Comparative studies of multi-photon induced emission by pyridine-based small molecular probes in biological media: Selective binding of bioactive molecules and in vitro imaging

Article abstract of DOI:10.1002/ejoc.201100570

A new class of organic molecular probes (1-3) based on a 1,3-disubstituted diethynylbenzene core has been developed. Both 1 and 2 (with the esters of 1 replaced by diethylamides in 2) show good linear and three-photon induced photophysical properties with

Full text of DOI:10.1002/ejoc.201100570

FULL PAPER
DOI: 10.1002/ejoc.201100570
Comparative Studies of Multi-Photon Induced Emission by Pyridine-Based
Small Molecular Probes in Biological Media: Selective Binding of Bioactive
Molecules and In Vitro Imaging
Wanqing Wu,[a] Hoi-Kuan Kong,[b] Hongguang Li,[a] Yu-Man Ho,[c] Yuan Gao,[a]
Jianhua Hao,[d] Margaret B. Murphy,*[c] Michael Hon-Wah Lam,[c] Ka-Leung Wong,*[b] and
Chi-Sing Lee*[a]
Keywords: Anions / Fluorescent probes / Imaging agents / Luminescence / Photochemistry
A new class of organic molecular probes (1–3) based on a
1,3-disubstituted diethynylbenzene core has been devel-
oped. Both 1 and 2 (with the esters of 1 replaced by diethyl-
amides in 2) show good linear and three-photon induced
photophysical properties with two-photon absorption cross-
sections (185–210 cm4 sphoton–1 molecule–1) that are suitable
for biological applications in live specimens. The propeller π-
conjugated systems of 3 (a C3 analogue of 1) shows threefold
enhancement for the two-photon absorption cross-section
(650 cm4 sphoton–1 molecule–1). Solvatochromism was ob-
served in the fluorescence spectra of all these molecular
probes; in acidic medium (pH = 4–5) their fluorescence emis-
sions are slightly blueshifted with a threefold enhancement
in intensity relative to those observed under basic conditions
(pH = 10–11). In the fluorometric titration study against a
variety of bioactive small molecules, only 2 shows strong
binding affinity (log KB Ͼ 7) towards citrates and bicarbon-
ates with approximately 30 nm redshift. The in vitro emission
spectra of 2 obtained show the same emission upon addition
of anions to the solution. The results of these studies could
provide new molecular-design strategies for two-photo ab-
sorption (TPA) chromophores and new materials for two/
multi-photon imaging in vitro.
even in vivo imaging; however, most have severe problems
regarding their toxicity and the requirement for non-pen-
etrative, ultraviolet emission excitation sources. Until the
2000s, fluorescence imaging mainly relied on UV excitation
because of the lack of available fluorescent probes that were
suitable for imaging in living cells using near-infrared (NIR)
two/multi-photon excitation. Recently, several two/multi-
photon absorption mechanisms have been established for
organic,[4] organometallic,[5] and organic-lanthanide com-
pounds.[6] Two/multi-photon excitation allows molecules
that typically absorb in the ultraviolet region to be excited
by red or NIR light. The potential applications of multi-
photon absorption processes (NIR excitation) shows prom-
ise in photonic and biomedical fields, and have recently
stimulated the scientific community to develop and charac-
terize new molecules with high multi-photon absorption
profiles.[7] NIR excitation is one way to improve the quality
of live-cell imaging, and the development of systems op-
erating with NIR excitation and emission (700–900 nm) is
a prime target, because NIR photons will not be absorbed
by the cell, even in blood media. NIR photons can pen-
etrate and be emitted from tissues without causing damage;
moreover, the sharp emission profile gives clear imaging
that can be differentiated from the usual biological auto-
fluorescence background.[8]
Introduction
Molecular imaging is used to detect, localize, and moni-
tor critical molecular processes in cells, tissue, and living
organisms, and requires highly sensitive instruments and
contrast mechanisms.[1] The technique of cell immunolabel-
ling and fluorescent imaging has been widely used in cell
biology and clinical applications,[2] and this has helped to
provide localization profiles and information about the
quantity of the molecules of interest with no genetic modifi-
cation required.[3] In general, there are many organic- and
metal-based commercial molecular probes for in vitro and
[a] Laboratory of Chemical Genomics, School of Chemical
Biology and Biotechnology, Peking University Shenzhen
Graduate School, Shenzhen University Town, Xili,
Shenzhen 518055, China
E-mail: lizc@szpku.edu.cn
[b] Department of Chemistry, Hong Kong Baptist University,
Kowloon Tong, Hong Kong SAR
E-mail: klwong@hkbu.edu.hk
[c] Department of Biology and Chemistry, City University of
Hong Kong,
Kowloon Tong, TatChee Avenue, Hong Kong SAR
E-mail: mbmurphy@cityu.edu.hk
[d] Department of Applied Physics, Hong Kong Polytechnic
University,
Hung Hum, Hong Kong SAR
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201100570.
