-emitting fluorophores and the DFT/TDDFT calculation aided probe design suggest that future application
of these results may prove useful toward the rational design of fluorophores or fluorescent probes with
predetermined photophysical properties.
Introduction
fluorescent probes. Concerning this aspect, the critical properties
of the fluorophores are excitation/emission wavelength, Stokes
shifts, fluorescent quantum yields (especially in aqueous solu-
tion), solvent polarity sensitivity of the emission, etc. Besides
appropriate fluorophores, proper sensing mechanism is also
essential for successful probe design. Popular fluorescence
sensing mechanisms include intramolecular charge transfer
(ICT), photoinduced electron transfer (PET) and metal chelation
Fluorescent molecular probes are in great interest due to their
versatile applications in chemical, environmental and biological
science.1 Fluorophores are the fundamental building blocks of
† School of Chemical Engineering.
‡ School of Biological and Environmental Science and Technology.
§ School of Physics and Optoelectronic Technology.
10.1021/jo900588e CCC: $40.75  2009 American Chemical Society
Published on Web 05/21/2009
J. Org. Chem. 2009, 74, 4855–4865 4855

Ji et al.
enhanced fluorescent, etc.1 Currently, design of fluorescence
probes is usually based on trial and error method, not a rational
approach, for example guided by theoretical calculations on the
photophysical properties (e.g., with density functional theory
calculations, DFT).2 This lack of rationale in the design makes
it difficult to prepare probes with predetermined photophysical
properties.
Pyrene is a versatile fluorophore, which shows featured
excimer/monomer emission and exceptionally long fluorescence
lifetime (τ is up to 400 ns in deaerated solution, compared to τ
< 10 ns for most organic fluorophores).1a,b,3-6 However, pyrene
suffers from short excitation/emission wavelength (emission is
less than 450 nm, in UV or blue range) and high oxygen
sensitivity of its emission (emission is dramatically quenched
in the presence of oxygen, O2).1a,b The blue emission makes it
difficult to be used for in vivo fluorescence bioimaging because
the background-fluorescence of the biological sample will exert
significant interference. Moreover, the significant quenching
effect of O2 on the emission of pyrene may diminish the probe’s
sensitivity on molecular sensing.1a,b Therefore, preparation of
pyrene derivatives with emission at longer wavelength and
oxygen-independent emission is of great interest.6 Recently,
pyrene derivatives with extended π-conjugation system and red-
shifted emission have been reported.3,6-9 Preliminary application
of these new pyrene derivatives as fluorescent probes is
promising.6 Inspired by all these works and the recent elegant
design of thiol probes with new sensing mechanism (thiol
induced deprotection of 2,4-dinitrobenzenesulfonyl protected
fluorophore),10-12 a sensing strategy which shows excellent
immunity to interference from nitrogen and oxygen nuleophiles,
we set out to synthesis new pyrene-based fluorophores and
design new thiol probes based on these new fluorophores, which
show fluorescence at 560 nm. Furthermore, we investigated the
sensing mechanism of this new kind of thiol probes from a point
of view of theoretical chemistry. Better understanding of the
sensing mechanism is achieved.
Herein we prepared 1,6- and 1,8-bis(phenylethynyl) pyrenes
(dyes 7-10) via Sonogashira coupling reaction. The new
derivatives give emission centered at 560 nm, which is red-
shifted by ca. 200 nm compared to pyrene. The photophysical
properties of the derivatives were found to be dependent on
the intramolecular charge transfer (ICT) feature. On the basis
of theoretical calculations, we designed new alkynylpyrene-
based fluorescent thiol probes. The calculations predict lowest-
lying dark excited states S1 for the thiol probes 11 and 12, which
make the probes nonfluorescent. With cleavage of the 2,4-
dinitrobenzenesulfonyl unit by thiol, thus elimination of the ICT
effect (2,4-dinitrobenzenesulfonyl unit as the electron acceptor),
the fluorophore is released, for which the TDDFT calculation
shows an emissive S1 state and as a result, the deprotected probe
becomes fluorescent. These theoretical predictions on the
fluorescence OFF-ON property of the probe were fully proved
by experimental results. Our finding demonstrate that the
fluorescence OFF-ON effect of the probe in the presence of
thiol is due to the elimination of the ICT effect (quenching
effect, with 2,4-dinitrobenzenesulfonyl unit as the electron
acceptor), not the recovery of the D-π-A feature of the
fluorophore.11 Our investigation on the photophysical properties
of the alkynylpyrenes and the DFT/TDDFT calculation aided
rational probe design approach will be helpful for future
development of organic fluorophores and molecular probes with
predetermined photophysical properties.
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Results and Discussion
Synthesis. Synthesis of the pyrene derivatives are outlined
in Scheme 1. First pyrene was iodized, but the resulted 1,6-
diioide and 1,8-diioide pyrene were difficult to be isolated from
each other. Therefore, a further alkynylation was carried out to
prepare a mixture of 3 and 4, which can be easily purified with
column chromatography to give 1,6-diethynyl and 1,8-diethy-
nylpyrene.13 Electron withdrawing as well as electron pushing
groups were appended on the phenylethynyl pyrenes to study
the ICT effect on the photophysical property of the derivatives.
We noticed the solubility of these compounds in normal organic
solvents is poor.
Guided by DFT and time-dependent DFT (TDDFT) calcula-
tions for the dyes,2 we designed fluorescent thiol probes 11 and
12, with phenylethynyl pyrene unit as the fluorophore and 2,4-
dinitrobenzenesulfonyl as the thiol reactive group (Scheme 1).
Amino group appended phenylethynyl pyrene was reacted with
2,4-dinitrobenzenesulfonyl chloride, with 2,6-dimethylpyridine
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A. Phys. Chem. Chem. Phys. 2004, 6, 51. (c) Benniston, A. C.; Harriman, A.;
Lawrie, D. J.; Mayeux, A.; Rafferty, K.; Russell, O. D. Dalton Trans. 2003,
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4856 J. Org. Chem. Vol. 74, No. 13, 2009

Intramolecular Charge Transfer of Alkynylpyrenes
SCHEME 1. Syntheses of 1,6- and 1,8-Diethynylpyrenes 5-10 and the Thiol Probes of 11 and 12a
a (a) I2/KIO3(1.0/0.4 equiv), CH3COOH/H2O/H2SO4 (100/10/1, V/V), 40 °C, 4 h. 22%. (b) 2-Mehyl-but-3-yn-2-ol (2.3 equiv), Et2NH, Pd(PPh3)2Cl2
(2.3% mol), CuI (2.7% mol), Ar, 50 °C, 20 h. (c) NaOH (9 equiv), toluene, reflux, 4 h, 40%. (d) NEt3-THF, Pd(PPh3)2Cl2 (2% mol), CuI (4% mol),
PPh3 (4% mol) Ar, 50 °C. 4 h. (e) Anhydrous CH2Cl2, 2,4-dinitrobenzenesulfonylchloride (2.4 equiv), 2,6-lutidine (3.0 equiv), r.t., 45 h.
