Glycosylated star-shaped conjugated oligomers for targeted two-photon fluorescence imaging

Article abstract of DOI:10.1002/chem.201200849

A glucopyranose functionalized star-shaped oligomer, N-tris{4,4',4"- [(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-2-amido-2- deoxy-1-thio-β-D-glucopyranose-hexyl)-9H-fluoren-7-yl)vinyl]phenyl} phenylamine (TVFVBN-S-NH₂), i

Full text of DOI:10.1002/chem.201200849

FULL PAPER
DOI: 10.1002/chem.201200849
Glycosylated Star-Shaped Conjugated Oligomers for Targeted Two-Photon
Fluorescence Imaging
Guan Wang,[a, b] Xinhai Zhang,[c] Junlong Geng,[a] Kai Li,[c] Dan Ding,[a] Kan-Yi Pu,[a]
Liping Cai,[a] Yee-Hing Lai,*[b] and Bin Liu*[a, c]
Abstract: A glucopyranose functional-
ized star-shaped oligomer, N-
tris{4,4’,4’’-[(1E)-2-(2-{(E)-2-[4-(ben-
zo[d]thiazol-2-yl)phenyl]vinyl}-9,9-
bis(6-2-amido-2-deoxy-1-thio-b-d-glu-
copyranose-hexyl)-9H-fluoren-7-yl)vi-
nyl]phenyl}phenylamine (TVFVBN-S-
NH2), is synthesized for two-photon
TVFVBN-S-NH2 has a two-photon ab-
sorption cross-section of ~1100 GM at
780 nm in water. The active amine
group on the glucopyranose moiety
allows further functionalization of
TVFVBN-S-NH2 with folic acid to
yield TVFVBN-S-NH2FA with similar
optical and physical properties as those
for TVFVBN-S-NH2. Cellular imaging
studies reveal that TVFVBN-S-NH2FA
has increased uptake by MCF-7 cells
relative to that for TVFVBN-S-NH2,
due to specific interactions between
folic acid and folate receptors on the
MCF-7 cell membrane. This study
demonstrates the effectiveness of gly-
cosylation as a molecular engineering
strategy to yield water-soluble materi-
als with a large two-photon absorption
(TPA) cross-section for targeted
cancer-cell imaging.
fluorescence
imaging.
In
water,
TVFVBN-S-NH2 self-assembles into
nanoparticles with an average diameter
of ~49 nm and shows a fluorescence
quantum yield of 0.21. Two-photon
fluorescence measurements reveal that
Keywords: fluorescence · glucopyr-
anose · oligomers · self-assembly ·
targeted imaging
Introduction
merits, such as high photostability and high brightness, the
intrinsic toxicity of QDs in the oxidative environment limits
their applications in long-term monitoring of cellular
events.[2] On the other hand, fluorescent proteins used for
TPM imaging generally show small two-photon absorption
(TPA) cross-sections (d) of less than 300 GM.[3] In addition,
their drawbacks related to maturation and monomeric state
were reported to show severe cytotoxicity effects.[4] The
shortcomings of QDs and fluorescent proteins have motivat-
ed researchers to develop alternative TPA chromophores,
such as organic p-conjugated materials for two-photon fluo-
rescence imaging applications.
Two-photon microscopy (TPM), which offers several advan-
tages over conventional one-photon confocal laser scanning
microscopy (CLSM), such as intrinsic high 3D resolution,
deeper penetration, less photodamage and less photobleach-
ing, has emerged as a powerful technology for non-invasive
biological imaging.[1] The advancement of TPM imaging is
highly dependent on the design and synthesis of highly fluo-
rescent two-photon absorbing materials. Although quantum
dots (QDs) as two-photon absorbing materials offer many
Organic p-conjugated materials with traditional linear
push–pull structures (donor–acceptor, D–A) generally ex-
hibit smaller TPA cross-sections (d) relative to those for
quadrupolar (D–A–D or A–D–A) and octupolar systems.[5]
However, star-shaped and dendritic chromophores, such as
three-branched octupolar structures, could bring excitonic
coupling between the dipolar branch and the central core to
induce large TPA d values.[6] Although materials with a TPA
d larger than 2000 GM have been reported,[5] most of them
are only soluble in organic solvents with limited biological
applications. In addition, water-soluble derivatives of these
TPA materials generally show a significantly decreased d
value relative to their neutral counterparts in organic sol-
vents.[7] To address this problem, we recently found that in-
tegration of a glucose moiety with a star-shaped p-conjugat-
ed oligomer is an effective strategy to yield a robust water-
soluble TPA material (structure shown as TFBS) with high
[a] G. Wang, J. Geng, Dr. D. Ding, Dr. K.-Y. Pu, Dr. L. Cai, Prof. B. Liu
Department of Chemical and Biomolecular Engineering
National University of Singapore
4 Engineering Drive 4, National University of Singapore
Singapore 117576 (Singapore)
Fax : (+65)6779-1936
E-mail: cheliub@nus.edu.sg
[b] G. Wang, Prof. Y.-H. Lai
Department of Chemistry
National University of Singapore
3 Science Drive 3, National University of Singapore
Singapore 117543 (Singapore)
E-mail: chmlaiyh@nus.edu.sg
[c] Dr. X. Zhang, Dr. K. Li, Prof. B. Liu
Institute of Materials Research and Engineering
3 Research Link, Singapore 117602 (Singapore)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200849: NMR spectra,
MALDI-TOF mass spectra.
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TPA d= ~1200 GM at 740 nm for two-photon fluorescence
rene vinylene is selected as the bridge due to its good mo-
lecular coplanarity, which is favourable to high d values and
redshifted absorption and emission maxima relative to those
for TFBS. The influence of glucopyranose functionalization
on the linear and TPA properties of TVFVBN-S-NH2 is
studied. The application of folic acid conjugated TVFVBN-
S-NH2FA for targeted two-photon fluorescence imaging of
MCF-7 cells is demonstrated.