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Selective Binding of Bioactive Molecules and In Vitro Imaging
Organic compounds are generally more suitable for clin- (1.3 dm3 mol–1 cm–1), which are attributed to π–π* transi-
ical use than metal complexes and nanomaterials, because tions. The absorption bands are similar to bands found in
they have no coordination instability problems and have the excitation spectra in a solution of DMSO (10–5 m) (Fig-
more flexible properties. We are particular interested in de- ure S1). The propeller π-conjugated system of 3 induces a
veloping organic molecular probes with structures that are significant redshift of the absorption bands (ca. 10 nm); in
suitable for two/multi-photon in vitro and in vivo imaging this case the bands are located at λ (ε) = 280 (6.1), 308 (4.6),
through NIR excitation. Herein, we report the development 340 nm (1.7 dm3 mol–1 cm–1), and a new shoulder band is
of a new class of organic molecular probes 1–3, based on present at λ (ε) = 370 nm (0.5 dm3 mol–1 cm–1).
the 1,3-disubstituted diethynylbenzene core (Scheme 1),
Molecular probes 1 and 2 gave very similar linear in-
with particular emphasis on their linear and two/multi-pho- duced emission band shapes at 420 and 440 nm, respectively
ton induced photo-physical properties, the dramatic change (Figure 1). The quantum yield of the emission bands of 1
of their cell-permeable properties with slight structural and 2 in DMSO were 17 and 19%, respectively; these values
modifications, and their use for in vitro imaging.
were measured by using an integrating sphere with linear
excitation at 350 nm. The quantum yield of 3 was slightly
increased to 21% (Table 1).
Figure 1. Emission spectra of 1 (squares), 2 (circles) and 3 (tri-
angles) in DMSO (10 μm, λex = 350 nm). The inset shows the emis-
sion intensity of 1–3 plotted against the pH of the buffer solutions
(pH = 3–10).
Scheme 1. Synthesis of organic molecular probes 1–3.
Table 1. Fluorescence quantum yields (Φ) and two-photon absorp-
tion cross sections (σ2) of 1–3 in DMSO (10 μm).
Results and Discussion
λabs [nm] (logε [dm3 mol–1 cm–1])
Φ [%][a] σ2 [GM][b]
1
2
270 (5.32), 310 (4.15), 330 (1.31)
272 (5.3), 308 (4.0), 331 (1.28)
280 (6.1), 308 (4.6), 340 (1.7), 370
(0.5)
17
19
182
210
Synthesis and Characterization of 1–3
The organic molecular probes 1–3 were readily synthe-
sized through a Sonogashira coupling strategy. As shown in
Scheme 1, Pd-catalyzed cross-coupling of 1,3-diethynyl-
benzene with 4 (prepared through triflation of diethyl 4-
hydroxypyridine-2,6-dicarboxylate[9] with Tf2O and pyr-
idine) and 5[10] afforded molecular probe 1 (75%) and 2
(95%), respectively. Sonogashira coupling between 1,3,5-tri-
ethynylbenzene and 4 under similar conditions provided
molecular probe 3 in good yield. These compounds were
characterized unambiguously b 1H, 13C NMR, and HRMS
analyses.