J. Org. Chem. Vol. 74, No. 13, 2009 4857

Ji et al.
FIGURE 1. (a) UV-vis absorption spectra and (b) normalized fluorescence emission spectra of dyes 7, 8, 9 and 10; 1.0 × 10-5 mol dm-3 of dyes
in methanol. The UV-vis absorption and emission of pyrene are included for comparison.
TABLE 1. Photophysical Properties of Alkynyl-Conjugated
(DMP) as the catalyst. The bis-protected and the monoprotected
fluorophore, that is, probes 11 and 12 were obtained and both
were studied for detection of thiol, either for in vitro detection
or in vivo fluorescence imaging of thiols in living cells and
different sensing property were found for probe 11 and 12.
UV-Vis Absorption: The Effect of ICT. The absorption
spectra of the pyrene derivatives in methanol are presented in
Figure 1a. The ethynylpyrenes show 80 nm of red-shifted
absorption compared to pyrene (320 nm). For 7, a derivative
with -CN appendent, a structured absorption band centered at
410 nm was observed. For 8, 9 and 10, however, the intense
absorption bands at about 400 nm are lack of vibronic structure.3
The intense and red-shifted absorption at 400 nm indicates that
there is strong electronic coupling between the appended
arylethynyl unit and the pyrene core, that is, efficient π-conjuga-
tion and ICT effects exist for the pyrene derivatives.5 We
propose that the structureless absorption bands are due to the
significant ICT character of the fluorophore (pyrene as electron
acceptor), conversely the structured absorption of 7 indicates
the lack of profound ICT effect.5 Isomers with 1,6- and 1,8-
disubstitution profiles give different UV-vis absorptions,
demonstrated by 8 and 10. Furthermore, the ethynylpyrenes
shows higher molar extinction coefficients (ε) than the parent
pyrene, together with the larger Stokes shifts, make these
derivatives ideal fluorophores for construction of molecular
probes. The UV-vis absorption of the dyes in different solvents
were studied and only minor solvatochromic shifts were found
(see Supporting Information). The photophysical data of the
ethynylpyrenes were listed in Table 1.
Pyrene Derivatives 7-12 in Different Solvents
c
d
dyes solvents λabs (/nm)a λem (/nm)b ∆ν (cm-1
)
ε (M-1 cm-1
)
Φe
7
8
9
hexane
DCM
MeOH
hexane
DCM
MeOH
hexane
DCM
329/412
332/412
327/405
327/421
429
434/461
447/474
443/469
457/485
530
560
441/469
480
551
456/485
530
559
539
1230
1900
2118
1871
4442
5562
2200
3147
5948
1823
3911
5047
6323
6515
57498
63936
58500
22224
21430
21338
93928
110848
107266
33830
34681
31021
13145
25144
0.80
0.73
0.73
0.63
0.53
0.26
0.47
0.65
0.20
0.69
0.53
0.37
0.003
0.016
427
329/402
331/417
415
421
439
436
402
406
MeOH
10 hexane
DCM
MeOH
11 MeOH
12 MeOH
552
a λabs
:
absorption wavelength (wavelength of first absorption
maximum). b λem: emission wavelength (at the maximum intensity).
c ∆ν: Stokes shifts. d ε, extinction coefficient. e Φ: fluorescence quantum
yields, with quinine sulfate as the standard (Φ ) 0.54 in 0.5 M H2SO4).
that electron donating group can induce substantially red-shifted
emission for the ethynylated pyrene dyes.
Dyes 8, 9 and 10, all appended with electron pushing group,
are characterized with broad emission bands, inferring the ICT
feature of the lowest-lying excited state.3,5 For 7, in which an
electron withdrawing group (-CN) is appended, however,
structured emission was observed (Figure 1b). The fluorescence
quantum yield of 7 is high and is independent of solvent polarity
(Table 1). For 8, 9 and 10, however, the fluorescence quantum
yields are lower in polar solvents than that in nonpolar solvents.
These results infer that ICT occurs for 8, 9 and 10 (with pyrene
unit as the electron acceptor), but not for 7.5 The fluorescence
quantum yields of the designed thiol probe 11 and 12 are very
low (predicted by DFT/TDDFT calculations, which were carried
out prior to the synthesis of the probes, will be discussed later).
Different from pyrene, no excimer emissions were observed for
these arylethynylpyrenes, which may be due to the lack of stable
π-π stacking effect. The increased molar extinction coefficients
and the bathochromic excitation/emission (shifts up to about
100 and 200 nm, respectively) are beneficial for the application
of these dyes for fluorescence analysis, for example, in vivo
fluorescent bioimaging.
Fluorescence Emission Spectra: The Effect of ICT on
the Solvatochromism Effect. The emission of the compounds
in methanol are presented in Figure 1b. The emission of
ethynylpyrene derivatives with electron pushing group is
centered at 560 nm (dye 8, 9 and 10). Compared to pyrene,
these arylethynylpyrenes show about 100 to 200 nm of red-
shifted emission. Emission at longer wavelength is beneficial
for fluorescent analysis, especially fluorescence bioimaging. For
the derivative with electron withdrawing group (dye 7), however,
the emission is centered at 450 nm, and the vibronic structure
of the pyrene emission is maintained. We noticed a monosub-
stituted ethynyl pyrene, which is an analogue of 8 or 10, gives
emission at 540 nm.5 An ethynyl pyrene with similar π-con-
jugation framework to compound 10, but without the dimethy-
lamino substituent, shows emission band centered at 423 nm.6
The emission of compound 10 (centered at 560 nm) demonstrate
The ICT effect can be better understood by performing
solvatochromic studies for all compounds in solvents with
different polarity.5 The emission spectra of the dyes, especially
for those with electron pushing groups (e.g., 8, 9 and 10), are
strongly dependent on solvent polarity (Figure 2b), which is
4858 J. Org. Chem. Vol. 74, No. 13, 2009

Intramolecular Charge Transfer of Alkynylpyrenes
FIGURE 2. Selected emission spectra of ethynyl-conjugated pyrene derivatives (a) dye 7 and (b) dye 9 (1.0 × 10-5 mol dm-3) in different solvents.