imaging of Hela cells.[8]
Results and Discussion
Synthesis and characterization: The synthetic route towards
TVFVBN-S-NH2 is depicted in Scheme 1. 2-p-Tolylben-
zo[d]thiazole (1) was obtained in 84% yield by heating 4-
methylbenzaldehyde with 2-aminothiophenol in N-methyl-2-
pyrrolidone (NMP) at 1108C for 72 h. Monobromination of
1 with N-bromosuccinimide (NBS) in CCl4 under light irra-
diation afforded 2-[4-(bromomethyl)phenyl]benzo[d]thiazole
(2) in 64% yield. Thiazole 2 was then converted to its phos-
Targeted cancer-cell imaging with fluorescent materials is
of vital importance in cancer
prognosis and treatment at an
early stage.[2c,9] A wide range of
targeting moieties, such as
folate,[10] epidermal growth
factor,[11] transferrin[12] or anti-
bodies,[13] which specifically
bind to the antigen or receptor
over expressed in cancer cells
have been functionalized onto
fluorescent materials for target-
ed fluorescence imaging. How-
ever, there are limited reports
on the conjugation of organic
TPA materials for targeted
TPM imaging.[14] Motivated by
the fact that chitosan, a linear
polysaccharide with a d-glucos-
amine unit, allows diverse func-
tionalization through the active
amine groups,[15–17] it occurs to
us that we could design a glyco-
sylated material for further con-
jugation to targeting moieties
to yield a robust two-photon
absorbing material for targeted
cancer-cell imaging.
In this contribution, we
report star-shaped glucopyra-
nose functionalized oligomers
(TVFVBN-S-NH2) with a large
TPA d value for targeted two-
photon fluorescence imaging of
cancer cells. In these molecules,
triphenyl amine serves as the
donor, and phenyl benzothia-
Scheme 1. The synthetic route towards TVFVBN-S-NH2 and TVFVBN-S-NH2FA. Reagents and conditions:
i) NMP, 1108C, 72 h; ii) NBS, CCl4, reflux, 1 h; iii) triethylphosphite, 1808C, 3 h; iv) POCl3, DMF (anhydrous),
08C, 1 h, followed by 1008C overnight; v) NaBH4, MeOH, reflux, 5 h; vi) HBr (g), CHCl3, RT, overnight; vii)
triethyl phosphite, 1208C, overnight; viii) nBuLi, DMF (anhydrous), THF (anhydrous), À788C to RT, over-
night; ix) 3, potassium tert-butoxide, THF (anhydrous), À108C, 4 h; x) 7, potassium tert-butoxide, THF (anhy-
drous), 08C, 6 h; xi) 2-acetamido-2-deoxy-1-thio-b-d-glucopyranose 3,4,6-triacetate, K2CO3, THF (anhydrous),
zole is the acceptor. The fluo- RT, 72 h; xii) hydrazine monohydrate, reflux, 48 h; xiii) folic acid, DCC/NHS, pyridine, RT, overnight.
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Glycosylated Star-Shaped Conjugated Oligomers
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phonate salt diethyl [4-(benzo[d]thiazol-2-yl)phenyl]methyl-
phosphonate (3) in 84% yield upon heating with triethyl-
phosphite at 1808C for 3 h.[18] Tris(4-formylphenyl)amine (4)
was synthesized from triphenylamine and POCl3 by using
a modified procedure from a literature report.[19] Amine 4
was then treated with NaBH4 to yield tris(4-hydroxymethyl-
phenyl)amine (5). Further bromination of 5 with HBr gas
afforded tris(4-bromomethylphenyl)amine (6), which upon
heating with triethylphosphite at 1208C yielded tris(4-dieth-
yl phosphonatemethylphenyl)amine (7) in an overall yield
of 67% for three steps. 9,9-Bis(6-bromohexyl)-9H-fluorene-
2,7-dicarbaldehyde (8) was synthesized in 39% yield from
2,7-dibromo-9,9-bis(6-bromohexyl)-9H-fluorene by using
nBuLi and anhydrous DMF at À788C. Coupling one equiva-
lent of 3 with 8 under Horner–Emmons conditions afforded
monoaldehyde 9 in 35% yield. Horner–Emmons coupling
between three equivalents of 7 and 9 gave TVFVBN in
65% yield. The correct structure of TVFVBN was affirmed
by NMR spectroscopy and MALDI-TOF mass spectroscopy
(see Figures S1a and S1b in the Supporting Information).
Further reaction between TVFVBN and 2-acetamido-2-
deoxy-1-thio-b-d-glucopyranose 3,4,6-triacetate with K2CO3
as the base afforded TVFVBN-S-NHAc in 80% yield. The
disappearance of the triplet at 3.29 ppm (CH2Br) in the
1H NMR spectrum (see Figure S2a in the Supporting Infor-
mation) of TVFVBN-S-NHAc indicates 100% attachment
of glucopyranose onto TVFVBN. The correct molecular
mass was confirmed by MALDI-TOF spectroscopy (see Fig-
ure S2b in the Supporting Information). Hydrolysis of
TVFVBN-S-NHAc in the presence of hydrazine under
reflux conditions[20] afforded TVFVBN-S-NH2 in 94% yield
Figure 1. DLS spectra of 2 mm TVFVBN-S-NH2 (a) and TVFVBN-S-
NH2FA (b) in water and TEM images of TVFVBN-S-NH2 (c) and
TVFVBN-S-NH2FA (d) nanoparticles.
TEM was performed to study the morphology of the self-
assembled nanoparticles. The samples were prepared by
drop-coating of TVFVBN-S-NH2 and TVFVBN-S-NH2FA
solutions (2 mm in water) onto a copper grid. As shown in
Figure 1c and d, both TVFVBN-S-NH2 and TVFVBN-S-
NH2FA form spherical nanoparticles with mean diameters
of ~20 nm. The smaller size in TEM relative to those in
DLS is due to the shrinking of samples when transformed
into a dry state from solution.[22] The fact that both
TVFVBN-S-NH2 and TVFVBN-S-NH2FA have nearly the
same nanoparticle size indicates that folic acid conjugation
does not obviously change the self-assembly behaviour of
TVFVBN-S-NH2 in water. However, it is noteworthy that
both TVFVBN-S-NH2 and TVFVBN-S-NH2FA are dis-
solved into the molecular level in DMSO with no DLS sig-
nals.
1
after dialysis and freeze drying. The H NMR spectrum for
TVFVBN-S-NH2 is shown in Figure S3 in the Supporting In-
1
formation. The singlet for acetyl groups from the H NMR
spectrum of TVFVBN-S-NHAc is almost unobservable,
which indicates the successful deacetylation of NHAc and
OAc groups to NH2 and OH, respectively. The folic acid
conjugation was conducted by using N,N-dicyclohexylcarbo-
diimide (DCC)/N-hydroxysuccinimide (NHS) at a molar
ratio of 1:10 for folic acid and TVFVBN-S-NH2 to give folic
acid functionalized TVFVBN-S-NH2FA. After purification
by dialysis with a 3.5 KDa molecular weight cut-off mem-
brane against Mill-Q water for two days, TVFVBN-S-
NH2FA was obtained in a quantitative yield as an orange
powder after freeze drying. The 1H NMR spectrum of
TVFVBN-S-NH2FA is shown in Figure S4 in the Supporting
Information.