3
21
620
[a] Emission quantum yields of 1–3 (λem = 400–700 nm, λex
350 nm). [b] Two-photon absorption cross-section (GM
10–50 cm4 sphoton–1 molecule–1, λex = 750 nm).
=
=
Spectrophotometric analysis of 1–3 at various pH values
revealed that their fluorescence emissions are pH-depend-
ent. The relationship between the emission intensity and the
pH is plotted in the inset of Figure 1. The emission spectra
of 1–3 exhibited an intense peak at 422, 440, and 420 nm,
respectively, in neutral pH, with bathochromic shifts to 430,
445, and 428 nm, respectively, at acidic pH. The emission
intensity had a twofold decrease as the pH increased from
3 to 6 and became minimal beyond pH = 8.
Linear Photophysical Properties of 1–3
All the molecular probes (1–3) show intense absorption
bands in the near-UV region. Molecular probes 1 and 2
in dimethyl sulfoxide (DMSO) have strong UV-absorption
Two/Multi-Photon Induced Photophysical Properties of 1–3
The emission maxima, band shape, and band width of
bands at λ (ε) = 270 (5.3), 310 (4.1), and 330 nm 1–3 upon linear excitation can be reproduced by NIR exci-
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M. B. Murphy, K.-L. Wong, C.-S. Lee et al.
FULL PAPER
tation (through multi-photon absorption). The linear and Cation and Anion Binding Affinity and Selectivity
three-photon induced emission spectra of 1–3 are shown
The binding properties of the organic molecular probes
in Figure 1 (with UV excitation at 350 nm) and Figure 2
1–3 in DMSO towards a variety of bioactive small mole-
(excitation at 900 nm by a femtosecond laser), respectively.
cules such as Zn2+, citrate, and hydrogencarbonate was
The linear absorption processes in 1–3 should be the same
studied by using fluorometric titration. Changes in the
as the two- or three-photon absorption processes. Power-
emission spectral behavior were not very significant for 1
dependence experiments were performed to confirm the
and 3, but were still sufficient to allow the variation of the
number of photons involved in the emission band under
emission intensity with anion/cation concentration to be
excitation at 900 nm (inset of Figure 2). In all cases, the
plotted and fitted to a 1:2 (analyte/probe) binding model.
output intensity of three-photon excited fluorescence
The affinity constants of 1–3 to several bioactive cations
(TPEF) was linearly dependent on the cube of the input
and anions were calculated and are summarized in Table 2.
laser intensity, thereby confirming three-photon absorption.
Adequate binding constants of 1 and 3 were observed for
several anions/cations (logKB ≈ 4, Table 2). Addition of
Zn2+ reduced and redshifted (30 nm) the emission from ap-
proximately 430 to 460 nm (emission diminished obviously)
for compound 3 (Figure 3).
Table 2. Affinity constants (logKB) of the organic molecular probes
for selected cations and anions (pH = 7.4, 0.1 m NaCl, 10 μm com-
pound 1–3, 298 K, λex = 350 nm).
Citrate
KHCO3NaHCO3Na2HPO4MgCl2 KCl NaCl ZnCl2
1
2
3
2.85
7.83
3.05
2.95
7.13
3.33
2.88
7.01
3.25
2.83
4.32
2.62
4.25 2.69 2.65 4.12
3.05 3.03 2.88 3.35
4.12 3.33 3.25 4.32
Figure 2. Three-photon induced emission spectra of 1 (squares), 2
(circles), and 3 (triangles) in DMSO. The inset shows the power-
dependence experiments with 1–3 [10 μm; λex = 900 nm; 1: λem
=
420 nm (n = 2.98), 2: λem = 440 nm (n = 3.05), 3: λem = 425 nm (n
= 2.95)].