In order to demonstrate the relative fluorescence intensity of the dyes in different solvents, the emission spectra were not normalized.
due to the general solvent polarity effect (see Supporting
Information).1a,b,14 On the contrary, the emission of 7 is
independent of solvent polarity and the vibronic structure of
the emission of pyrene is maintained (Figure 2a). We noticed a
monosubstituted ethynyl pyrene with electron withdrawing
substituent, however, shows structureless emission band.5
We carried out DFT and TDDFT calculations for these
arylethynylpyrenes to investigate the relation between the
polarity-dependent emission and the ICT feature of the dyes
(see Supporting Information). The geometry of these molecules
are coplanar according to the optimization result, which is sup-
ported by the intense UV-vis absorption at 400 nm, this 100
nm of red-shifted absorption indicates efficient electronic
communication between the pyrene core and the peripheral
appendents.5 The emissive state of 7 is basically a locally excited
(LE) state as suggested by the HOMO-LUMO distributions.
For 9, however, the emissive state is characterized with more
significant electron redistribution, i.e. ICT feature. Similar results
were found for 8 and 10 (see Supporting Information). The
calculated excitation energy for the transition of S1rS0 of 7
and 9 are 473 and 472 nm (in vacuum), respectively. These
values are close to the experimental result of 412 and 402 nm
(absorption in hexane). These calculations rationalized the
explanation of ICT origin of the solvent polarity dependency
of the dyes’ emission (Figure 2).
The ICT feature of the emission of the dyes was also
demonstrated by the emission-pH profile of 9 (Figure 3). At
neutral and basic pH, the fluorescence was characterized with
ICT emission, that is, broad and structureless emission band
centered at 560 nm. Protonation of the amino group will inhibit
the formation of the ICT state, but generate the LE state, which
eventually leads to a blue-shifted emission.15 By decreasing the
pH of the solution, that is, with protonation of the N atom, the
emission intensity at 560 nm decreased and a new structured
emission band at 420 nm developed (Figure 3). This spectral
change upon pH variation is in full agreement with the proposed
ICT mechanism. The emissive state at neutral and basic pH
range (free amine) is in ICT feature, but at acidic pH (the amino
unit is protonated), the ICT emissive state is not accessible and
the emissive state is basically in LE feature. These results infer
that pyrene unit is slightly electron deficient. This finding is
important for future design of new pyrene-based fluorophores.
No isosbestic point was observed and two different pKa values
can be derived from the titration (Figure 3, apparent pKa1 and
FIGURE 3. pH-dependency of the fluorescence emission of 9. λex
)
334 nm, 1.0 × 10-5 mol dm-3 of 9 in MeOH-H2O (4/1, V/V). Apparent
pKa1 ) 3.00 ( 0.01 (r2 ) 0.99) by emission at 560 nm, pKa2 ) 1.70
( 0.12 (r2 ) 0.99) by emission at 427 nm. (Inset) Emission intensity
at 427 and 560 nm vs the pH of the solution, the solid lines are the
fitting results. *The minor emission shoulder at 668 nm is due to the
second-order transmission of the spectrophotometer’s monochromator.
pKa2). These observations may be due to the fact that more than
two species were involved in the equilibrium, for example, the
free dye 9, the monoprotonated dye 9 and the bis-protonated
dye 9. We propose the first pKa1 ) 3.00 is due to the first step
of the protonation of the dye 9 ([9-H]+ as the product) and the
second pKa2 ) 1.70 is due to the second step of the protonation
([9-H2]2+ as the product).
As the emission of the dyes are strongly dependent on solvent
polarity (Figure 2b), the solvent effect of the dyes were studied
indetailwiththeLippert-Matagaplots(eq1).1a,14 Lippert-Mataga
plots can be used to evaluate the dipole moment changes of the
dyes with photoexcitation, and to clarify the solvent effect as a
general polarity effect or a specific solvent effect, such as
hydrogen bonding.1a,b Linear Lippert-Mataga plot infers general
solvent effect, conversely curves with upward or downward
curvature infer specific solvent effect, such as hydrogen bonding
between the solvent molecules and the dye molecules.1a,14
A
specific solvent effect, for example, hydrogen bond, can possibly
quench the fluorescence.1a,14,16
2∆f
4πε0pca3
∆ν )
(µe - µg)2 + b
(1)
(14) Han, F.; Chi, L.; Wu, W.; Liang, X.; Fu, M.; Zhao, J. J. Photochem.
Photobio. A: Chem. 2008, 196, 10.
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Ji et al.
n2 - 1
thiol probes, which are based on ICT effect. In this kind of
probes, -CHO group, usually as the electron acceptor of the
ICT, can be transformed to thiazolidine, which is electron-
donating. Thus ratiometric as well as fluorescent OFF-ON
signal transduction were observed for these ICT based probes
in the presence of cysteine.19 Recently, fluorophores protected
by 2,4-dinitrobenzenesulfonyl ester have been reported as new
thiol sensing mechanism.10-12 These new thiol probes inspired
us to prepared pyrene based thiol probe and to investigate the
sensing mechanism from a point of view of quantum chemistry.
ε - 1
2ε + 1
∆f )
-
2n2 + 1
where ∆ν ) νabs - νem stands for Stokes shift, νabs and νem are
absorption and emission (cm-1), p is the Planck’s constant, c is
the velocity of light in vacuum, a is the radius of the solvent
cavity in which the fluorophore resides (Onsager cavity radius)
and b is a constant. ∆f is the orientation polarizability, µe and
µg is the ground-state dipole in the ground-state geometry and
the excited dipole in the excited-state geometry and ꢀ0 is the
permittivity of the vacuum. (µe-µg)2 is proportional to the slope
of the Lippert-Mataga plot.
Lippert-Mataga plots were constructed for 7 and 9 (Figure
4) and linear curves were observed, infers that no specific solvent
effect exist.1a,14 To secure this postulation, we carried out
fluorescent MeOH titration of the PhMe solution of the 9.14
Linear plot, instead of a curve with burst phase (i.e., a sharp
change at the beginning of the titration) was observed (Figure
4). This linear plot unambiguously demonstrated that the
quenching effect of the alcohols on the emission of 9 (see
Supporting Information) is mainly due to the general solvent
effect.1a,14 8 and 10 show similar solvent sensitivity, which
means the regioisomers do not show different photophysical
properties.