Linear optical properties: The UV/Vis absorption and PL
spectra of TVFVBN in different solvents at a concentration
of 2 mm are shown in Figure 2. The absorption spectra of
Self-assembly in water: The self-assembly behaviours of
TVFVBN-S-NH2 and TVFVBN-S-NH2FA were studied by
using dynamic light scattering (DLS). As shown in Figure 1,
the mean diameter for TVFVBN-S-NH2 in water is ~49 nm
with a polydispersity of 0.23 (Figure 1a), whereas that for
TVFVBN-S-NH2FA is ~50 nm with a polydispersity of 0.27
(Figure 1b). The polydispersities for both (0.23 and 0.27) are
less than 0.3, which indicates that they form nanoparticles
that are medium-dispersed in water.[21]
Figure 2. UV/Vis absorption and PL spectra of TVFVBN in toluene, di-
chloromethane and DMF.
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TVFVBN in toluene, dichloromethane and dimethylforma-
mide (DMF) have the same shape and maxima, which indi-
cate that the ground state of TVFVBN is not sensitive to
solvent polarity.[23] On the other hand, the emission maxi-
mum of TVFVBN in toluene is at 486 nm, which is redshift-
ed to 564 nm in dichloromethane. The quantum yields of
TVFVBN in toluene and dichloromethane are 0.99 and
0.68, respectively, measured by using Rhodamine 6G in
methanol as the reference. However, under the same experi-
mental conditions, no fluorescence is observed for TVFVBN
in DMF. Our previous molecular-orbital simulation indicates
that charge transfer from electron-rich triphenylamine to
electron-deficient benzothiazole units exists in the excited
state.[8] Similarly, the redshifted emission maxima and de-
creased fluorescence quantum yields with increased solvent
polarity for TVFVBN are due to the charge-transfer charac-
teristics of its excited state. As a result, bright greenish/blue
and orange fluorescent colours are observed for the toluene
and dichloromethane solutions of TVFVBN, whereas its
DMF solution is not emissive (see Figure S5 in the Support-
ing Information).
with folic acid, the optical properties of TVFVBN-S-NH2FA
are nearly the same as those for TVFVBN-S-NH2, which in-
dicate that folic acid conjugation does not change the linear
optical properties of TVFVBN-S-NH2 in water. As shown in
Figure S5 in the Supporting Information, the aqueous solu-
tion of TVFVBN-S-NH2 shows bright-yellow fluorescence,
which indicates a high potential for optical applications.
TPA properties: The TPA spectra of TVFVBN in toluene
and TVFVBN-S-NH2 in water were collected by using
a standard two-photon excited fluorescence (TPEF) tech-
nique with a femtosecond pulsed laser source.[24] To avoid
the interference on TPEF by laser excitation, TPA spectra
were measured by starting at the wavelength at which each
individual oligomer has nearly no emission. In addition, lim-
ited by the laser availability in our experiments, TVFVBN
in toluene was investigated in the range from 740 to 900 nm,
whereas TVFVBN-S-NH2 in water was studied from 750 to
900 nm.
The TPA spectrum of TVFVBN in toluene (see Figure S6
in the Supporting Information) shows a maximum cross-sec-
tion (d) of 3031 GM at 820 nm. This value is ~500 GM
larger than TFBN[8] (2494 GM), which is ascribed to the in-
creased coplanarity with an ethylene linker for TVFVBN.
This d value is also larger than those for octupolar tri-
branched molecules when using triphenylamine as the core,
with a fluorenyl-ethylene linker and SO2CF3 (2080 GM)[6a]
or CHO (1265 GM)[25] as the peripheries, respectively. This
agrees with the fact that an elongated conjugated backbone
and a proper degree of intramolecular charge transfer (ICT)
favour larger TPA d values.[26]
The absorption and emission spectra of TVFVBN-S-NH2
in DMSO and pure water are presented in Figure 3. The ab-
sorption spectra of TVFVBN-S-NH2 in DMSO and water
The TPA spectrum of TVFVBN-S-NH2 in water is shown
in Figure 4. The maximum TPA d value is 1098 GM at
780 nm, giving an action cross-section value (hd) of 230 GM.
Figure 3. UV/Vis absorption and PL spectra of TVFVBN-S-NH2 in
DMSO and H2O and TVFVBN-S-NH2FA in H2O.
are similar, with the maximum at 393 nm. The emission
maxima of TVFVBN-S-NH2 in DMSO and water locate at
494 and 542 nm, respectively. The quantum yield of
TVFVBN-S-NH2 in water is 0.21, which is substantially
higher than that in DMSO (0.07). The higher quantum yield
of TVFVBN-S-NH2 in water relative to that in DMSO is as-
cribed to the formation of nanoparticles, as supported by
the DLS and TEM measurements. In DMSO, TVFVBN-S-
NH2 is dissolved into the molecular level as aforementioned,
and the charge-transfer between TVFVBN-S-NH2 and
DMSO quenches the fluorescence. However, the nanoparti-
cles formed in water provide a hydrophobic microenviron-
ment for TVFVBN-S-NH2, which reduces its interaction
with water for fluorescence quenching.[22a] After conjugation
Figure 4. TPA cross-sections of TVFVBN-S-NH2 in water.
As TVFVBN and TVFVBN-S-NH2 share the same back-
bone, the decrease of the TPA d value relative to that in tol-
uene is due to the solvent effect, because water can influ-
ence the electronic structure of donor–acceptor chromo-
phores through hydrogen bonding, which leads to a de-
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Glycosylated Star-Shaped Conjugated Oligomers
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creased ICT.[7b] A further study of TVFVBN-S-NH2FA
under the same conditions yielded a similar hd value (see
Figure S7 in the Supporting Information). As a consequence,
the large TPA hd of TVFVBN-S-NH2 in water would ensure
a high brightness in two-photon biological imaging applica-
tions. Moreover, the lmax(TPA) of 780 nm makes TVFVBN-S-
NH2 more appealing for biological applications relative to
TFBS, since excitation at longer wavelength can lead to less
photodamage and photobleaching.
