Fabrication of fluorescent silica nanoparticles hybridized with AIE luminogens and exploration of their applications as nanobiosensors in intracellular imaging

Article abstract of DOI:10.1002/chem.200901823

Highly emissive inorganicorganic nanoparticles with core-shell structures are fabricated by a one-pot, surfactant-free hybridization process. The surfactant-free sol-gel reactions of tetraphenylethene- (TPE) and silolefunctionalized siloxanes followed by

Full text of DOI:10.1002/chem.200901823

DOI: 10.1002/chem.200901823
Fabrication of Fluorescent Silica Nanoparticles Hybridized with AIE
Luminogens and Exploration of Their Applications as Nanobiosensors
in Intracellular Imaging**
Mahtab Faisal,[a, b] Yuning Hong,[a, b] Jianzhao Liu,[a, b] Yong Yu,[a, b] Jacky W. Y. Lam,[a, b]
Anjun Qin,[a, c] Ping Lu,[a, b] and Ben Zhong Tang*[a, b, c]
Abstract: Highly emissive inorganic–
organic nanoparticles with core–shell
structures are fabricated by a one-pot,
surfactant-free hybridization process.
The surfactant-free sol–gel reactions of
tetraphenylethene- (TPE) and silole-
functionalized siloxanes followed by re-
actions with tetraethoxysilane afford
fluorescent silica nanoparticles FSNP-1
and FSNP-2, respectively. The FSNPs
are uniformly sized, surface-charged
and colloidally stable. The diameters of
the FSNPs are tunable in the range of
45–295 nm by changing the reaction
conditions. Whereas their TPE and
silole precursors are non-emissive, the
FSNPs strongly emit in the visible
vision, as a result of the novel aggrega-
tion-induced emission (AIE) character-
istics of the TPE and silole aggregates
in the hybrid nanoparticles. The FSNPs
pose no toxicity to living cells and can
be utilized to selectively image cyto-
plasm of HeLa cells.
Keywords: aggregation-induced
emission
fluorescence
sol–gel reaction
·
cell
nanoparticles
imaging
·
·
·
Introduction
the area of cellular marking or imaging. QDs enjoy such ad-
vantages as size-tunable emission color, long luminescence
lifetime and resistance to photobleaching. Their surfaces,
however, need to be modified in order to improve their hy-
drophilicity. Recent investigations have revealed that a large
portion (>40%) of QDs used under biological conditions
show dark state. The use of high concentrations of QDs may
solve this problem but causes more serious problems such as
enhanced cytotoxicity. This is easy to understand, taking
into consideration that QDs are usually chalcogenides of
heavy metals (e.g., ZnSe, CdS and PdTe), which are well
known toxicants or carcinogens.[3–6]
Fluorescent nanoparticles have been found useful as visuali-
zation tools for biological sensing, probing, imaging, moni-
toring.[1,2] Among nanoparticles, semiconductor quantum
dots (QDs) have attracted much attention, particularly in
[a] M. Faisal, Y. Hong, J. Liu, Y. Yu, Dr. J. W. Y. Lam, Dr. A. Qin,
Dr. P. Lu, Prof. Dr. B. Z. Tang
Department of Chemistry, Bioengineering Graduate Program
Nano Science and Technology Program
Institute of Molecular Functional Materials
The Hong Kong University of Science & Technology (HKUST)
Clear Water Bay, Kowloon, Hong Kong (China)
Fax : (+852)2358-1594
In contrast, silica nanoparticles (SNPs) are hydrophilic
and biocompatible. SNPs have found a variety of applica-
tions in areas spanning from information technology to bio-
logical engineering. Besides being cytophilic, SNPs are
transparent but nonfluorescent and hence ideal host materi-
als for the fabrication of fluorescent silica nanoparticles
(FSNPs) for imaging purposes. FSNPs can be prepared by
incorporating fluorophores into silica networks via physical
processes or chemical reactions. The silica matrix acts as a
protective shield, reducing the likelihood of penetrations of
oxygen and other harmful species that may cause photo-
bleaching of the embedded fluorophores.[7–9]
E-mail: tangbenz@ust.hk
[b] M. Faisal, Y. Hong, J. Liu, Y. Yu, Dr. J. W. Y. Lam, Dr. P. Lu,
Prof. Dr. B. Z. Tang
HKUST Fok Ying Tung Research Institute
Nansha, Guangzhou 511458 (China)
[c] Dr. A. Qin, Prof. Dr. B. Z. Tang
Department of Polymer Science and Engineering
Institute of Biomedical Macromolecules, Zhejiang University
Hangzhou 310027 (China)
[**] AIE = aggregation-induced emission.