In general, the two-photon absorption cross-section of a
material in solution is used to evaluate the strength of two-
photon absorption and hence the possibility of two-photon
induced visible imaging in vitro.[9] Thus, the two-photon ab-
sorption cross-section of 1 and 2 at 750 nm was measured
and compared to that of Rhodamine 6G to establish their
two-photon photophysical efficiency.[10] Molecular probe 1
(210 GM; GM = 10–50 cm4 sphoton–1 molecule–1) demon-
strated a higher two-photon absorption cross-section than
2 (185 GM). Both compounds were found to have much
higher two-photon absorption values than the minimum
suggested by Furuta et al. for biological applications
Figure 3. Responsive emission quenching of 3 (squares) in the pres-
ence of 250 mm of ZnCl2 (circles). The inset shows emission inten-
sity ratio vs. amount of added zinc chloride (0.32 μm to 240 mm),
which was used to determine the apparent binding constant (pH =
7.4, 0.1 m NaCl, 10 μm compound 3, 298 K, λex = 350 nm).
Significant optical changes were found for 2 upon the
(Ͼ 0.1 GM) in live specimens,[11] and the values were also addition of anions, especially upon addition of citrate and
higher than those of most organometallic compounds used bicarbonates. The partial positive charge of the amide nitro-
for in vitro imaging (10–100 GM).[12] A difference (de- gen atoms of 2 could be responsible for the difference in
crease) in the two-photon absorption cross-section could be anion-binding behavior compared with 1 and 3. A relatively
induced by modifying the structure of the conjugated sys- strong binding affinity of 2 was observed for hydrogen
tem (donor–π–acceptor) in 2 by introducing a stronger elec- phosphate, citrate, and hydrogencarbonate (Table 2). Ad-
tron-donating group. Significant enhancements of the TPA dition of citrate and bicarbonates led to a four-fold en-
cross-section were observed in 3, which is a derivative of hancement of emission, accompanied by a 30 nm (ca.
compound 1 with an additional branch. The co-operative 3333 cm–1) redshift, which allowed the emission intensity to
effects in the three branches of 3 significantly enhanced the be plotted as a function of added [citrate] or [hydrogen-
two-photon absorption cross-section from 210 to 650 GM. carbonate]; thus, an apparent 1:2 binding constant of
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Selective Binding of Bioactive Molecules and In Vitro Imaging
107.58(Ϯ0.05) for citrate, and 107.13(Ϯ0.05) for hydrogencarbon- tation that was localized within the cytoplasm of the cells
ate were estimated for the citrate/hydrogencarbonate associ- (Figure 5a). After 6 and 12 h of incubation, more than 90%
ation (Figure 4).
of cells emitted under NIR excitation (Figure 5c and Fig-
ure 5d, respectively). The most interesting finding for our
molecular probes was their NIR excited induced visible
photophysical properties. The in vitro imaging and emission
spectra of 2 were recorded and are shown in Figure 6. After
3 h of dosage time, strong green emission in the 400–650 nm
region could be observed in the cytoplasm of HeLa cells
under excitation at 900 nm (50 mW). A lambda scan of a
confocal microscope (resolution 6 nm) was recorded that
showed green emission bands inside the cytoplasm of HeLa
cells. The emission band shape and maximum shown in Fig-
ure 6 were the same as the emission spectra shown in Fig-
ure 4; a 30 nm redshift was obtained that indicated that the
molecular probe was bonded to the anions, and that a
strong three-photon induced emission with a fingerprint
redshifted emission was established. This experiment
showed that, in the presence of anions, molecular probe 2
can reproduce the same emission spectra in solution (Fig-
ure 4) and in vitro (Figure 6) under either linear or two-
photon excitation.
Figure 4. (Top) Emission spectra of 2 upon addition of sodium
citrate (pH = 7.4, 0.1 m NaCl, 10 μm compound 2, 298 K, λex
=
350 nm), the inset shows the binding isotherm. (Bottom) Emission
intensity ratio of compound 2 versus added analytes: sodium citrate
(squares), sodium hydrogencarbonate (circles), potassium hydro-
gencarbonate (triangles-up), potassium chloride (triangles-down),
magnesium chloride (diamonds), sodium chloride (triangles-left),
zinc chloride (triangles-right), and sodium hydrogen phosphate
(hexagons) to determine the apparent binding constants (pH = 7.4,
0.1 m NaCl, 10 μm compound 2, 298 K, λex = 350 nm).