To confirm the folate receptor-mediated targeting effect
of TVFVBN-S-NH2FA, firstly, we studied the competitive
effect of folic acid on the endocytosis of TVFVBN-S-
NH2FA on MCF-7 cells. As shown in Figure S10 in the Sup-
porting Information, the fluorescence image of free folic
acid pretreated MCF-7 cells shows lower intensity relative
to that in Figure 5c. This indicates that the uptake of
TVFVBN-S-NH2FA is greatly inhibited by the free folic
acid treatment, which effectively blocks the interaction be-
tween TVFVBN-S-NH2FA and the folate receptor on the
cell surface.[10b] In addition, NIH/3T3 fibroblast normal cells,
which lack a folate receptor on the cell membrane, were
also used as a negative control. The CLSM images of NIH/
3T3 cells treated with TVFVBN-S-NH2FA and TVFVBN-S-
NH2 are shown in Figure S11 in the Supporting Information.
The fluorescence intensities in Figure S11A (stained with
TVFVBN-S-NH2) and Figure S11C (stained with TVFVBN-
S-NH2FA) are comparable to each other, which are weaker
than that in Figure 5c (estimated by using ImageJ, see Fig-
ure S12 in the Supporting Information). These data indicate
the specific binding between TVFVBN-S-NH2FA and the
folate receptor on the cell surface, which shows the ability
of TVFVBN-S-NH2FA to discriminate folate-positive cancer
cells from others.
Targeted one- and two-photon fluorescence imaging: To
demonstrate the potential of glycosylated materials in tar-
geted cellular imaging, single (CLSM) and two-photon fluo-
rescence (TPEF) cellular imaging of MCF-7 cancer cells
were investigated with TVFVBN-S-NH2FA and TVFVBN-
S-NH2. MCF-7 cancer cells are known to over express folate
receptors, and, therefore, TVFVBN-S-NH2FA with folate
targeting ligands is expected to offer a higher uptake by
MCF-7 cells relative to TVFVBN-S-NH2. MCF-7 cells were
incubated with TVFVBN-S-NH2 and TVFVBN-S-NH2FA at
the molar concentration of 1 mm before fixation and image.
As shown in Figure 5, both CLSM images of MCF-7 cells in-
The TPEF images MCF-7 cells incubated with TVFVBN-
S-NH2 and TVFVBN-S-NH2FA are shown in Figure 6a and
b, respectively. Green fluorescence was obtained in both
images upon excitation at 800 nm with an average laser
power of 10 mW. The fluorescence from Figure 6b is sub-
stantially brighter than that from Figure 6a, which is as-
cribed to the increased cellular uptake of TVFVBN-S-
NH2FA relative to TVFVBN-S-NH2. In addition, the bright-
green fluorescence from Figure 6b should benefit from the
large TPA cross-section of TVFVBN-S-NH2 in the 750–
830 nm region (up to 1098 GM), which is substantially
larger than a number of fluorescent probes designed for
TPEF imaging applications.[14c,27] These data clearly illustrate
the potential of using TVFVBN-S-NH2FA as a two-photon
fluorescent probe for targeted TPEF cellular imaging.
Figure 5. CLSM images of MCF-7 breast cancer cells after incubation
with TVFVBN-S-NH2 (a) and TVFVBN-S-NH2FA (c); images a and c to-
gether with propidium iodide nucleus stain are shown in b and d. a–d
share the same scale bar.
cubated with TVFVBN-S-NH2 (Figure 5a) and TVFVBN-S-
NH2FA (Figure 5c) show green fluorescence. In addition,
the fluorescence from Figure 5c is about three times (esti-
mated by using ImageJ, Figure S8 in the Supporting Infor-
mation) more intense relative to that from Figure 5a, which
indicates the increased cellular uptake of TVFVBN-S-
NH2FA by MCF-7 cells. Figure 5b and d are the overlapped
images with nuclei stained, which clearly indicate that both
TVFVBN-S-NH2 and TVFVBN-S-NH2FA are localized in
the cytoplasm of MCF-7 cells. The experiments have also
been tested in living MCF-7 cells, which offered essentially
similar results as those for fixed cells (see Figure S9 in the
Supporting Information).
Figure 6. a) TPEF images of MCF-7 breast cancer cells after incubation
with TVFVBN-S-NH2 (a) and TVFVBN-S-NH2FA (b). a and b share the
same scale.
Cytotoxicity and photostability study: The cytotoxicity of
TVFVBN-S-NH2 and TVFVBN-S-NH2FA was evaluated for
MCF-7 cells by using a methylthiazolyldiphenyl-tetrazolium
(MTT) cell viability assay. Figure S13 in the Supporting In-
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Y. H. Lai, B. Liu et al.
carried out on a Perkin–Elmer LS-55 instrument equipped with a xenon-
lamp excitation source and a Hamamatsu (Japan) 928 photomultiplier
tube (PMT), by using 908 angle detection for solution samples. Quantum
yields were measured by using Rhodamine 6G in methanol as the refer-
ence.
formation shows the in vitro MCF-7 cell viabilities after
being cultured with TVFVBN-S-NH2 and TVFVBN-S-
NH2FA solution at concentrations of 1, 10 and 100 mm for
24, 48 and 72 h, respectively. Within the test period of time,
the cell viabilities are close to 100% at 10 mm each (10 times
the concentration used for imaging studies shown in
Figure 5), which indicates the low cytotoxicity of both
TVFVBN-S-NH2 and TVFVBN-S-NH2FA. When the con-
centration is increased to 100 mm, both oligomeres exhibit
slight cytotoxicity (the cell viabilities drop to ꢀ80%). These
studies indicate that both oligomers are suitable for in vitro
and in vivo cellular imaging studies. The changes in the
CLSM and TPM images of MCF-7 cells treated with
TVFVBN-S-NH2FA before and after continuous laser scan-
ning for 10 min were also monitored. As shown in Fig-
ure S14 in the Supporting Information, the intensity decreas-
es in both the CLSM and TPM fluorescence images are less
than 20% of its original intensity (estimated by using
ImageJ), which indicate good photostability of TVFVBN-S-
NH2FA in the cellular environment.