For sensitive detection, trace analysis, diagnostic assay,
and real-time monitoring, FSNPs should emit intense visible
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901823.
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light upon photoexcitation. However, light emission from
most of the FSNPs prepared thus far have been rather
weak. This is due to the emission quenching caused by the
aggregation of fluorophores in the solid state.[10] A low fluo-
rophore loading in the nanoparticle may be free of aggrega-
tion but can offer only weak fluorescence signals. The light
emission can further be weakened, rather than enhanced,
when more fluorophores are loaded into the nanoparticles,
because of the notorious aggregation-caused quenching
(ACQ) effect.[11]
We have discovered an “abnormal” phenomenon that is
exactly opposite to the ACQ effect, namely aggregation-in-
duced emission (AIE). Nonemissive luminogens, such as tet-
raphenylethene (TPE) and hexaphenylsilole, are induced to
emit efficiently by aggregate formation.[12–14] The AIE effect
dramatically boosts fluorescence quantum yields (FF) of the
luminogens, turning them from faint fluorophores to strong
emitters. An extreme example of such AIE luminogen is
4,4’-bis(1,2,2-triphenylvinyl)biphenyl: its emission efficien-
cies in the solution (FF,S) and aggregate (FF,A) states are ~0
and 100%, respectively, resulting in an infinitely large AIE
effect (aAIE =FF,A/FF,S ! 1).[12] Mechanistic investigations
reveal that the AIE effect is caused by the restriction to the
intramolecular rotations of the luminogens in the aggregate
state.[15–19] The AIE effect has enabled the luminogens to
find an array of technological applications as chemosensors,
bioprobes, PAGE visualizers, active layers in the construc-
tion of organic light-emitting diodes.[15]
Results and Discussion
Fabrication of FSNPs: With a view to fabricating FSNPs
with strong light emissions in the solid state, we prepared di-
brominated TPE and silole derivatives 3 and 12 with AIE
attribute (Schemes 1 and 2). While 3 was prepared following
our previously published experimental procedures,[13,18] the
synthetic route to 12 is given in Scheme 2. Compound 6 is
first prepared by the etherization of 4-iodophenol (5) with
1,2-dibromoethane. The lithiation of phenyl-acetylene (7)
followed by reaction with dichlorosilane 8 produces bis(phe-
nylethynyl)silane (9), which reacts with an excess amount of
lithium 1-naphthalenide to form 2,5-dilithio-silole 10. Trans-
metallation of 10 with ZnCl2·TMEDA affords 2,5-dizincated
silole 11, treatment of which with 6 in the presence of a pal-
ladium catalyst yields 12.
In this work, we explored the possibility of hybridizing
the AIE luminogens with silica gels with the aim of fabricat-
ing emissive FSNPs. Although microemulsion process has
been widely used for the preparations of FSNPs, it involves
tedious syntheses of dye-doped silica cores followed by dep-
osition of pure silica shells.[20] Surfactants are needed in the
process to generate nanodimensional templates. The resul-
tant FSNPs thus must be thoroughly washed prior to biolog-
ical applications, because surfactants are harmful to living
cells.[21] Unfortunately, however, the surfactant coatings on
the FSNPs are extremely difficult, if not impossible, to clean
up.