The Lippert-Mataga plot of 7 gave much smaller slop than
the other dyes (Figure 4 and Supporting Information), which
infers smaller dipole moment changes for 7 with photoexci-
tation.1a,14 The dipole moment changes of the dyes 7, 8, 9 and
10 were calculated as 11.9 D, 31.7 D, 35.7 and 34.8 D,
respectively (eq 1). These large values may be due to the size
of the molecules and the efficient charge separation.1a,b
Dark Lowest-Lying Excited State Induced by ICT Ef-
fect: Theoretical Calculation Aided Design of Fluorescent
Thiol Probes 11 and 12. Changes in the levels of cellular thiols
are closely related to clinical abnormalities, thus, fluorescent
molecular probes for detection of thiol compounds is of great
interest.10,17-19 For these 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 fluorophore attached with
maleimides unit are used as thiol probes.1a,18 Fluorophores
containing -CHO group are also popular for construction of
Herein we designed new fluorescent thiol probes with the
2,4-dinitrobenzenesulfonyl motif.10-12 We wish to clarify
several questions with the design of the probes. First, we wish
to reveal the new sensing mechanism of these thiol sensor from
a point of view of quantum chemistry, which will be helpful
for future probe design. Second, we wish to extend the emission
wavelength of the thiol probes based on ethynylpyrene. Recently
a phenylethynyl pyrene based thiol probe was reported and show
emission at 420 nm (with maleimide-thiol sensing motif),
which is not an ideal candidate to be used for in vivo
fluorescence imaging of thiols in living cells, due to the short
emission wavelength.6
We designed thiol probe 11 and 12 with 1,8-diaminophenyl-
ethynyl pyrene 9 as the fluorophore (Scheme 1 and Figure 5).
We envision that 11 and 12 will show low background emission
but intensified emission in the presence of thiols, due to the
cleavage of 2,4-dinitrosulfonyl group from the fluorophore by
thiols, such as cysteine (Figure 5).10-12 Herein with theoretical
calculations, we will clarify what is responsible for the
fluorescence OFF-ON switching effect, it is the quenching
effect of the 2,4-dinitrobenzenesulfonyl unit, or the re-establish-
ment of the D-π-A feature of the free fluorophore. The
elucidation of the sensing mechanism will be helpful for
selection of proper fluorophore for future design of molecular
probes.
First we examined the possibility of 11 and 12 (with 9 as the
fluorophore) to be used as thiol probes. The main purpose of
the quantum calculation is to examine the lowest-lying singlet
excited state, which is responsible for the emissive property of
the probes. Higher electronic excited states are usually not
directly involved in the emission of excited fluorophores,
according to Kasha’s rule.1a-c,16 The critical issue is to prove
the free probe is nonfluorescent but the deprotected probe is
fluorescent (Figure 5). The quantum calculation of the designed
probes 11, 12 and the free fluorophore 9 were summarized in
Figure 5 and Table 2. The emissive property (as well as the
excitation) of a dye can be evaluated by the quantum mechanics
selection rule, e.g. the symmetry and the overlapping of the
molecular orbitals (MOs), the change of the spin state and
the oscillator strength (f) of the electronic transitions, etc.1a-c,16
The calculated excitation energy for the transition of S1rS0 of
9 is 472 nm (in vacuum), which is close to the experimental
result of 402 nm (absorption in hexane). This S1rS0 transition
is a fully allowed transition, indicated by the overlapping of
the HOMO and LUMO, as well as the transition oscillator
strength (f) of 0.9776.1b,c This means the reverse transition, that
is, S1fS0 transition is also fully allowed, thus this dye is
potentially fluorescent.1b,c,16 For probe 11, however, the S1rS0
transition is excited at much longer wavelength of 852 nm (1.45
eV), with f value of 0.0086. This small f value and the lack of
overlapping for the HOMO and LUMO (Figure 5) indicates
that transition from ground state to the lowest-lying excited state
FIGURE 4. Lippert-Mataga regressions of the dyes 7 and 9. Stokes
shifts (cm-1) vs the Lippert solvent parameters of orientation polariz-
ability (∆f). For the solvents used, see experimental section. The change
of the Stokes shift of 9 in binary solvent (PheMe/MeOH, with increasing
MeOH) is also included. Solid lines are the fitting result with the
Lippert-Mataga equation (eq 1). 7, r2 ) 0.80; 9, in neat solvents r2 )
0.95, in binary solvents r2 ) 0.99.
4860 J. Org. Chem. Vol. 74, No. 13, 2009

Intramolecular Charge Transfer of Alkynylpyrenes
FIGURE 5. Design of thiol probes with predetermined photophysical properties. Theoretical prediction of the fluorescence OFF-ON switching
effect of the designed thiol probes 11 in the presence of thiol. HOMO and LUMO of 11 and the free fluorophore 9 are presented. Note the S1 and
S2 states of the probe 11 are isoenergetic dark states. Calculated with DFT/TDDFT at the B3LYP/6-31G(d) level using Gaussian 03.
is strongly forbidden, i.e. S1 state is not directly accessible with
photoexcitation, instead it can only be populated with inner
conversion (IC) of the higher excited states, such as the allowed
excited state of S7 (f ) 1.0289, Table 2).1a-c An isoenergetic
electronic transition of S2rS0 (847 nm, 1.46 eV) was found
(Table 2). This degenerated low-lying excited states S1 and S2
are reasonable because the ET to both the 2,4-dinitrobenzene-
sulfonyl units is featured with equal opportunity. The low-lying
allowed transition for 11 is S7rS0 (464 nm). According to the
Kasha’s rule, the IC process is fast and S7 will relaxes to S1
before any possible relaxation to S0 state.1a-c However, the
lowest-lying excited state S1 is dark state, i.e. nonemissive state,
no radiative decay to the ground state S0 will occur (in this
case a nonradiative deactivation channel will act as a drain pipe
for the excited state energy). Similar dark states of S1 and S2
are found for 12 (Table 2). This means dye 11 and 12 are not
fluorescent. Inspection of the HOMO-LUMO (Figure 5) of 11
and 12 indicate that the dark state is due to the ICT effect, with
the pyrene core as the electron donor and the 2,4-dinitroben-
zenesulfonyl unit as the electron acceptor.