2-p-Tolylbenzo[d]thiazole (1):
A mixture of 4-methylbenzaldehyde
(7 mL, 58 mmol), 2-aminothiophenol (7 mL, 65 mmol), and NMP
(50 mL) was heated in an oil bath at a temperature of 1108C for 72 h,
which was then poured into 1:1 ethanol/water. The precipitates were col-
lected and recrystallized from ethanol to afford 1 as yellow needle crys-
tals (11 g, 84%). 1H NMR (500 MHz, CDCl3): d=8.06 (d, J=8 Hz, 1H),
7.99 (d, J=8.5 Hz, 2H), 7.89 (d, J=8 Hz, 1H), 7.49 (t, J=7 Hz, 1H), 7.37
(t, J=8 Hz, 1H), 7.29 (d, J=8 Hz, 2H), 2.43 ppm (s, 3H); 13C NMR
(125 MHz, CDCl3): d=168.25, 154.19, 141.44, 134.98, 131.00, 129.74,
127.52, 126.26, 125.02, 123.08, 121.58, 21.54 ppm; MS (EI): m/z: 225.1
[M+].
2-(4-(Bromomethyl)phenyl)benzo[d]thiazole (2): 2-p-Tolylbenzo[d]thia-
zole (1) (1.5 g, 6.7 mmol) and NBS (1.2 g, 6.7 mmol) were mixed in CCl4
(40 mL). The suspension was irradiated to reflux under a 120 W lamp for
1 h. Completion of reaction was confirmed by TLC. After cooling down
to room temperature, the succinimide salt was filtered off and solvent
was removed under reduced pressure. The residue was dissolved in di-
chloromethane and washed with water three times. The organic layer was
dried with MgSO4 and solvent was subsequently removed under reduced
pressure. The crude product was recrystallized from ethanol to yield 2 as
white crystals. (1.4 g, 64%). 1H NMR (500 MHz, CDCl3): d=8.09–8.06
(m, 3H), 7.90 (d, J=8 Hz, 1H), 7.51–7.48 (m, 3H), 7.40 (t, J=8 Hz, 1H),
4.53 ppm (s, 2H); 13C NMR (125 MHz, CDCl3): d=167.13, 154.16,
140.58, 135.13, 133.66, 129.68, 126.43, 125.37, 123.37, 121.65, 32.55 ppm;
MS (EI): m/z: 303.0 [M+], 305.0 [M+], 224.1 [M+ÀBr].
Diethyl (4-(benzo[d]thiazol-2-yl)phenyl)methylphosphonate (3): 2-(4-
(Bromomethyl)phenyl)benzo[d]thiazole (2) (1.2 g, 4 mmol) and triethyl-
phosphite (1.0 g, 6 mmol) were mixed and heated at 1808C for 3 h under
an argon atmosphere. After cooling down to 508C, excess of triethylphos-
phite was distilled off under vacuum. The crude yellow solid product was
dissolved in diethyl ether and precipitated in hexane to afford 3 as white
needle crystals (1.2 g, 84%). 1H NMR (500 MHz, CDCl3): d=8.11–8.07
(m, 3H), 7.93 (d, J=8 Hz, 1H), 7.51 (t, J=7.5 Hz, 1H), 7.47–7.45 (m,
2H), 7.42 ppm (t, J=7 Hz, 1H); 13C NMR (125 MHz, CDCl3): d=167.68,
154.16, 135.09, 135.05, 135.02, 132.37, 132.34, 130.46, 130.41, 127.72,
127.69, 126.33, 125.19, 123.21, 121.60, 62.30, 62.25, 34.47, 33.38, 16.41,
16.36 ppm; MS (EI): m/z: 361.1 [M+], 224.0 [M+ÀC4H10O3P].
Tris(4-formylphenyl)amine (4): Phosphorus oxychloride (9.5 mL,
101.9 mmol) was added dropwise to dry DMF (7.3 mL, 94.3 mmol) at
08C under an argon atmosphere. The mixture was stirred at 08C for 1 h
and additionally stirred at room temperature for 1 h. After the addition
of triphenylamine (5.0 g, 20.4 mmol) in chloroform (5 mL), the mixture
was stirred at 1008C overnight. After cooling, the solution was poured
into ice water (400 mL) and the resulting mixture was neutralized to
pH 7 with 5% NaOH aqueous solution. After extraction with dichloro-
methane, the organic layer was washed with brine and water three times.
The organic layer was dried with MgSO4 and solvent was removed under
reduced pressure. The residue was filtered through a short column with
hexane/dichloromethane (1:2, v/v) to produce yellowish solids. After
drying, the solid was added into an ice-cooled mixture of phosphorus
oxychloride (9.5 mL, 101.9 mmol) and dry DMF (7.3 mL, 94.3 mmol)
under an argon atmosphere. The resulting mixture was heated at 1008C
overnight. After cooling, the solution was poured into ice water (400 mL)
and the resulting mixture was neutralized to pH 7 with 5% NaOH aque-
ous solution, which was then extracted with dichloromethane. The organ-
ic layer was washed with brine and water and then dried over MgSO4.
After the solvent was removed under reduced pressure, the residue was
columned by using hexane/dichloromethane (1:4, v/v) to yield 4 as yellow
crystals (2.7 g, 40%). 1H NMR (500 MHz, CDCl3): d=9.96 (s, 3H), 7.85
(d, J=8.5 Hz, 6H), 7.27 ppm (d, J=7 Hz, 6H); 13C NMR (125 MHz,
CDCl3): d=190.51, 151.21, 132.59, 131.52, 124.55 ppm; MS (EI): m/z:
329.0 [M+].
Conclusion
In summary, we have synthesized glucopyranose functional-
ized star-shaped conjugated oligomers with a push–pull
structure for two-photon fluorescence imaging. The precur-
sor oligomer TVFVBN-S-NH2 has been found to self-assem-
ble into nanoparticles with a quantum yield of 0.21 in water.
These nanoparticles have a large TPA cross-section of
~1100 GM at 780 nm in aqueous solution when calculated
based on a molecule. More importantly, this glycosylated
material is born with a reactive amine group on each gluco-
pyranose substituent, which allows further modification with
versatile biocompatible elements or bio-recognition moiet-
ies. As a demonstration, folic acid was conjugated to
TVFVBN-S-NH2 to yield TVFVBN-S-NH2FA, which shows
similar self-assembly behaviour and optical properties as
those of TVFVBN-S-NH2. Cellular studies reveal that both
nanoparticles show low cytotoxicity and TVFVBN-S-
NH2FA is more favourable to MCF-7 cells due to specific
folic acid–folate receptor interactions on cell membranes.