In this paper, we report our work on the syntheses of
AIE-active FSNPs with core–shell structures through a sur-
factant-free sol–gel process in a one-pot experimental proce-
dure. In our approach, the luminogens are chemically bound
to, rather than physically blended with, the silica networks.
The AIE luminogens thus do not leak out from the
FSNPs.[22–24] We obtained FSNPs with uniform sizes, high
surface charges and excellent colloidal stability. Thanks to
the AIE nature of the luminogen nanoaggregates and the
biocompatibility of the silica gels, the FSNPs emit strong
visible lights and pose no cytotoxicity, which make them
promising fluorescent biomarkers for cell imaging.
Scheme 1. Fabrication of TPE-containing fluorescent silica nanoparticles
FSNP-1. Abbreviation: TPE=tetraphenylethene, DMSO=dimethyl sulf-
oxide.
To covalently bind the AIE luminogens to SNPs at molec-
ular level, we employed (3-aminopropyl)triethoxysilane
(APS or 4) as a chemical linker. As shown in Scheme 1, the
APS–TPE adduct 1 is prepared by stirring a mixture of APS
and 3 in distilled DMSO at room temperature overnight
under stringently dry conditions. The reaction product gives
an [M++1] peak at m/z 799.4536 in its high-resolution mass
spectrum (HRMS), as shown in Figure S1 in the Supporting
Information, confirming the occurrence of the coupling re-
action and the formation of expected adduct
1 (m/z
798.4459 [M+]). The sol–gel reaction of 1 catalyzed by am-
monium hydroxide is carried out in an aqueous mixture at
room temperature for 3h. The resultant TPE-silica nanocore
is then subjected to further sol–gel reaction with tetraethyl
orthosilicate (TEOS), yielding FSNP-1 with the luminogen
core coated with a silica shell. Similarly, coupling of APS
with 12 affords adduct 2 and its two-step sol–gel reaction in
a one-pot procedure results in the formation of FSNP-2 with
a core–shell structure (Scheme 2).
The atomic compositions of the obtained FSNPs were es-
timated by energy dispersive X-ray spectroscopy (EDX) and
the results are given in Table S1 in the Supporting Informa-
tion. Analysis by x potential analyzer at room temperature
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B. Z. Tang et al.
pure SNPs containing no luminogens prepared under the
similar conditions are shown in the figures for comparison.
Nearly no fluorescence signals are recorded when the SNPs
are photoexcited. Similarly the fluorescence spectra of 1 and
2 are almost flat lines parallel to the abscissas. In the dilute
ethanol solutions, the multiple peripheral aryl rings in the
isolated molecules of luminogens 1 and 2 undergo active in-
tramolecular rotations, which effectively annihilate their ex-
cited states and hence render them nonemissive.[27–30]
Scheme 2. Synthesis of silole derivative 2 and fabrication of its fluores-
cent silica nanoparticles FSNP-2. Abbreviation: Naph=1-naphthyl,
Figure 2. Fluorescence spectra of a) FSNP-1 and 1 and b) FSNP-2 and 2
in ethanol. The spectra of SNPs are shown for comparison. Concentra-
tion of luminogen: a) 4 mm, b) 3 mm; excitation wavelength: a) 353 nm, b)
371 nm.
THF=tetrahydrofuran,
TMEDA=N,N,N’,N’-tetramethylethylenedi-
amine.
shows that the FSNPs are monodispersed with polydispers-
ity down to 0.005 (Figure 1). The average diameters of
FSNP-1 and FSNP-2 are 206.3 and 225.0 nm, respectively,
which are somewhat larger than those measured by trans-
mission electron microscopy (TEM): 175 and 200 nm for
FSNP-1 and FSNP-2, respectively, due to swelling of the
FSNPs in aqueous mixtures and their shrinkage under the
high vacuum in the TEM chamber.[25,26]
When the molecules of 1 and 2 are covalently incorporat-
ed into, and aggregate in, the silica networks, strong fluores-
cence spectra peaked at 465 and 485 nm, respectively, are
recorded, confirming that 1 and 2, similar to their respective
congeners 3 and 12, are AIE active.[13,18] The rigid silica net-
works largely restrict intramolecular rotations in the lumino-
gens. This blocks the nonradiative relaxation channel and
populates the radiative decay, thus making the FSNPs highly
luminescent.