We demonstrated that a reported fluorescent thiol probe with
similar sensing mechanism and fluorescence OFF-ON effect
can be fully rationalized by the above theoretical considerations
(see Supporting Information for detail).11
To interpret the quantum calculations in a more intuitive
manner, the possible fluorescence lifetimes of dye 9, probe 11
(15) Zhao, G.-J.; Chen, R.-K.; Sun, M.-T.; Liu, J.-Y.; Li, G.-Y.; Gao, Y.-L.;
Han, K.-L.; Yang, X.-C.; Sun, L. Chem.sEur. J. 2008, 14, 6935.
(16) Zhao, G.-J.; Liu, J.-Y.; Zhou, L.-C.; Han, K.-L. J. Phys. Chem. B 2007,
111, 8940.
(17) Fujikawa, Y.; Urano, Y.; Komatsu, T.; Hanaoka, K.; Kojima, H.; Terai,
T.; Inoue, H.; Nagano, T. J. Am. Chem. Soc. 2008, 130, 14533.
(18) (a) Matsumoto, T.; Urano, Y.; Shoda, T.; Kojima, H.; Nagano, T. Org.
Lett. 2007, 9, 3375. (b) Weh, J.; Duerkop, A.; Wolfbeis, O. S. ChemBioChem
2007, 8, 122.
J. Org. Chem. Vol. 74, No. 13, 2009 4861

Ji et al.
TABLE 2. Selected Electronic Excitation Energies (eV) and
Corresponding Oscillator Strengths (f), Main Configurations and CI
Coefficients of the Low-lying Electronically Excited States of 9, 11
and 12a
bond, i.e. reaction of 11 and 12 with thiol leads to dye 9 (Figure
5),11,12 correspondingly the dark state of probe is replaced by
emissive state, thus a fluorescence OFF-ON response is
predicted for probes 11 and 12 in the presence of thiols.11,16
This finding is of great interest because it relieve the restriction
on using D-π-A type of fluorophore to construct this kind of
thiol probes,11 as it is known that D-π-A fluorophores usually
show low fluorescence quantum yields in high polarity solvents,
such as aqueous solution. Our findings offers more diversity
for the selection of fluorophores in molecular probe design.
Synthesis of 11 and 12 as ICT Modulated Fluorescent
Thiol Probes. Inspired by the theoretical calculations which
predicts the fluorescence OFF-ON switch property of the
designed thiol probes, we prepared the two probes 11 and 12
and the absorption/emission spectra with and without thiol (e.g.,
L-cysteine) were recorded (Figure 6).
The maximal absorption of 11 is located at ca. 400 nm. The
calculated excitation energy (464 nm, S7rS0) is close to the
experimental result.20 The UV-vis absorption did not change
significantly in the presence of thiol (Figure 6a), inferring that
the D-π-A feature of the fluorophore 9 does not change
profoundly with cleavage of the 2,4-dinitrobenzenesulfonyl unit.
This UV-vis absorption profile is in contrast to a reported thiol
probe with similar sensing mechanism,11 and suggests that the
fluorescence OFF-ON switching effect is not due to the intrinsic
ICT feature of the fluorophore, instead, the 2,4-dinitrobenze-
nesulfonyl unit is essential.
Theoretical calculations predict a dark state S1 for 11.
Experimentally, 11 is nonfluorescent (Figure 6b). In the presence
of thiol compound, e.g. L-cysteine, however, the fluorescence
is switched ON, which is in full agreement with the theoretically
predicted emissive 9 (Figure 6b). The fluorescence intensity of
11 is enhanced by 53 folds. We propose the emission in the
presence of cysteine is due to the deprotected amine, that is, 9
(Figure 5).10-12 This is demonstrated by the emission spectra
of 9 under similar conditions.
For the monoprotected 12, similar fluorescence OFF-ON
switch is observed. In the presence of L-cysteine, 26-folds of
fluorescence intensifying is observed (see Supporting Informa-
tion). This lower enhancement may be due to the lower ET
efficiency for this monoprotected probe.
The reaction kinetics of probe 11 and 12 with cysteine were
also investigated and the result show that the reaction is fast at
room temperature and finished within 5 min (inset of Figure
6b). The reaction of probe 11 is slightly faster than that of probe
12. We propose that dinitrophenylsulfonylamide deprotection
is likely to proceed via a two-step mechanism: 11 f 12 f 9,
the slow one is assumed to be the second step.
We noticed phenylethynylpyrene has been used for fluores-
cent thiol probes, based on the maleimide approach.6 However,
the emission wavelength of that reported probe (centered at
about 400 nm) is shorter than the probes 11 and 12 described
herein (emission centered at 560 nm). The blue-shifted emission
of the reported probe is probably due to the electron withdrawing
effect of the maleimide group.5,6
As the alkynylpyrene show much shorter luminescence
lifetime than pyrene, any possible interference from oxygen on
the fluorescence can be eliminated.5,6 Our experiments show
the fluorescence pyrene can be profoundly quenched by oxygen
(with I0/I100 ) 64.1, where I0 is the emission intensity in
deaerated solution, I100 is the emission intensity in oxygen
saturated solution, see Supporting Information). But the fluo-
TDDFT//B3LYP/6-31G(d)
electronic
dyes transitions
energy (eV)b
fc
compositiond
CIe
9
S0fS1 2.63 eV (472 nm) 0.9776 HOMOfLUMO
S0fS2 3.05 eV (406 nm) 0.1048 HOMO-1fLUMO
HOMOfLUMO+1
S0fS3 3.47 eV (357 nm) 0.5213 HOMO-3fLUMO
HOMOfLUMO+1
0.6411
0.6072
0.3417
0.3127
0.4722
0.3780
0.3320
0.4262
0.7030
0.7027
0.6221
S0fS5 3.83 eV (324 nm) 0.5976 HOMO-3fLUMO
HOMOfLUMO+1
HOMOfLUMO+2
11
12
S0fS1 1.45 eV (853 nm) 0.0086 HOMOfLUMO
S0fS2 1.46 eV (847 nm) 0.0091 HOMOfLUMO+1
S0fS7 2.67 eV (464 nm) 1.0289 HOMOfLUMO+4
S0fS25 3.60 eV (344 nm) 1.1588 HOMO-1fLUMO+4 0.3814
HOMOfLUMO+7
S0fS1 1.13 eV (1090 nm) 0.0065 HOMOfLUMO
S0fS2 1.60 eV (774 nm) 0.0004 HOMOfLUMO+1
S0fS6 2.56 eV (484 nm) 0.8528 HOMOfLUMO+2
0.4602
0.7038
0.7045
0.6406
S0fS16 3.41 eV (364 nm) 0.5139 HOMO-1fLUMO+2 0.3294
HOMOfLUMO+4 0.4713
a Calculated by TDDFT//B3LYP/6-31G(d), based on the DFT//
B3LYP/6-31G(d) optimized ground state geometries. b Only the
low-lying excited states and some allowed transions were presented.
c Oscillator strength. d Only the main configurations with configuration
interaction (CI) coefficients >0.3 are presented. e CI coefficients are in
absolute values.
and probe 12 were calculated based on the calculated data (Table
2) by using Einstein transition probabilities of the spontaneous
transitions, which is described by the eq 2.20
c3
τ )
(2)
2 · (E)2 · f
where τ is the fluorescence lifetime, c stands for the light
velocity, E is the transition energy and f is the oscillator strength.
