Star-shaped glycosylated conjugated oligomer for two-photon fluorescence imaging of live cells

Article abstract of DOI:10.1021/cm201377u

A star-shaped glycosylated conjugated oligomer, 4,4′,4″-tris(4- (2-(4-(benzo[d]thiazol-2-yl)phenyl)-9,9′-bis(6-thiol-β-d-glucose) -hexyl)-fluoren-7-yl)phenylamine (TFBS), is synthesized for two-photon fluorescence imaging of live cells. The high density o

Full text of DOI:10.1021/cm201377u

ARTICLE
pubs.acs.org/cm
Star-Shaped Glycosylated Conjugated Oligomer for Two-Photon
Fluorescence Imaging of Live Cells
Guan Wang,†,‡ Kan-Yi Pu,† Xinhai Zhang,§ Kai Li,† Long Wang,† Liping Cai,† Dan Ding,† Yee-Hing Lai,*,‡
and Bin Liu*,†,§
†Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576
‡Department of Chemistry, 3 Science Drive 3, National University of Singapore, Singapore 117543
§Institute of Materials Research and Engineering, Singapore 117602
S Supporting Information
b
ABSTRACT: A star-shaped glycosylated conjugated oligomer, 4,40,400-tris(4-(2-(4-(benzo-
[d]thiazol-2-yl)phenyl)-9,90-bis(6-thiol-β-D-glucose)-hexyl)-fluoren-7-yl)phenylamine (TFBS), is
synthesized for two-photon fluorescence imaging of live cells. The high density of hydrophilic sugar
side groups induces self-assembly of TFBS into nanoparticles in water with an average diameter of
61 nm. Because of the self-assembled nanostructure, TFBS has a higher quantum yield in water (Φ =
0.10), compared to its cationic counterpart, 4,40,400-tris(4-(2-(4-(benzo[d]thiazol-2-yl)phenyl)-9,90-
bis(6-N,N,N-trimethylammonium)-hexyl)-fluoren-7-yl)phenylamine (TFBC) (Φ = 0.03). In addi-
tion, TFBS has a large TPA cross section (δmax) of∼1200 GM at 740 nm in aqueous media, which is
significantly higher than that for TFBC. TFBS can be effectively internalized by the human cervical
cancer cell line and accumulates in the cytoplasm, allowing for live cell two-photon fluorescence
imaging upon 800-nm excitation. TFBS has also shown low cytotoxicity, which is essential for in vitro
and in vivo cellular imaging and other clinical applications. This study demonstrates the significant advantages of glycosylation in molecular
engineering of water-soluble fluorescent molecules for two-photon fluorescence imaging applications.
KEYWORDS: star-shaped, water-soluble conjugated oligomer, two-photon fluorescence imaging, live cells, glucose
’ INTRODUCTION
soluble in organic solvents, which make them unsuitable for
TPM imaging or other biological applications.