Figure 5. In vitro cytoplasmic staining microscopy with 2 (50 μm,
λex = 900 nm, λem = 450–750 nm) in A549 after (a) 1 h, (b) 6 h,
and (c) 12 h. (d) Bright-field image of panel (c).
In Vitro Imaging and Cytotoxicity Studies
The performance of 1–3 in vitro was evaluated with a
variety of assays including linear, two/multi-photon confo-
cal imaging, and toxicity tests (MTT assay and IC50 deter-
mination). For in vitro imaging, molecular probes 1–3 were
added to the same amounts of two carcinoma cell lines
(Human cervical carcinoma HeLa and Human lung carci-
noma A549), and the samples were examined at various
points of time. For 1 and 3, no emission could be observed
in the cells; only a strong bluish green emission could be
obtained from the tissue culture medium (Figure S2). How-
ever, molecular probe 2 demonstrated strong green emis-
sions in the in vitro imaging (Figure 5); 1 h after dosage,
Figure 6. (Left) Multi-photon (NIR) induced in vitro imaging of
HeLa cells after 12 h of incubation with 2. (Right) In vitro emission
spectra of the two regions in the visible and NIR region obtained
by using the λ scan of a Leica SP5 laser fluorescent microscope
(resolution ca. 6 nm, λex = 900 nm); (squares) channel 1, green emis-
weak emission could be observed under UV and NIR exci- sion in HeLa cells; (circles) channel 2, background.
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M. B. Murphy, K.-L. Wong, C.-S. Lee et al.
FULL PAPER
We extended the application of these three organic mole- organic molecules to improve cell permeability. The results
cules as potential citrate or hydrogencarbonate probes for also confirm NIR induced emission occurs after two/multi-
in vitro imaging. Toxicity tests with the three compounds photon absorption, which has potential applications both
were performed, and no evidence of cytotoxicity on HeLa in vitro and in vivo, especially for 2. These findings can be
cells was found upon exposure to 1–3 (50 μm) for 24 h (Fig- applied in the design of new materials that make use of
ure 7). MTT assays on cells exposed to 50 μm 1–3 (as high highly penetrating NIR excitation sources and more target-
as 20 times the dosage concentration required for in vitro specific emissions, and should advance investigations on hu-
imaging) for prolonged periods revealed no significant de- man cells.
crease in the number of viable cells. In addition, the non-
toxic IC50 values (ca. 22 mm, ca. 25 mm and ca. 18 mm for
1–3, respectively) of the complexes in HeLa cells were found
to distribute inside the cytoplasm and can be further devel-
oped as specific imaging probes with connection to vectors
Experimental Section
Synthesis of Organic Molecular Probes 1–3
such as peptides. It is worth noting that the molecular General Information: All air- and water-sensitive reactions were car-
ried out under nitrogen with anhydrous solvents under anhydrous
conditions, unless otherwise noted. All chemicals were purchased
and used without further purification. Anhydrous THF was dis-
tilled from sodium/benzophenone, and dichloromethane was dis-
tilled from calcium hydride. Yields refer to chromatographically
pure products, unless otherwise stated. NMR spectra were recorded
with either a Bruker Avance 300 (1H: 300 MHz; 13C: 75.5 MHz) or
a Bruker Avance 500 (1H: 500 MHz; 13C: 125.8 MHz) spectrometer.
The following abbreviations were used for multiplicities: s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, br. = broad.
High-resolution mass spectra were obtained with an Applied Biosy-
stems (ABI) Q-Star Elite MALDI-TOF mass spectrometer.
probes were mostly distributed inside the cytoplasm and
could be further developed as specific imaging probes with
connection to vectors such as peptides.
Diethyl
4-(Trifluoromethylsulfonyloxy)pyridine-2,6-dicarboxylate
(4): To a solution of diethyl 4-hydroxypyridine-2,6-dicarboxylate[13]
(0.14 g, 0.59 mmol) in CH2Cl2 (5 mL) at –78 °C, were added pyr-
idine (0.30 mL, 3.56 mmol) and Tf2O (0.30 mL, 1.78 mmol). After
stirring at –78 °C for 2 h, the reaction was quenched with water.