As a result, this study highlights the glycosylation strategy
as an effective molecular engineering approach to develop
robust materials with good water dispersibility, self-assembly
ability and large TPA cross-sections for targeted cancer cell
imaging.
Experimental Section
Materials and instruments: Chemicals and reagents were purchased from
Sigma–Aldrich unless otherwise stated. NMR spectra were collected on
a Bruker AMX 500 spectrometer with [D]chloroform as the solvent
(unless otherwise stated) and tetramethylsilane as the internal standard.
Elemental analyses were carried out by the Microanalysis Laboratory of
the National University of Singapore. UV/Vis spectra were recorded on
a Shimadzu UV-1700 spectrometer. Fluorescence measurements were
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Glycosylated Star-Shaped Conjugated Oligomers
FULL PAPER
Tris(4-hydroxyphenyl)amine (5): Tris(4-formylphenyl)amine (4) (1.0 g,
3.0 mmol) and NaBH4 (1.6 g, 42.6 mmol) were suspended into methanol
(50 mL), which was then refluxed for 5 h. After cooling down to room
temperature, the reaction solution was quenched with water. The mixture
was further diluted with water (200 mL) and neutralized to pH 7 by using
diluted acetic acid. White solid precipitated after neutralization, which
was collected via centrifugation and dried under vacuum to give 5 as
a white solid (1 g, 98%). 1H NMR (500 MHz, CDCl3): d=7.25 (d, J=
8.5 Hz, 6H), 7.06 (d, J=8.5 Hz, 6H), 4.65 ppm (s, 6H); 13C NMR
(125 MHz, CDCl3): d=147.27, 135.31, 128.30, 124.17, 65.08 ppm; MS
(EI): m/z: 335.2 [M+].
(125 MHz, CDCl3): d=192.19, 167.64, 154.21, 152.48, 151.47, 147.09,
139.91, 139.69, 137.80, 135.43, 135.03, 132.79, 130.87, 130.49, 128.23,
128.00, 127.10, 126.42, 126.31, 125.25, 123.20, 122.84, 121.64, 121.42,
121.08, 120.14, 55.19, 40.14, 33.82, 32.58, 28.99, 27.76, 23.57 ppm; MS
(EI): m/z: 755.3 [M+].
N-Tris{4,4’,4’’-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-
9,9-bis(6-bromohexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylnamine
(TVFVBN): 7-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-bro-
mohexyl)-9H-fluorene-2-carbaldehyde (9) (100 mg, 0.13 mmol) and
tris(4-diethylphosphonatemethylphenyl)amine 7 (26 mg, 0.037 mmol) was
mixed in dry THF (10 mL) under an argon atmosphere, and then the so-
lution was cooled down to 08C. A suspension of potassium tert-butoxide
(19 mg, 0.17 mmol) in dry THF (5 mL) was added dropwise into the solu-
tion. The reaction was kept at 08C for 6 h before it was quenched with
water. The solvent was removed under reduced pressure and dichlorome-
thane was used for extraction. The organic layer was washed with brine
and water and then dried with MgSO4. After solvent removal, the residue
was columned by using dichloromethane/hexane (1:1, v/v) to give
TVFVBN as a yellow solid (60 mg, 65%). 1H NMR (500 MHz, CDCl3):
d=8.12–8.07 (m, 9H), 7.91 (d, J=8.5 Hz, 3H), 7.69–7.67 (m, 12H), 7.56–
7.47 (m, 21H), 7.40 (t, J=6 Hz, 3H), 7.33 (d, J=15.5 Hz, 3H), 7.24–7.17
(m, 15H), 3.28 (t, J=7.5 Hz, 12H,), 2.07–2.04 (m, 12H), 1.69–1.64 (m,
12H), 1.24–1.13 (m, 24H), 0.88–0.86 ppm (m, 12H); 13C NMR (125 MHz,
CDCl3): d=167.71, 154.28, 151.32, 151.28, 141.21, 140.34, 140.25, 138.92,
136.85, 135.96, 135.06, 132.52, 130.98, 129.06, 127.98, 127.05, 126.97,
126.39, 126.16, 125.71, 125.19, 124.35, 123.19, 121.64, 120.93, 120.60,
120.18, 120.10, 114.11, 54.96, 40.38, 34.02, 32.65, 29.09, 27.77, 23.59 ppm;
MS (MALDI-TOF): m/z: calcd for C144H138Br6N4S3 [M+]: 2500.5; found:
2500.5.
Tris(4-bromomethylphenyl)amine (6): HBr gas was bubbled into a sus-
pension of tri(4-hydroxyphenyl)amine 5 (500 mg, 1.5 mmol) in CHCl3
(200 mL) for 20 min. The resulting solution was allowed to stir at room
temperature for 2 days in a sealed round-bottomed flask. Nitrogen gas
was then bubbled into the solution to remove excess of HBr gas. The re-
sulting solution was washed with saturated NaHCO3 (150 mL) and then
washed with brine before drying with MgSO4. Solvent removal under re-
duced pressure gave
6 as a
white solid (708 mg, 90%). 1H NMR
(500 MHz, CDCl3): d=7.27 (d, J=8.5 Hz, 6H), 7.03 (d, J=8 Hz, 6H),
4.49 ppm (s, 6H); MS (EI): m/z: 524.9 [M+], 444.0 [M+ÀBr].
Tris(4-diethylphosphonatemethylphenyl)amine (7): Tri(4-bromomethyl-
phenyl)amine (6) (500 mg, 0.95 mmol) and triethylphosphite (712 mg,
4.3 mmol) were mixed under an argon atmosphere. The resulting mixture
was heated at 1208C overnight. After cooling down to 508C, the excess
of triethylphosphite was removed by using vacuum distillation. The resi-
due was dissolved in diethyl ether and poured into hexane (30 mL) to
give 7 as a colourless oil (500 mg, 76%). 1H NMR (500 MHz, CDCl3):
d=7.16–7.14 (m, 6H), 6.97 (d, J=8.5 Hz, 6H), 4.04 (quintet, 12H), 3.09
(d, J=21.5 Hz, 6H), 1.25 ppm (t, J=7.5 Hz, 18H); 13C NMR (125 MHz,
CDCl3): d=146.45, 146.43, 130.66, 130.60, 125.78, 125.70, 124.08, 124.06,
62.13, 62.08, 33.64, 32.54, 16.36, 16.32 ppm; MS (EI) m/z: 695.4 [M+].