All the parameters are in atomic unit (au). The f and E values
are based on the DFT/TDDFT calculation results (Table 2). The
calculated fluorescence lifetimes for dye 9, probe 11 and probe
12 are 3.4 ns, 1270 and 2775 ns, respectively. The reasonable
predicted lifetime of dye 9 infers that it may be fluorescent.1a-c
For probe 11 and 12, however, the exceptionally long lifetimes
are far beyond the allowed lifetime range for the relaxation of
a singlet excited state (usually singlet excited state is the
emissive state of most organic fluorophores, with lifetimes
shorter than 10 ns). These results show that probe 11 and 12
are nonfluorescent. It should be pointed out that this is a
approximation because the excitation energy is used in the
calculation, not the energy corresponding to the emission
(optimization of the excited states are required).
Thus 2,4-dinitrophenylsulfonyl substituent quenches the
fluorescence but thiols hydrolyses the phenylsulfonyl amide
(19) (a) Lin, W.; Long, L.; Yuan, L.; Cao, Z.; Chen, B.; Tan, W. Org. Lett.
2008, 10, 5577. (b) Toutchkine, A.; Nalbant, P.; Hahn, K. M. Bioconjugate Chem.
2002, 13, 387. (c) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am.
Chem. Soc. 2007, 129, 11666. (d) Tanaka, F.; Mase, N.; Barbas, C. F., III Chem.
Commun. 2004, 1762. (e) Zhang, D.; Zhang, M.; Liu, Z.; Yu, M.; Li, F.; Yi, T.;
Huang, C. Tetrahedron Lett. 2006, 47, 7093. (f) Kim, T.-K.; Lee, D.-N.; Kim,
H.-J. Tetrahedron Lett. 2008, 49, 4879. (g) Duan, L.; Xu, Y.; Qian, X.; Wang,
F.; Liu, J.; Cheng, T. Tetrahedron Lett. 2008, 49, 6624. (h) Lee, K.-S.; Kim,
T.-K.; Lee, J. H.; Kim, H.-J.; Hong, J.-I. Chem. Commun. 2008, 6173. (i) Zhang,
X.; Ren, X.; Xu, Q.-H.; Loh, K. P.; Chen, Z.-K. Org. Lett. 2009, 11, 1257.
(20) (a) Yang, L.; Ren, A.-M.; Feng, J.-K.; Wang, J.-F. J. Org. Chem. 2005,
70, 3009. (b) Liu, Y.-L.; Feng, J.-K.; Ren, A.-M. J. Phys. Chem. A 2008, 112,
3157.
4862 J. Org. Chem. Vol. 74, No. 13, 2009

Intramolecular Charge Transfer of Alkynylpyrenes
FIGURE 6. (a) UV-vis absorption and (b) fluorescence emission spectra (λex ) 441 nm) of probe 11 (1.0 × 10-5 mol dm-3) before and after
addition of L-cysteine in methanol/water (4/1, V/V) solution. The final concentrations of the probe and L-cysteine are 1.0 × 10-5 mol dm-3 and 2.0
× 10-3 mol dm-3, respectively. Inset of (b): fluorescence emission intensity changes of probes 11 and 12 (1.0 × 10-5 mol dm-3) as a function of
elapsed time upon addition of L-cysteine (2.0 × 10-3 mol dm-3) in MeOH-H2O (4/1, V/V). λex ) 441 nm; λem ) 562 nm; 25 °C.
propose that 11 is less cell-permeable, possible due to its high
hydrophobicity. In order to prove that the fluorescence switch
on effect was due to the reaction between the probe and the
intracellular thiols, we used thiol-reactive N-methylmaleimide
to pretreat the cells to remove the intracellular thiol,11,12 no
fluorescence was observed (Figure 8, C and E). The discrepancy
between the color of the bioimaging photo (Figure 8) and the
emission wavelength of the dyes in solution (Table 1) is due to
the different polarity of the microenvironment in which the dye
molecules dwell.1a We also used onion epidermal cells to do
the control experiment (Supporting Information). We found that
12 is onion epidermal cell-permeable but 11 is not. Yellow-
green fluorescence was observed for 12 within the cell. The
strong signal was localized to cytoplasm. On the other hand,
nuclei showed weak fluorescence signals (see Supporting
Information). In a control experiment, the onion epidermal cells
FIGURE 7. Response of probe 11 to different analytes. Relative
were pretreated with N-methylmaleimide to react with the
fluorescence intensity of 10 µM probe at 562 nm (λex) 441 nm) before
intracellular thiols. Then the onion epidermal cells were
and after incubation in the presence of 2 mM analytes at room
incubates with 12, no fluorescence signal was observed. These
results confirm 12 is specific for thiols over other analytes in
living cells.
temperature for 15 min. Twenty-five °C, pH 7.4, methanol/water (4/1,
V/V) solution.
rescence intensity of 9 is less sensitive to oxygen (I0/I100 ) 1.9,
see Supporting Information).
Conclusion
The selectivity of probe 11 for thiols over biologically relevant
analytes was studied at physiological conditions (Figure 7). 11
shows a positive fluorescence response only in the presence of
thiols (cysteine, reduced glutathione and 3-mercaptopropoinic
acid), for which fluorescence signals were increased by 20- to
53-fold. No significant positive fluorescence response were
observed in the presence of amines (lysine), reactive oxygen
species (hydrogen peroxide), or reducing agents (ascorbic acid).
Probe 12 shows similar thiol selectivity (see Supporting
Information).
It is worth to note that the fluorescence quantum yield of 9
is 0.20 in MeOH (0.018 in MeOH-H2O) because the fluoro-
phore is without any profound D-π-A feature. D-π-A type of
fluorophores usually show decreased emission intensity in high
polar solvents, such as aqueous solution.