The aqueous phase was extracted with ethyl acetate (3ϫ), and the
combined extracts were washed with brine, dried with MgSO4, fil-
tered, and concentrated under reduced pressure. Silica gel flash col-
umn chromatography (hexanes/ethyl acetate = 5:1 to 3:1) of the
Figure 7. MTT assay for cytotoxicity of 1 (white bars), 2 (black
bars) and 3 (hatched bars) in HeLa cells.
1
residue gave the product as a white solid (0.18 g, 80%). H NMR
(500 MHz, CDCl3): δ = 8.17 (s, 2 H), 4.53 (q, J = 7.2 Hz, 4 H),
1.48 (t, J = 7.2 Hz, 6 H) ppm. 13C NMR (125 MHz, CDCl3): δ =
163.1, 157.7, 151.8, 120.2, 117.4, 63.0, 14.2 ppm. HRMS (ESI):
calcd. for C12H13NO7F3S [M + H]+ 372.0365; found 372.0362.
Conclusions
Tetraethyl 4,4Ј-[1,3-Phenylenebis(ethyne-2,1-diyl)]dipyridine-2,6-di-
carboxylate (1): To a solution of 1,3-diethynylbenzene (0.20 mL,
1.49 mmol), 4 (1.0 g, 2.70 mmol), and CuI (26 mg, 0.135 mmol) in
THF (10 mL) was added Et3N (10 mL), followed by [PdCl2-
(PPh3)2] (28 mg, 0.041 mmol). After stirring at 40 °C overnight, the
reaction was quenched with saturated aqueous NH4Cl. The aque-
ous phase was extracted with diethyl ether (3ϫ), and the combined
extracts were washed with brine, dried with Na2SO4, filtered, and
concentrated under reduced pressure. Silica gel flash column
chromatography (dichloromethane/methanol = 100:1 to 50:1) of
the residue gave the product as a pale-yellow solid (0.58 g, 75%).
M.p. 116–124 °C. 1H NMR (500 MHz, CDCl3): δ = 8.32 (m, 4 H),
7.77 (m, 1 H), 7.58 (m, 2 H), 7.42 (m, 1 H), 4.50 (q, J = 7.1 Hz, 8
H), 1.46 (t, J = 7.1 Hz, 12 H) ppm. 13C NMR (125 MHz, CDCl3):
δ = 167.3, 149.2, 135.9, 135.5, 133.9, 133.8, 133.7, 133.1, 132.8,
129.5, 129.2, 129.0, 122.5, 95.0, 86.6, 62.6, 14.3 ppm. HRMS (ESI):
calcd. for C32H29N2O8 [M + H]+ 569.1918; found 569.1913.
We have developed a new class of organic molecular
probes 1–3 based on the 1,3-disubstituted diethynylbenzene
core. Both 1 and 2 (with the esters of 1 replaced by diethyl-
amides in 2) show good linear and three-photon induced
photophysical properties with two-photon absorption
cross-section (185–210 GM) that are suitable for biological
applications in live specimens. Molecular probe 3 has an
additional π-conjugated branch forming a C3 analogue of
1. The propeller π-conjugated system of 3 shows a co-oper-
ative effect that induces a threefold enhancement of the
two-photon absorption cross-section compared with 1 and
2, which contain only two π-conjugated systems. More
interestingly, molecular probe 2, with the partial positive
charged amides, shows specific binding to anions, particu-
larly citrate and hydrogencarbonate, exhibits more cell per-
meability, and is capable of producing emission spectra in
vitro upon excitation with NIR irradiation within the “bio-
logical window” (700–1000 nm) in cellular studies. These
4,4Ј-[1,3-Phenylenebis(ethyne-2,1-diyl)]bis(N2,N2,N6,N6-tetraethyl-
pyridine-2,6-dicarboxamide) (2): To a solution of 1,3-diethynylben-
zene (30 μL, 0.21 mmol), N2,N2,N6,N6-tetraethyl-4-iodopyridine-
results indicate the possibility of structurally modifying the 2,6-dicarboxamide[14] (0.16 g, 0.40 mmol), and CuI (13 mg,
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Selective Binding of Bioactive Molecules and In Vitro Imaging
0.065 mmol) in THF (5 mL), was added Et3N (5 mL), followed by
[PdCl2(PPh3)2] (23 mg, 0.03 mmol). After stirring at 40 °C over-
night, the reaction was quenched with saturated aqueous NH4Cl.