By dissolving 1 and 2 and dispersing FSNP-1 and FSNP-2
with the same molar quantities of luminogens in ethanol,
their emission intensities are compared. The light emissions
from FSNP-1 and FSNP-2 are 135- and 60-fold more intense
than those from 1 and 2, respectively. The FF values of
FSNPs with different loadings of luminogens manifest the
salient feature of AIE (Table 1): they do not suffer from the
ACQ effect and their fluorescence efficiencies are increased
with an increase in the luminogen loading. The light emis-
Table 1. Fluorescence quantum yields (FF) of fluorescent silica nanopar-
ticles[a]
Figure 1. Particle size distributions of a) FSNP-1 and b) FSNP-2. Abbre-
viation: de =effective diameter, dm =mean diameter, PD=polydispersity.
Content of luminogen
[mmol]
FF [%]
G
FSNP-1
FSNP-2
4
8
12
17.6
21.9
30.1
13.5
23.3
39.0
Light emission: Figure 2 shows fluorescence spectra of solu-
tions of 1 and 2 and suspensions of their core–shell nanopar-
ticles FSNP-1 and FSNP-2 in ethanol. The spectra of the
[a] Absolute values determined by integrating sphere.
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sion is very stable, with no change in the fluorescence spec-
tra detectable after the FSNPs have been put on shelves for
several months without protecting from light and air.
TEM and scanning electron microscope (SEM) images
show that all the nanoparticles prepared are uniform in di-
ameters with narrow size distributions and smooth surfaces
(Figures 4 and 5). These results are consistent with those ob-
tained from the x potential analyses of their suspensions in
the aqueous media (Table 2), in which the FSNPs practically
do not agglomerate and thus show their intrinsically low
polydispersities (down to 0.005).
Figure 3 shows images of the solutions of 1 and 2 and the
suspensions of FSNP-1 and FSNP-2 as well as SNPs taken
upon exposure to the irradiation with a UV lamp. Whereas
the solutions of 1 and 2 and the suspensions of SNPs are in-
visible under the UV illumination, intense blue and green
light is emitted from FSNP-1 and FSNP-2, respectively. This
visual observation further supports that the intramolecular
rotations of 1 and 2 are restricted by the covalent melding
of the AIE luminogens with the silica matrix.
Figure 3. Solutions and suspensions of a) 1, SNPs and FSNP-1 and b) 2,
SNPs and FSNP-2 in ethanol; photographs taken upon irradiation with a
UV light of 365 nm. Whereas 1, 2 and SNPs are invisible under the UV
illumination, strong blue and green lights are emitted from FSNP-1 and
FSNP-2, respectively.
Figure 4. a)–c) TEM and d)–f) SEM images of monodispersed FSNP-1
with different particle sizes: a) and d) 60 nm, b) and e) 175 nm, and c)
and f) 250 nm.
Colloidal stability: The x potential analyses reveal that the
FSNPs, similar to SNPs, possess high surface charges and
hence excellent colloidal stability.[31] The x potential of pure
SNPs is negatively signed, whose absolute value is increased
with an increase in pH of the aqueous medium (Supporting
Information, Figure S3). At high pH value or in the basic
medium, the acidic hydroxyl groups on the nanoparticle sur-
faces are converted into basic form, imparting high surface
charges and hence good stability to the SNPs.
The FSNPs, however, exhibit positively signed x potentials
at pH 2 due to the protonation of the amino groups contrib-
uted from the APS moieties.[32,33] At higher pH values, this
event is less likely to occur but the dissociation of hydroxyl
groups is favored. This explains why the x potentials of the
FSNPs change to negative sign and are increased in the
aqueous media with high or low pH values. Although the x
potential of FSNP-1 is different from that of FSNP-2 be-
cause some of the free APS moieties are present on the par-
ticle surfaces but the general trend is the same for both of
the FSNPs.