The development of organic materials with large two-photon
absorption (TPA) cross sections (δ) in the near-infrared (NIR)
regions (700ꢀ1000 nm) has attracted intensive attention in the
past two decades, because of their vital applications in three-
dimensional (3D) optical data storage, optical power limiting,
two-photon microscopy (TPM), and photo dynamic therapy.1ꢀ3
TPM is a powerful technique for noninvasive live cell imaging,4
because the NIR excitation enables deeper penetration, less photo
bleaching, and higher spatial resolution, compared to that for one-
photon microscopy (OPM).5 To obtain a high signal-to-noise
ratio in TPM, materials with large TPA action cross sections
(defined as ηδ, where η is the fluorescence quantum yield) are
essential. The majority of currently used fluorescent imaging
reagents are mainly designed for OPM and are not ideal for TPM,
which is characterized by small δ values and low brightness.6
Dipolar or quadrupolar models with donorꢀbridgeꢀdonor
(D-π-D), donorꢀbridgeꢀacceptor (D-π-A), and donorꢀ
acceptorꢀdonor (D-A-D) architectures have been proven effec-
tive in yielding large δ values.1ꢀ3 In comparison with their linear
counterparts, multibranched octupolar chromophores are more
promising to yield large δ values, because of the excitonic
coupling between each branch.7 By taking this advantage, several
octupolar TPA chromophores have been designed and synthe-
sized. Although large δ values of more than ∼2000 GM have
been obtained among these chromophores,7ꢀ12 they are only
Water-soluble π-conjugated TPA molecules generally show
significantly decreased δ in water, relative to that for their neutral
counterparts in organic solvents.13 In addition, their η value in
water is also low, because of fierce interaction between polar water
molecules and chromophores in their excited states through
nonradiative decay.14 Until now, very limited efforts have been
made to address these problems. Bazan et al. have reported that
the addition of sodium odecyl sulfate (SDS) surfactant into
water-soluble paracyclophane chromophores could increase
the δ and η values, because of micelle formation.13 Similarly,
Jen et al.12 reported that the encapsulation of TPA fluorophores
with an amphiphilic block copolymer, poly(methacrylic acid)-
block-polystyrene (PMAA-b-PS), led to increased δ values of the
fluorophore in micelles. The micellization-enhanced TPA cross
sections are associated with the incorporation of optically active
units within the hydrophobic microenvironment in the interior
of micelles.
On the other hand, glycolipids and carbohydrate compounds
have been reported to self-assemble into regular layers or spherical
micelles in aqueous media.15 It occurs to us that the attachment
Received: May 16, 2011
Revised:
September 1, 2011
Published: September 26, 2011
r
2011 American Chemical Society
4428
dx.doi.org/10.1021/cm201377u Chem. Mater. 2011, 23, 4428–4434
|

Chemistry of Materials
ARTICLE
1
ethanol to afford 1 as yellow needle-shaped crystals (2.8 g, 80%). H
NMR (CDCl3, 500 MHz, ppm): δ 8.07 (d, 1H, J = 8.2 Hz), 7.94 (d, 2H,
J = 8.5 Hz), 7.89 (d, 1H, J = 7.9 Hz), 7.61 (d, 2H, J = 8.6 Hz), 7.50 (t, 1H,
J = 7.7 Hz), 7.39 (t, 1H, J = 7.6 Hz). 13C NMR (CDCl3, 125 MHz, ppm):
δ 166.68, 154.08, 135.06, 132.55, 132.23, 128.91, 126.51, 125.46, 125.44,
123.34, 121.67. HRMS (EI, m/z): calcd 288.9561 (for C13H8N179Br132S1)
and 290.9540 (for C13H8N181Br132S1); found: 288.9566, 290.9548.
Synthesis of 2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)phenyl)benzo[d]thiazole (2). 2-(4-Bromophenyl)benzo[d]-
thiazole (2.4 g, 8.3 mmol), bis(pinacolato)diborane (2.5 g, 10 mmol),
KOAc (2.9 g, 29 mmol), and dioxane (60 mL) were mixed together in a
100-mL flask. After degassing, [Pd(dppf)Cl2] (250 mg, dppf = 1,10-
bis(diphenylphosphanyl)ferrocene) was added. The reaction mixture
was kept at 85 °C overnight, which was then cooled to room tempera-
ture. After the organic solvent was removed, the residual was dissolved in
dichloromethane and washed with water. After drying with MgSO4, the
solvent was distilled out. The crude product was purified by chroma-
tography using ethylacetate/hexanes (1:9) as eluent to give 2 as a white
solid (2.4 g, 86%). 1H NMR (CDCl3, 500 MHz, ppm): δ 8.02 (dd, 4H,
J = 8.1 Hz, J = 79.7 Hz), 7.99 (dd, 2H, J = 8.0 Hz, J = 95.8 Hz), 7.49
(t, 1H, J = 7.7 Hz), 7.38 (t, 1H, J = 7.6 Hz), 1.37 (s, 12H). 13C NMR
(CDCl3, 125 MHz, ppm): δ 167.97, 154.18, 135.87, 135.39, 135.16,
126.69, 126.38, 125.33, 123.36, 121.65, 84.11, 25.04. HRMS (EI, m/z):
calcd for C19H20O2N111B132S1, 337.1308; found, 337.1312.