N-Tris{4,4’,4’’-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-
9,9-bis(6-2-acetamido-2-deoxy-1-thio-b-d-glucopyranose 3,4,6-triacetate-
hexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylnamine (TVFVBN-S-NHAc):
N-Tris{4,4’,4’’-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-
bis(6-bromohexyl)-9H-fluoren-7-yl)vinyl]phenyl}phenylnamine
9,9-Bis(6-bromohexyl)-9H-fluorene-2,7-dicarbaldehyde (8): 2,7-Dibormo-
9,9-di(6-bromohexyl)fluorene (3.9 g, 6.0 mmol) in freshly distilled THF
(60 mL) was cooled to À788C with a dry ice/acetone bath under an
argon atmosphere. At À788C, nBuLi in hexane (9 mL, 12.4 mmol) was
added dropwise into the solution, which was stirred for 30 min. Anhy-
drous DMF (1.2 mL, 15 mmol) was subsequently added and the solution
was stirred for another 2 h at À788C before being kept at room tempera-
ture overnight. The resulting mixture was quenched with water and the
solvent was removed by evaporation. After extraction with dichlorome-
thane, the organic phase was separated and dried over MgSO4. After sol-
vent removal under reduced pressure, the residue was purified with silica
gel column by using hexane/dichloromethane (3:2, v/v) to afford 8 as
a colourless oil (1.3 g, 39%), which solidified after standing in a fridge
overnight. 1H NMR (500 MHz, CDCl3): d=10.07 (s, 2H), 7.92–7.88 (m,
6H), 3.20 (t, J=7 Hz, 4H), 2.07–2.04 (m, 4H), 1.69–1.56 (m, 4H), 1.11–
1.02 (m, 8H), 0.88–0.53 ppm (m, 4H); 13C NMR (125 MHz, CDCl3): d=
191.93, 152.51, 145.56, 136.58, 130.37, 123.33, 121.43, 55.45, 39.83, 33.69,
32.52, 28.87, 27.70, 23.59 ppm; MS (EI) m/z: 548.2 [M+].
(TVFVBN) (30 mg, 0.012 mmol), 2-acetamido-2-deoxy-1-thio-b-d-gluco-
pyranose 3,4,6-triacetate (52 mg, 0.14 mmol) and K2CO3 (200 mg,
1.4 mmol) were mixed in THF (5 mL). The mixture was stirred at room
temperature for 3 days. The solvent was removed and the residue was
dissolved with dichloromethane. After washing with brine and water and
drying with MgSO4, the solvent was removed under reduced pressure.
Gradient column chromatography firstly by using ethyl acetate to
remove an excess of 2-acetamido-2-deoxy-1-thio-b-d-glucopyranose 3,4,6-
triacetate, then ethyl acetate/methanol (10:1, v/v) to give TVFVBN-S-
NHAc as a yellow solid after precipitation from methanol (40 mg, 80%).
1H NMR (500 MHz, CDCl3): d=8.11 (d, J=8 Hz, 2H), 8.08 (d, J=
8.5 Hz, 1H), 7.91 (d, J=8 Hz, 1H), 7.69 (m, 4H), 7.56–7.46 (m, 7H),
7.40–7.36 (m, 2H), 7.25–7.17 (m, 5H), 5.57–5.51 (m, 2H), 5.14–5.01 (m,
4H), 4.47–4.43 (m, 2H), 4.18–3.99 (m, 6H), 3.58–3.54 (m, 2H), 2.58–2.52
(m, 4H), 2.00–1.98 (m, 22H), 1.87 (m, 6H), 1.43–1.40 (m, 4H), 1.17–1.11
(m, 8H), 0.69–0.66 ppm (m, 4H); 13C NMR (125 MHz, CDCl3): d=
171.04, 170.63, 170.01, 169.29, 167.69, 154.16, 151.42, 140.24, 134.99,
128.00, 127.01, 126.42, 125.22, 123.14, 121.63, 120.93, 84.41, 75.80, 73.87,
68.49, 62.31, 54.97, 53.27, 40.47, 33.39, 29.80, 29.37, 29.28, 28.38, 28.27,
23.46, 23.20, 20.69, 20.62 ppm; MS (MALDI-TOF): m/z: calcd for
7-{(E)-2-[4-(Benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6-bromohexyl)-
9H-fluorene-2-carbaldehyde (9): Diethyl [4-(benzo[d]thiazol-2-yl)phe-
nyl]methylphosphonate (3) (200 mg, 0.55 mmol) and 9,9-bis(6-bromohex-
yl)-9H-fluorene-2,7-dicarbaldehyde (8) (274 mg, 0.50 mmol) were dis-
solved in freshly distilled THF (10 mL) under an argon atmosphere. The
solution was cooled to À108C and a suspension of potassium tert-butox-
ide (140 mg, 1.24 mmol) in dry THF (5 mL) was added dropwise. The re-
action was kept at À108C for 4 h and then quenched with water. The sol-
vent of the mixture was removed by evaporation. After extraction with
dichloromethane, the organic layer was washed with brine and water,
dried with MgSO4, and the solvent was removed under reduced pressure.
The residue was columned with dichloromethane/hexane (1:1, v/v) to
give 9 as a yellow solid (132 mg, 35%). 1H NMR (500 MHz, CDCl3): d=
10.07 (s, 1H), 8.13–8.08 (m, 3H), 7.92 (d, J=8 Hz, 1H), 7.88–7.83 (m,
3H), 7.78 (d, J=7.5 Hz, 1H), 7.69 (d, J=8.5 Hz, 2H), 7.60 (d, J=8 Hz,
1H), 7.54–7.49 (m, 2H), 7.40 (t, J=8 Hz, 1H), 7.34 (d, J=16.5 Hz, 1H),
7.26 (d, J=16.5 Hz, 1H), 3.27 (t, J=7 Hz, 4H), 2.08–2.06 (m, 4H), 2.05–
1.68 (m, 4H), 1.63–1.08 (m, 8H), 0.65–0.59 ppm (m, 4H); 13C NMR
C
228H258N10O48S9: 4193.56 [M+]; found: 4193.34.