Figure 5. a)–c) TEM and d)–f) SEM images of monodispersed FSNP-2
with different particle sizes: a) and d) 45 nm, b) and e) 200 nm, and c)
and f) 270 nm.
Table 2. Particle sizes and polydispersities of fluorescent silica nanoparti-
cles.[a]
FSNP-1
FSNP-2
d [nm]
PD
d [nm]
PD
86.3
206.3
295.0
0.027
0.011
0.005
58.1
225.0
280.6
0.167
0.005
0.005
Size tunability: It is important to tailor the sizes of nanopar-
ticles to meet the requirements of different technological
applications. By varying the reaction conditions, we can ma-
nipulate the sizes of the FSNPs. The FSNPs with smaller di-
ameters are obtained when the sol–gel reactions are carried
out in the presence of smaller amounts of ammonium hy-
droxide, while higher concentrations of TEOS and ammoni-
um hydroxide promote the formation of FSNPs with larger
particle sizes.
[a] Determined by x potential analysis; d=diameter, PD=polydispersity.
Cell imaging: To serve as a useful biological tracer, a lumi-
nogen should neither inhibit nor promote the growth of
living cells.[15] As can be seen from Figures S4 and S5 (Sup-
porting Information), in the presence of the TPE and silole
derivatives 3 and 12, the HeLa cells grow similar as in the
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B. Z. Tang et al.
control experiments in the absence of the luminogens. Evi-
dently, 3 and 12 exert no toxicity on the living cells. In other
words, they are cytocompatible without interfering with the
metabolisms of the cells.
The TPE and silole derivatives 3 and 12 are the precur-
sors to the AIE luminogens 1 and 2, respectively, and their
biocompatibilities prompt us to explore the possibility of
utilizing FSNP-1 and FSNP-2 for cell-imaging applications.
As shown in Figure 6, the AIE-active FSNPs selectively
stain the cytoplasmic regions of the living cells. The bright-
ness of the fluorescence images of the cells is increased with
an increase in the luminogen loading in the FSNPs.
smooth surfaces. They possess high surface charges and
hence excellent colloidal stability. Whilst the solutions of 1
and 2 in ethanol are nonemissive, the suspensions of FSNP-
1 and FSNP-2 in the aqueous medium emit strong blue and
green lights, respectively, upon photoexcitation, thanks to
their novel AIE attributes. The particle diameters and emis-
sion efficiencies of the FSNPs are manipulable by changing
the reaction conditions and luminogen loadings. The FSNPs
pose no toxicity to living cells and function as fluorescent
visualizers for intracellular imaging.
We are now investigating multi-photon luminescence pro-
cesses of the FSNPs and their surface modifications via bio-
conjugations with (macro)molecules of biological origin to
enhance their binding specificities and sensing selectivities.
We are also working on the introduction of magnetic com-
ponents to the inner cores of the core–shell particles, in an
effort to prepare nanostructured materials that show high
fluorescence efficiency and magnetic susceptibility. The re-
sults on the fabrication of fluorescent and magnetic particles
and their biotechnoligical applications have been briefly dis-
cussed in one of our recent reviews;[38] full experimental de-
tails will be reported in a separate paper in due course.
Figure 6. Fluorescence images of HeLa cells stained by a)–c) FSNP-1 and
d)–f) FSNP-2 with different luminogen loadings. Concentration of lumi-
nogen [mm]: a) 2, b) 4, c) 8, d) 2, e) 4, f) 6.