Synthesis of 2-(4-(2-Bromo-9,90-bis(6-bromohexyl)-fluoren-
7-yl)phenyl)benzo[d]thiazole (4). 2-(4-(4,4,5,5-Tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)benzo[d]thiazole (216 mg, 0.64 mmol)
and 2,7-dibromo-9,90-bis(6-bromohexyl)fluorene (1.3 g, 1.9 mmol),
Pd(PPh3)4 (37 mg, 0.02 mmol), tetrabutylammonium bromide (cat.
amount), toluene (8 mL), 2 M Na2CO3 aqueous solution (1 mL)
were mixed together ina 100-mLround-bottomflask, and themixture was
then degassed. The reaction was kept at 100 οC overnight before it
was cooled to room temperature. Dichloromethane was added for
extraction, and the organic layer was washed successively with brine
and water, and then dried with MgSO4. After solvent removal, the
crude product was purified with gradient column chromatography
using ethyl acetate/hexane (1:100) and then ethyl acetate/hexane
(1:9) to afford 4 as a white solid (166 mg, 50%). 1H NMR (CDCl3,
500 MHz, ppm): δ 8.21 (d, 2H, J = 8.3 Hz), 8.11 (d, 1H, J = 8.1 Hz),
7.93 (d, 1H, J = 7.9 Hz), 7.80 (d, 2H, J = 8.3 Hz), 7.76 (d, 1H, J = 7.9
Hz), 7.66 (dd, 1H, J = 1.4 Hz, J = 7.9 Hz), 7.60 (m, 2H), 7.51 (m, 4H),
7.41 (t, 1H, J = 7.6 Hz), 3.28 (t, 4H, J = 6.8 Hz), 2.06ꢀ1.96 (m, 4H),
1.70ꢀ1.64 (m, 4H), 1.25ꢀ1.07 (m, 8H), 0.70ꢀ0.64 (m, 4H). 13C
NMR (CDCl3, 125 MHz, ppm): δ 167.35, 154.08, 152.78, 150.71,
143.52, 139.83, 139.43, 139.18, 134.90, 132.34, 130.10, 127.88,
127.45, 126.23, 126.00, 125.07, 123.06, 121.49, 121.27, 121.20,
121.04, 120.20, 55.30, 39.93, 33.76, 32.46, 28.82, 27.59, 23.43. HRMS
(EI, m/z): calcd 777.0275 (for C38H38N179Br332S1), 781.0234 (for
C38H38N179Br181Br232S1), and 779.0255 (for C38H38N179Br281Br132S1);
found: 777.0255, 781.0214, 779.0223.
Figure 1. Chemical structures of TFBN, TFBC, TFBS-OAc, and TFBS.
of glycose groups could be an effective strategy to develop
amphiphilic multibranched conjugated molecules with intrinsic
self-assembly properties. Unlike the micelle-assisted systems, this
molecular design could integrate the hydrophilic terminals and
the backbone to yield robust water-soluble TPA materials for
two-photon fluorescence imaging. Although several examples of
metal complexes have been developed as probes for two-photon
fluorescence imaging of live cells,16ꢀ18 these probes are generally
highly toxic to cells and possess small δ values (<100 GM). To
the best of our knowledge, there are only a limited number of
organic fluorophores designed for two-photon imaging of live
cells,19ꢀ22 of which the δ values are also very small (<20 GM).