The aqueous phase was extracted with diethyl ether (3ϫ), and the
combined extracts were washed with brine, dried with Na2SO4, fil-
tered, and concentrated under reduced pressure. Silica gel flash col-
umn chromatography (dichloromethane/methanol = 100:1 to 50:1)
of the residue gave the product as a pale-yellow solid (95 mg, 70%).
M.p. 83–84 °C. 1H NMR (500 MHz, CDCl3): δ = 7.70 (m, 5 H),
7.54 (m, 2 H), 7.38 (m, 1 H), 3.56 (q, J = 7.1 Hz, 8 H), 3.35 (q, J
the reference. The two-photon absorption cross-section σ2 of 1–3
was determined by using Rhodamine 6G as a reference. For spec-
trofluorometric titrations, all the solvents used were of analytical
grade, and the water used was purified by double distillation. Mea-
surements were taken after equilibrium was attained, and the emis-
sion of the compound was monitored. Luminescent responses in
terms of I0/(I – I0) were plotted as a function of analyte concentra-
tion. For the determination of binding strengths of the various ana-
lyte adducts, a series of analyte solutions of known concentrations
were mixed with the anion solutions at various concentrations. The
= 7.1 Hz, 8 H), 1.26 (t, J = 7.1 Hz, 12 H), 1.15 (t, J = 7.1 Hz, 12 titration curve was then fitted either with the 1:1, 1:2, or 1:3 Be-
H) ppm. 13C NMR (125 MHz, CDCl3): δ = 167.6, 154.1, 135.3,
nesi–Hildebrand equations to establish which type of donor–ac-
133.4, 132.9, 129.0, 125.5, 122.7, 94.2, 87.0, 43.4, 40.4, 14.4,
ceptor interaction existed.[16]
12.9 ppm. HRMS (ESI): calcd. for C40H49N6O4 [M
677.3810; found 677.3814.
+
H]+
Microscopy Imaging: To study the behavior of compounds 1–3 in
vitro, experiments were conducted by using a commercial multi-
photon confocal microscope. For two-photon imaging in vitro, the
cells were imaged in a tissue culture chamber (5% CO2, 37 °C)
by using a Leica SP5 (upright configuration) confocal microscope
equipped with a femtosecond-pulsed Ti:Sapphire laser (Libra II,
Coherent). The excitation beam was produced by the femtosecond
laser, which was tuneable in the range 680–1050 nm (λex = 900 nm,
ca. 5 mW), and focused on coverslip-adherent cells by using a 40ϫ
oil-immersion objective.
Hexaethyl 4,4Ј,4ЈЈ-[Benzene-1,3,5-triyltris(ethyne-2,1-diyl)]tris(pyr-
idine-2,6-dicarboxylate) (3): To a solution of 1,3,5-triethynylben-
zene (0.33 g, 2.16 mmol), 4 (2.0 g, 5.41 mmol), and CuI (34 mg,
0.18 mmol) in THF (10 mL), was added Et3N (10 mL), followed
by [PdCl2(PPh3)2] (38 mg, 0.054 mmol). After stirring at 45 °C
overnight, the reaction was quenched with saturated aqueous
NH4Cl. The aqueous phase was extracted with diethyl ether (3ϫ),
and the combined extracts were washed with brine, dried with
Na2SO4, filtered, and concentrated under reduced pressure. Silica
gel flash column chromatography (dichloromethane/methanol =
100:1 to 50:1) of the residue gave the product as a pale-yellow solid
(1.05 g, 72%). M.p. 195–200 °C (dec). 1H NMR (300 MHz,
CDCl3): δ = 8.36 (s, 6 H), 7.83 (s, 3 H), 4.52 (q, J = 7.1 Hz, 12 H),
1.48 (t, J = 7.1 Hz, 18 H) ppm. 13C NMR (75.5 MHz, CDCl3): δ =
164.2, 149.2, 136.0, 133.4, 129.6, 123.2, 93.6, 87.4, 62.8, 14.4 ppm.