Experimental Section
Materials: THF was purchased from Labscan and purified by simple dis-
tillation from sodium/benzophenone under nitrogen immediately prior to
use. TEOS, DMSO, APS, phenylacetylene, n-butyllithium, naphthalene,
decylmethyldichlorosilane, lithium, dichloro(N,N,N,N’-tetramethylethyle-
nediamine)zinc, 4-iodophenol, 1,2-dibromoethane, dichlorobis(triphenyl-
phosphine)palladium(II) and other reagents were all purchased from Al-
drich and used as received. 1,2-Bis(4-bromomethylphenyl)diphenylethene
(3) was prepared using our previously reported procedures.[13, 18]
Instrumentations: 1H and 13C NMR spectra were recorded on a Bruker
ARX 400 spectrometer with tetramethylsilane (TMS; d=0) as internal
standard. HRMS spectra were recorded on a Finnigan TSQ 7000 triple
quadrupole spectrometer operating in a MALDI-TOF mode. The mor-
phologies of the FSNPs were investigated using JOEL 2010 TEM and
JOEL 6700F SEM at an accelerating voltage of 200 and 5 kV, respective-
ly. Samples were prepared by drop-casting dilute dispersions of the
FSNPs onto copper 400-mesh carrier grids covered with carbon-coated
formvar films. The solvent was evaporated at room temperature in open
air. Fluorescence spectra were taken on a Perkin–Elmer LS 50B spectro-
fluorometer with a Xenon discharge lamp excitation. x potentials and
particle sizes of the FSNPs were determined at room temperature by a
ZetaPlus Potential Analyzer (Brookhaven Instruments Corporation,
USA).
The major route for the FSNPs to enter the living cells is
through endocytosis, with the amino groups on the particle
surfaces further facilitating the cellular uptake.[20] The
FSNPs are enclosed by the cell membranes to form small
vesicles, which are then internalized by the cells. The FSNPs
are further processed in endosomes and lysosomes and are
eventually released to the cytoplasm.[34–36] When bound to
the biomacromolecules, the FSNPs may emit even more in-
tensely, because the intramolecular rotations in the lumino-
genic moieties are further restricted if some of them are lo-
cated on the nanoparticle surfaces.[37] Although the silica
shells are hydrophilic, no fluorescence is observed in the cell
nucleus, probably due to the “large” sizes of the FSNPs.
The AIE luminogens can be excited by a two-photon pro-
cess, which enables the use of excitation wavelengths with
lower energies and higher benignancy to living cells. Further
studies on the two-photon fluorescence processes of the
FSNPs and their bioimaging applications are in progress in
collaborations with our physicist and bioengineering col-
leagues.
1-(2-Bromoethoxy)-4-iodobenzene (6): To
a mixture of 4-iodophenol
(5.0 g, 22.7 mmol) and potassium carbonate (4.7 g, 34.1 mmol) in acetone
was added 12.8 g (68.2 mmol) of 1,2-dibromoethane. The mixture was
stirred and heated to reflux for 24 h. After filtration and solvent evapora-
tion, the crude product was purified by silica gel chromatography using
chloroform/hexane 1:4. The product was obtained as white powder (5.8 g,
78%). 1H NMR (400 MHz, CDCl3, TMS): d = 7.57 (d, 2H), 6.69 (d,
2H), 4.25 (t, 2H), 3.62 ppm (t, 2H); 13C NMR (100 MHz, CDCl3, TMS):
d = 158.0, 138.3, 117.1, 83.6, 67.9, 28.8 ppm.