N-Tris{4,4’,4’’-[(1E)-2-(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-
9,9-bis(6-2-amido-2-deoxy-1-thio-b-d-glucopyranose-hexyl)-9H-fluoren-7-
yl)vinyl]phenyl}phenylnamine (TVFVBN-S-NH2): N-Tris{4,4’,4’’-[(1E)-2-
(2-{(E)-2-[4-(benzo[d]thiazol-2-yl)phenyl]vinyl}-9,9-bis(6–2-acetamido-2-
deoxy-1-thio-b-d-glucopyranose 3,4,6-triacetate-hexyl)-9H-fluoren-7-yl)-
vinyl]phenyl}phenylnamine (TVFVBN-S-NHAc) (20 mg, 0.0047 mmol)
and hydrazine monohydrate (1 mL) were heated at 1208C in a sealed
tube for 2 days. After cooling down to room temperature, the reaction
mixture was purified by dialysis against Mill-Q water by using a 3.5 kDa
molecular weight cut-off dialysis membrane for 3 days. It was then
lyophilized to give TVFVBN-S-NH2 as a yellow solid (14 mg, 94%).
1H NMR (500 MHz, DMSO): d=8.10 (d, J=7.5 Hz, 1H), 8.01 (d, J=
7.5 Hz, 1H), 7.49 (d, J=7 Hz, 2H), 7.65–7.60 (m, 4H), 7.52 (t, J=7.5 Hz,
Chem. Eur. J. 2012, 00, 0 – 0
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Y. H. Lai, B. Liu et al.
1H), 7.45 (t, J=8 Hz, 1H), 7.35 (d, J=6.5 Hz, 2H), 7.15 (m, 8H), 6.81
(m, 2H), 4.98–4.94 (m, 2H), 4.45–4.10 (m, 4H), 3.62–3.60 (m, 2H), 3.06–
2.83 (m, 14H), 2.41–2.40 (m, 4H), 1.82–1.80 (m, 4H), 1.23 (m, 4H), 0.99–
0.91 (m, 8H), 0.40 ppm (m, 4H).
Acknowledgements
The authors are grateful to the Singapore National Research Foundation
(R279-000-323-281), the Temasek Defence Systems Institute (R279-000-
305-232/422/592) and the Institute of Materials Research and Engineer-
ing (IMRE/11-1C0213) for financial support.
Synthesis of TVFVBN-S-NH2FA : The conjugation of TVFVBN-S-NH2
with folic acid was carried out through a DCC/NHS coupling reaction. In
brief, folic acid (1 mmol) in DMSO (20 mL) was activated by using DCC
(1.2 mmol) and NHS (2 mmol) at 508C for 6 h. After the reaction, the in-
soluble salt was filtered off. The resulting folate-NHS solution (2 mL, con-
taining 1ꢁ10À4 mmol of folate-NHS) was added into TVFVBN-S-NH2
(3 mg, 1ꢁ10À3 mmol) in DMSO (1 mL), together with a catalytic amount
of pyridine. The reaction mixture was stirred overnight at room tempera-
ture. The resulting solution was dialyzed by using a 3.5 kDa molecular
weight cut-off dialysis membrane for 2 days to eliminate the unreacted
folate-NHS. TVFVBN-S-NH2FA was finally collected after lyophilisation
in a quantitive yield.
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fetal bovine serum and 1% penicillin-streptomycin at 378C in a humidi-
fied environment containing 5% CO2. Before experiments, the cells were
precultured until confluence was reached.
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Cell viability: MTT assays were performed to assess the metabolic activi-
ty of MCF-7 breast cancer cells. MCF-7 cells were seeded in 96-well
plates (Costar, IL, USA) at an intensity of 2ꢁ104 cellsmLÀ1. After 48 h
incubation, the medium was replaced by TVFNBN-S-NH2 and
TVFNBN-S-NH2FA solutions at concentrations of 1 and 3 mm, and the
cells were then incubated for 24, 48 and 72 h, respectively. After the des-
ignated time intervals, the wells were washed twice with 1ꢁPBS buffer
and freshly prepared MTT (100 mL, 0.5 mgmLÀ1) solution in culture
medium was added into each well. The MTT medium solution was care-
fully removed after 3 h incubation in the incubator.
Isopropanol (100 mL) was then added into each well and the plate was
gently shaken for 10 min at room temperature to dissolve all the precipi-
tate formed. The absorbance of MTT at 570 nm was monitored by the
microplate reader (Genios Tecan). Cell viability was expressed by the
ratio of the absorbance of the cells incubated with TVFNBN-S-NH2 and
TVFNBN-S-NH2FA solutions to that of the cells incubated with culture
medium only.
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One- and two-photon fluorescence imaging: MCF-7 cells and NIH/3T3
cells were cultured in a chamber (LAB-TEK, Chambered Coverglass
System) at 378C for qualitative study. After 80% confluence, the
medium was removed and the adherent cells were washed twice with 1ꢁ
PBS buffer. TVFNBN-S-NH2 and TVFNBN-S-NH2FA solutions (0.8 mL,
1 mm) were then added to the chambers. After incubation for 2 h, cells
were washed three times with 1ꢁPBS buffer and then fixed by 75% eth-
anol for 20 min, which was further washed with 1ꢁPBS buffer twice. The
nuclei were stained with propidium iodide (PI) for 40 min. The cells were
then imaged by CLSM (Zeiss LSM 410, Jena, Germany) with imaging
software (Fluoview FV1000). TVFNBN-S-NH2 and TVFNBN-S-NH2FA
were excited at 405 nm (3% laser power), and the fluorescence was col-
lected from 505–560 nm. PI was excited at 543 nm and the fluorescence
was collected at 575–635 nm. To study the competitive effect of free folic
acid on the endocytosis of TVFVBN-S-NH2FA, 50 mgmLÀ1 free folic acid
in medium was first added into the chamber. After 30 min, the medium
was replaced by 1 mm TVFVBN-S-NH2FA and incubated for 2 h before
imaging.
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Received: March 13, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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These are not the final page numbers!

Y. H. Lai, B. Liu et al.
Self-Assembly
G. Wang, X. Zhang, J. Geng, K. Li,
D. Ding, K.-Y. Pu, L. Cai, Y.-H. Lai,*
B. Liu* ......................... &&&& — &&&&
Glycosylated Star-Shaped Conjugated
Oligomers for Targeted Two-Photon
Fluorescence Imaging
Biological imaging: A star-shaped two-
photon absorbing material (see
scheme) with folic acid functionaliza-
tion has been synthesized for targeted
two-photon fluorescence imaging of
MCF-7 cancer cells.
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www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!