In this contribution, we report a star-shaped glycosylated con-
jugated oligomer (TFBS) with high TPA cross sections in aqueous
solution for two-photon fluorescence imaging of live cells. A cationic
conjugated oligomer (TFBC) as the counterpart of TFBS has
also been synthesized to investigate how the terminal group
of conjugated oligomers affects their self-assembly behavior in
aqueous media and, in turn, determines their linear and nonlinear
properties. The chemical structures of TFBC and TFBS are
shown in Figure 1. To fulfill a good charge transfer feature from
center to terminals, triphenylamine was used as the donor, while
fluorene was chosen as the bridge, with phenylbenzo[d]thiazole
as the terminal acceptor.23
’ EXPERIMENTAL SECTION
Materials and Instruments. Chemicals and reagents were pur-
chased from Aldrich Chemical Co. unless otherwise stated. NMR
spectra were collected on a Bruker Model ACF 300 or Model AMX
500 spectrometer with chloroform-d as the solvent (unless otherwise
stated) and tetramethylsilane as the internal standard. Elemental analysis
were carried out by the Microanalysis Laboratory of the National
University of Singapore. UVꢀvis spectra were recorded on a Shimadzu
Model UV-1700 spectrometer. Fluorescence measurements were carried
out on a PerkinꢀElmer LS-55 instrument equipped with a xenon-lamp
excitation source and a Hamamatsu (Japan) 928 photomultiplier tube
(PMT), using 90ο angle detection for solution samples. Quantum yields
were measured using quinine sulfate as the standard, with a quantum
yield of 55% in H2SO4 (0.1 M).
Synthesis of 4,40,400-Tris(pinacolatoborane)phenylamine (5).
Tris(4-bromophenyl)amine (1.93 g, 4 mmol), bis(pinacolato)diborane
(3.75 g, 15 mmol), KOAc (7.0 g, 70 mmol), and dioxane (60 mL) were
mixed together in a 100-mL flask. After degassing, [Pd(dppf)Cl2] (0.5 g,
dppf = 1,10-bis(diphenylphosphanyl)ferrocene) was added. The reac-
tion mixture was kept at 85 °C overnight before it was cooled to room
temperature. The organic solvent was removed, and the residual was
dissolved in dichloromethane and washed with water. After drying the
organic layer with MgSO4, the solvent was removed. The crude product
was purified by flash chromatography, using hexane and dichloro-
methane (2:1) as an eluent, to give 5 as a white solid (4.2 g, 59%). 1H
NMR (CDCl3, 500 MHz, ppm): δ 7.68 (d, 6H, J = 8.3 Hz), 7.07 (d, 6H,
J = 8.4 Hz), 1.34 (s, 36H). 13C NMR (CDCl3, 125 MHz, ppm): δ 149.80,
135.94, 123.50, 83.70, 24.90.
Synthesis of 2-(4-bromophenyl)benzo[d]thiazole (1). A
mixture of 4-bromobenzaldehyde (3.7 g, 20 mmol), 2-aminothiophenol
(2.5 g, 20 mmol), and N-methyl-2-pyrrolidone (NMP) (30 mL) was
heated in an oil bath at 110 °C for 72 h, which was then poured into
1:1 ethanol/water. The precipitates were collected, recrystallized from
4429
dx.doi.org/10.1021/cm201377u |Chem. Mater. 2011, 23, 4428–4434

Chemistry of Materials
ARTICLE
Synthesis of 4,40,400-Tris(4-(2-(4-(benzo[d]thiazol-2-yl)-
phenyl)-9,90-bis(6-bromohexyl)-fluoren-7-yl)phenylamine
(TFBN). 2-(4-(2-Bromo-9,90-bis(6-bromohexyl)-fluoren-7-yl)phenyl)-
benzo[d]thiazole (395 mg, 0.5 mmol) and 4,40,400-tris(pinacolatoborane)-
phenylamine (88 mg, 0.14 mmol), Pd(PPh3)4 (37 mg, 3% mmol),