HRMS (ESI): calcd. for C45H40N3O12 [M + H]+ 814.2607; found
814.2607.
Cell Culture: Human lung carcinoma A549 cells were purchased
from the American Type Culture Collection (ATCC) (#CCL-185,
ATCC, Manassas, VA, USA). Cells were cultured in Ham’s F12K
medium with l-glutamine and phenol red (N3520, Sigma, St.
Louis, MO, USA) supplemented with 10% fetal bovine serum at
37 °C and 5% CO2. Cells were passaged every 3–5 d. Human cervi-
cal carcinoma (HeLa) cells were maintained in an RMPI 1640 me-
dium supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin and streptomycin in 5% CO2. The MTT viability assay
was performed as reported previously.[17] Briefly, three thousand
HeLa cells were seeded in 96-well plates 24 h prior to exposure to
compounds 1–3 or DMSO as control. After various exposure time
points, 20 μL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide] solution (5 mg/mL) was added to the culture
medium in each well and incubated at 37 °C for 5 h. The media
was removed, 200 μL of DMSO solubilizing reagent was added,
and incubation was continued for a further 1 h to dissolve the
formazan crystals. The absorbance was measured at 570 nm with a
Labsystem Multiskan microplate reader (Merck Eurolab, Switzer-
land). MTT assays were conducted in triplicate wells, and repeated
twice. Each data point represents the ratio of mean values between
the compounds versus the DMSO control.
Spectroscopic and Photophysical Measurements: UV/Vis absorption
spectra in the spectral range 200–1100 nm were recorded with an
HP UV-8453 spectrophotometer. Single-photon luminescence spec-
tra were recorded with an Edinburgh Instrument FLS920 Com-
bined Fluorescence Lifetime and Steady-state spectrophotometer
that was equipped with a red-sensitive single-photon counting pho-
tomultiplier in a Peltier Cooled Housing. The spectra were cor-
rected for detector response and stray background light phospho-
rescence. The quantum yields of compounds 1–3 were measured
with a demountable 142 mm (inner) diameter barium sulfide coated
integrating sphere supplied with two access ports. For multi-photon
experiments, the 900 nm pump source was from a femtosecond
mode locked Ti:Sapphire laser system (output beam ca. 150 fs du-
ration and 1 kHz repetition rate). The lasers were focused to a spot
size of approximately 50 μm by an f = 10 cm lens onto the sample.
The emitting light was collected with a backscattering configura-
tion into a 0.5 m spectrograph and detected by a liquid nitrogen
cooled CCD detector. A power meter was used to monitor the uni-
form excitation. The theoretical framework and experimental pro-
tocol for the two-photon cross-section measurement have been out-
lined by Webb and Xu.[15] In this approach, the two-photon exci-
tation (TPE) ratios of the reference and sample systems are given
by:
Supporting Information (see footnote on the first page of this arti-
cle): UV/Vis absorption, excitation spectra, in vitro imaging of 1
1
and 3, H NMR and 13C NMR spectra of molecular probes 1–3.
Acknowledgments
This work was funded by grants from the Peking University
Shenzhen Graduate School, the Hong Kong Research Grants
Council, the City University of Hong Kong, and the Hong Kong
Baptist University (FRG1/10-11/037).
where φ is the quantum yield, C is the concentration, n is the refrac-
tive index, and F(λ) is the integrated photoluminescent spectrum.
In our measurements, we have ensured that the excitation flux and
the excitation wavelengths are the same for both the sample and
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