Conclusion
Bis(phenylethynyl)decylmethylsilane (9): nBuLi (25.0 mL, 40.1 mmol,
1.6m solution in hexane) was added at ꢀ788C to a THF solution of 7
(4.0 mL, 36.4 mmol). After stirring at the same temperature for 2 h, de-
cylmethyldichlorosilane (4.8 mL, 18.2 mmol) was added at ꢀ788C. The
mixture was warmed to room temperature and stirred overnight. The sol-
vent was removed under reduced pressure. The mixture was dissolved in
In this work, we have developed a simple process for the
syntheses of highly emissive FSNPs with core–shell struc-
tures. The process does not use surfactant stabilizers and in-
volves a two-step sol–gel reaction in a one-pt experimental
procedure. The resultant FSNPs are monodispersed with
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Nanobiosensors in Intracellular Imaging
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dichloromethane and washed with water. The organic layer was dried
over magnesium sulfate and filtered. The filtrate was evaporated and the
crude product was purified by a silica gel column using hexane. Product 9
was obtained as colorless liquid (12.42 g, 82%). 1H NMR (400 MHz,
CDCl3, TMS): d = 7.60 (m, 4H). 7.40 (m, 6H), 1.73–1.69 (t, 2H), 1.56
(m, 2H), 1.38 (m, 12H), 1.03–0.98 (m, 5H), 0.57 ppm (s, 3H); 13C NMR
(100 MHz, CDCl3, TMS): d = 132.1, 128.7, 128.1, 122.8, 106.3, 90.0, 32.9,
31.9, 29.6, 29.5, 29.3, 29.2, 23.6, 22.7, 16.2, 14.1, 1.2 ppm.
different concentrations and incubated for 24 h. The cells were imaged
under an inverted fluorescence microscope (Nikon Eclipse TE2000-U;
lex =330–380 nm, diachronic mirror=400 nm). The images of the cells
were captured using a digital CCD camera.
1-Decyl-1-methyl-2,5-bis[4-(2-bromoethoxy)phenyl]-3,4-diphenylsilole
(12): A mixture of lithium (0.056 g, 8 mmol) and naphthalene (1.04 g,
8 mmol) in THF (8 mL) was stirred at room temperature under nitrogen
for 3 h to form a deep dark green solution of lithium 1-naphthalenide.
The viscous solution was added dropwise to a solution of 9 (0.77 g,
2 mmol) in THF (5 mL) over 2 min at room temperature. After stirring
for 1 h, the mixture was cooled to 08C with an ice bath and diluted with
Acknowledgements
This work was partially supported by the Hong Kong Research Grants
Council (603008, HKUST13/CRF/08 and 602706), the National Science
Foundation of China (20974028 and 20634020), the University Grants
committee of Hong Kong (AoE/P-03/08) and the Ministry of Science &
Technology (2009CB623605). B.Z.T. thanks the support from Cao Guang-
biao Foundation of Zhejiang University.
.
THF (10 mL). ZnCl2 TMEDA (2 g, 8 mmol) was then added to the mix-
ture to give a black suspension. After stirring for an additional hour at
room temperature, a solution of 6 (1.63 g, 4.9 mmol) and [PdCl2ACTHNUTRGEN(UNG PPh3)2]
(0.08 g, 0.1 mmol) in THF (10 mL) was added. The mixture was refluxed
overnight. After cooling to room temperature, 3m HCl solution (10 mL)
was added and the mixture was extracted with dichloromethane. The
combined organic layer was washed with brine and dried over magnesi-
um sulfate. After solvent evaporation under reduced pressure, the resi-
due was purified by a silica gel column using ethyl acetate/hexanes 1:9.
The product was obtained as a yellow liquid (0.78 g, 50%). 1H NMR
(400 MHz, CDCl3, TMS): d = 7.03 (t, 6H), 6.87–6.81 (m, 8H), 6.70–6.66
(m, 4H), 4.24–4.15 (m, 4H), 3.77 (t, 1H), 3.60 (t, 1H), 3.38 (t, 2H), 1.46–
1.21 (m, 16H), 1.03–0.99 (t, 2H), 0.91–0.88 (t, 4H), 0.47 ppm (s, 3H);
13C NMR (100 MHz, CDCl3, TMS): d = 156.0, 155.9, 155.8, 153.9, 139.5,
139.3, 133.2, 133.1, 130.2, 129.9, 127.5, 126.1, 114.3, 114.2, 114. 1, 68.5,
67.7, 41.9, 32.9, 31.9, 29.6, 29.5, 29.3, 29.1, 23.6, 22.7, 14.1, 14.0, 1.2,
ꢀ5.0 ppm; HRMS (TOF): m/z: calcd for 788.1906; 788.3618 [M]+.
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