Fluorescence Imaging of Thiols in Living Cells. The
monitoring of thiols in living pheochromocytoma cells (PC12
cells) was carried out (Figure 8). The pictures of bright field
images and fluorescence images were taken by fluorescence
microscope. The cells were incubated with the probes and green
fluorescence was observed with 12 (Figure 8D). Weaker
fluorescence image was observed with 11 (Figure 8B). We
We synthesized 1,8- and 1,6-bis(phenylethynyl) pyrenes with
different intramolecular charger transfer (ICT) feature and the
ICT effect on the photophysical properties of these dyes were
studied. For the dyes with profound ICT feature (dye 8 and 10,
appended with -N(CH3)2), broad and solvatochromic emission
bands were observed. Conversely, for alkynylpyrenes with minor
ICT feature (dye 7, appended with -CN), however, solvent
polarity independent and structured emission spectra were
observed. Phenylethynylated pyrene derived fluorescent thiol
probes with 2,4-dinitrobenzenesulfonyl amide structure were
designed and were predicted by TDDFT calculations to show
fluorescence OFF-ON switching effect in the presence of thiol.
The TDDFT calculations predict lowest-lying dark states of S1
for the probes, induced by ICT effect (electron transfer from
fluorophore to 2,4-dinitrobenzenesulfonyl unit). This dark state
infers the probe is nonfluorescent. Cleavage of the 2,4-
dinitrobenzenesulfonyl unit by thiol releases the free fluorophore
(the ICT effect is terminated), for which the lowest-lying excited
state S1 is no longer a dark state, therefore the product will
potentially be fluorescent. These theoretical predictions on the
photophysical properties of the probes were fully proved by
experiments, fluorescence OFF-ON switching effect was
J. Org. Chem. Vol. 74, No. 13, 2009 4863

Ji et al.
FIGURE 8. Fluorescence images of PC12 cells. (A) Fluorescence images of cell. (B) Fluorescence images of cells incubated with probe 11 (25
µM) for 20 min at 37 °C. (C) Fluorescence images of cells pretreated with N-methylmaleimide (0.1 mM) for 60 min at 37 °C and then incubated
with probe 11 (25 µM) for 20 min at 37 °C. (D) Fluorescence images of cells incubated with probe 12 (25 µM) for 20 min at 37 °C. (E) Fluorescence
image of PC12 cells pretreated with N-methylmaleimide (0.1 mM) for 60 min at 37 °C and then incubated with probe 12 (25 µM) for 20 min at
37 °C. (F-J) are the corresponding bright field images of (A-E).
were added under argon. The reaction mixture was heated at 60
°C for 7 h. The solvent was removed under vacuum and the residue
was subjected to column chromatography (silica gel, petroleum
methanol/dichloromethane, 1/150, V/V). Compound 9 was obtained
observed for the probes 11 and 12 in the presence of thiols.
The probes were successfully used for bioimaging of thiols in
living cells. Our results show that the fluorescence OFF-ON
switching effect of the fluorescent probe is due to the re-
establishment of the emissive S1 state of the probe, after
termination of the ICT effect by cleavage of the 2,4-dinitroben-
zenesulfonyl unit, instead of the recovery of the D-π-A character
of the fluorophore. Our finding infers that modular fluorophore
unit for this kind of thiol probes is not restricted to the D-π-A
type of fluorophore, instead, dyes with diverse structure can be
used for construction of fluorescent thiol probes with 2,4-
dinitrobenzenesulfonyl as the thiol reactive unit. We envision
that these investigation on the photophysical properties of the
pyrene derived fluorophores and the successful application of
DFT/TDDFT calculations in the rational design of fluorescent
molecular probes are of great interest for development of new
fluorophores and molecular probes with predetermined photo-
physical properties.
1
as a orange solid (90 mg, 82.0%). Mp 213.1-214.3 °C. H NMR
(400 MHz, DMSO-d6) δ 8.75 (s, 2H), 8.17(d, 2H, J ) 8.0 Hz),
8.10 (d, 2H, J ) 8.0 Hz), 8.02 (s, 2H), 7.54(d, 4H, J ) 8.4 Hz),
6.73(d, 4H, J ) 8.4 Hz), 3.88(s, 4H), HRMS: m/z calcd for
C32H20N2 ([M + H]+) 432.1626, found 432.1630.
Compound 11 and 12. To a stirred solution of 9 (60.0 mg, 0.14
mmol) and 2,6-lutidine (50 µL, 0.39 mmol) in anhydrous dichlo-
romethane (10 mL) was added 2,4-dinitrobenzeneslfonyl chloride
(100.0 mg, 0.34 mmol) in portions at 0 °C. The reaction mixture
was allowed to stir for 45 h as it slowly warms up to room
temperature. The solvent was removed under vacuum to give a
mixture of 11 and 12. The crude product was then subjected to
column chromatography (silica gel, dichloromethane/methanol, 100/
1, V/V). Compound 11 and 12 was obtained as yellow brown solid.
The yield of 11 and 12 are 25.0 mg (20.0%) and 40.0 mg (43.0%),
1
respectively. 11: H NMR (400 MHz, DMSO-d6) δ 8.90 (s, 2H),
8.73(s, 2H), 8.61-8.63(d, 2H, J ) 8.0 Hz), 8.37(d, 2H, J ) 8.0
Hz), 8.25-8.30 (q, 6H, J ) 8.0 Hz), 7.70 (d, 4H, J ) 8.0 Hz),
7.24(d, 4H, J ) 8.4 Hz). 13C NMR(100 MHz, DMSO-d6): δ 149.8,
147.8, 132.7, 131.6, 131.0, 130.9, 129.8, 128.1, 127.1, 126.2, 125.8,
123.3, 120.7, 120.2, 117.6, 95.5, 88.0, 79.1, 28.9. HRMS: m/z calcd
Experimental Section
Compound 7. 1-Cyano-4-iodobenzene (58.0 mg, 0.25 mmol)
was dissolved in 5 mL Et3N. The solution was purged with argon,
then Pd(PPh3)2Cl2 (3.8 mg, 5.4 µmol), PPh3 (2.7 mg, 0.01 mmol),
6 (50.0 mg, 0.12 mmol) and CuI (1.9 mg, 0.01 mmol) were added.
The reaction mixture was heated at 60 °C for 7 h. The solvent was
removed under vacuum and subjected to column chromatography
(silica gel, dichloromethane/petroleum ether, 2/1, V/V). Compound
7 was obtained as a orange solid (20.0 mg, yield: 35.0%). Mp
293.7-294.5 °C.1H NMR (400 MHz, CDC13) δ 8.75 (s, 2H),
8.27(d, 2H, J ) 12.0 Hz), 8.21(d, 2H, J ) 8.0 Hz), 8.13(s, 2H),
7.81 (d, 4H, J ) 7.6 Hz), 7.73 (d, 4H, J ) 8.4 Hz), HRMS: m/z
calcd for C34H16N2 ([M + H]+) 452.1313, found 452.1306.
Compounds 8 and 10 were synthesized by the same method. 8:
orange powder, yield: 40.0%. Mp 249.1-250.4 °C. 1H NMR (400
MHz, CDC13) δ 8.78 (s, 2H), 8.18 (d, 2H, J ) 8.0 Hz), 8.11 (d,
2H, J ) 8.0 Hz), 8.03 (s, 2H) 7.64 (d, 4H, J ) 8.8 Hz), 6.82 (m,
4H), 3.06 (s, 12H). HRMS: m/z calcd for C36H28N2 ([M + H]+)
488.2252, found 488.2252. 10: orange powder, yield: 32.0%. Mp
>300 °C. 1H NMR (400 MHz, CDC13) δ 8.68 (d, 2H, J ) 9.6 Hz),
8.13-8.21 (m, 6H), 7.68 (d, 4H, J ) 8.0 Hz), 7.30 (m, 4H), 3.10
(s, 12H). HRMS: m/z calcd for C36H28N2 ([M + H]+) 488.2252,
found 488.2256.
1
for C44H23N6O12 ([M - H]-) 891.0815, found 891.0836. 12: H
NMR (400 MHz, DMSO-d6) δ ) 8.94 (s, 1H), 8.72 (d, 2H, J )
5.6 Hz), 8.62-8.65 (d, 1H, J ) 6.8 Hz), 8.19-8.34 (q, 7H,), 7.75
(d, 2H, J ) 8.8 Hz), 7.45 (d, 2H, J ) 8.0 Hz), 7.28 (d, 2H, J ) 8.4
Hz), 6.66 (d, 2H, J ) 8.0 Hz), 5.75 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 150.6, 150.4, 148.3, 136.5, 133.4, 132.1, 131.7, 131.6,
130.8, 130.7, 130.3, 129.8, 128.8, 128.1, 127.8, 127.0, 126.3, 125.9,
123.9, 121.1, 120.9, 119.6, 119.2, 117.5, 114.2, 108.5, 98.9, 95.5,
88.8, 86.0. HRMS: m/z calcd for C36H21N4O6 ([M - H]-) 661.1182,
found 661.1171.
UV-Vis and Fluorescence Spectra. The concentration of the
dyes was fixed at 1.0 × 10-5 mol dm-3 for the pH titration. pH
measurements were carried out with a Delta 320 Microprocessor
pH meter (Mettler Toledo). The fluorescence spectra of the dyes
were recorded as the pH was changed from pH 2 to 11 in
approximate intervals of approximately 0.5 pH units. The pH was
controlled using minimum volumes of sodium hydroxide and
hydrochloric acid solutions. For the Lippert-Magata plots (Figure
4), the solvents used were hexane, toluene, dioxane, dioxane/ethyl
acetate (1/1, V/V), ethyl acetate, dichloromethane, acetone, 1-hex-
anol, ethanol and methanol (in the order of increasing ∆f values).
Curves were constructed using the Origin 5.0 (Microcal software).
Compound 9. 4-Iodoaniline (121.5 mg, 0.56 mmol) was
dissolved in 8 mL Et3N. The solution was degassed and purged
with argon, then Pd(PPh3)2Cl2 (7.1 mg, 0.01 mmol), PPh3 (5.3 mg,
0.02 mmol), 6 (100.0 mg, 0.25 mmol) and CuI (4.5 mg, 0.02 mmol)
4864 J. Org. Chem. Vol. 74, No. 13, 2009

Intramolecular Charge Transfer of Alkynylpyrenes
Theoretical Calculations. The ground state structure of the dyes
and probes were optimized using density functional theory (DFT)
with B3LYP functional and 6-31G (d) basis set. The excited state
related calculations were carried out with the Time dependent
density functional theory (TD-DFT) with the optimized structure
of the ground state (DFT/6-31G(d)). There are no imaginary
frequencies in frequency analysis of all calculated structures,
therefore each calculated structure is an local energy minimum.
All these calculations were performed with Gaussian 03.
Fluorescence Microscopy and Cell-Uptake Studies. PC12 cells
were grown in DMEM (Dulbecco’s Modified Eagle’s Medium) in
an atmosphere of 5% CO2, 95% air at 37 °C. Cells were plated on
24-well plate and allowed to adhere for 24 h. After 24 h, the cells
were washed with PBS (phosphate buffered saline) and then
incubated at 37 °C in the presence probe 11 or 12 (25 µM, 0.5:100
DMSO/DPBS, V/V, pH 7) for 20 min. Fluorescence imaging was
performed after washing the cells three times with PBS buffer. The
fluorescence images were obtained using an OLMPUS IX-71
fluorescence microscope with blue excitation module. The exposure
times for PC12 cells were 0.02 s (with 640× magnification), for
onion epidermal cells, the exposure times were 0.25 s (with 160×
magnification). The images for the thiol-dependent activation and
corresponding maleimide controls were taken with identical settings.
Acknowledgment. We thank the NSFC (20642003 and
20634040), Ministry of Education (SRF for ROCS, SRFDP-
200801410004 and NCET-08-0077), PCSIRT(IRT0711), Cul-
tivation Fund of the Key Scientific and Technical Innovation
Project (707016), State Key Laboratory of Fine Chemicals
(KF0710 and KF0802), State Key Laboratory of Chemo/
Biosensing and Chemometrics (2008009) and Dalian University
of Technology (SFDUT07005) for financial support. We are
also grateful to the reviewers for their critical and helpful
comments on the manuscript.
Supporting Information Available: General experimental
methods, 1H and 13C NMR data of the compounds, photophysi-
cal data and solvent effect of the dyes, oxygen sensitivity of
the emission intensity, response of probe 12 to thiols, theoretical
calculations details (including z-matrix), theoretical rationaliza-
tion of the sensing mechanism of a reported thiol probe,
fluorescence imaging of thiol in living cells. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO900588E
J. Org. Chem. Vol. 74, No. 13, 2009 4865