tetrabutylammonium bromide (cat. amount), toluene (5 mL), 2 M
Na2CO3 aqueous solution (1 mL) were mixed together in a 50-mL
Schlenk flask, and the mixture was degassed. The reaction was kept at
100 °C for 48 h before it was cooled to room temperature. Dichloro-
methane was added for extraction; the organic layer was washed successively
with brine and water, and then dried with MgSO4. After the solvent
was removed under reduced pressure, the crude product was purified
by gradient column chromatography, using toluene/hexanes (5:1),
followed by toluene to afford TFBN as a yellowish green solid (60 mg,
18%). 1H NMR (CDCl3, 500 MHz, ppm): δ 8.21 (d, 6H, J = 8.0 Hz),
8.11 (d, 3H, J = 8.0 Hz), 7.93 (d, 3H, J = 7.5 Hz), 7.83 (m, 12H), 7.65 (m,
18H), 7.52 (t, 3H, J = 7.5 Hz), 7.41 (d, 3H, J = 7.5 Hz), 7.34 (d, 6H,
J = 8.0 Hz), 3.28 (t, 12H, J = 6.8 Hz), 2.11ꢀ2.08 (m,12H), 1.69ꢀ
1.65 (m,12H), 1.24ꢀ1.10 (m, 24H), 0.89ꢀ0.77 (m, 12H). 13C NMR
(CDCl3, 125 MHz, ppm): δ151.51, 146.84, 144.07, 140.92, 139.80, 139.64,
138.92, 135.99, 135.10, 132.42, 129.07, 128.26, 128.09, 128.03, 127.67,
126.43, 126.32, 125.87, 125.34, 125.26, 124.56, 123.24, 121.67, 121.29,
120.92, 120.38, 120.28, 55.30, 40.35, 34.00, 32.67, 29.10, 27.80, 23.69. MS
(MALDI-TOF, m/z): cald for C132H126Br6N4S3, 2344.4; found, 2344.3.
Synthesis of 4,40,400-Tris(4-(2-(4-(benzo[d]thiazol-2-yl)-
phenyl)-9,90-bis(6-N,N,N-trimethylammonium)-hexyl)-fluoren-
7-yl)phenylamine (TFBC). Trimethylamine (2 mL) was added
dropwise to a solution of TFBN (30 mg) in THF (5 mL) at ꢀ78 °C.
The mixture was stirred for 12 h and then allowed to warm to room
temperature. The precipitate was redissolved via the addition of
methanol (5 mL). After the mixture was cooled to ꢀ78 °C, additional
trimethylamine (2 mL) was added, and the mixture was stirred at room
temperature for 24 h. After solvent removal, acetone was added to
precipitate the quaternized salt TFBC as yellow powders (27 mg, 86%).
1H NMR (MeOD, 500 MHz, ppm): δ 8.11ꢀ7.25 (m, 18H), 3.19 (m,
4H), 3.02 (s, 18H), 2.20 (m, 4H), 1.53 (m, 4H), 1.15 (m, 8H), 0.76 (m,
4H). 13C NMR (MeOD, 125 MHz, ppm): δ 167.86, 153.84, 146.83,
141.05, 139.56, 134.72, 132.04, 127.87, 127.75, 127.25, 126.49, 125.98,
125.40, 124.48, 122.52, 121.66, 121.02, 120.51, 66.27, 55.37, 52.14,
39.47, 28.83, 25.37, 23.53, 22.29. MS (MALDI-TOF, m/z): [M ꢀ 2Br]+
cald for C150H180Br4N10S3, 2538.0; found, 2537.8.
Synthesis of 4,40,400-Tris(4-(2-(4-(benzo[d]thiazol-2-yl)-
phenyl)-9,90-bis(6-thiol-β-D-glucose)-hexyl)-fluoren-7-yl)-
phenylamine (TFBS). TFBS-OAc (30 mg, 0.007 mmol) was dis-
solved in 2 mL of dry dichloromethane and 4 mL of anhydrous methanol
under nitrogen, 0.3 mL of 0.5 M NaOMe in methanol was subsequently
added into the solution. The resulting mixture was allowed to react
overnight under vigorous stirring at room temperature. After removing
the solvent under reduced pressure, water was added to the residue, and
the solution was dialyzed against Mill-Q water for 12 h before it was
lyophilized to give TFBS as yellow solid (21 mg, 93%). 1H NMR
(DMSO, 500 MHz, ppm): δ 8.22ꢀ7.27 (m, 18H), 4.98 (m, 4H), 4.39
(m, 2H), 4.16 (m, 2H), 3.61 (m, 2H), 3.36 (m, 2H), 3.05 (m, 4H), 2.17
(m, 4H), 1.34 (m, 16H), 0.64 (m, 4H). 13C NMR (DMSO, 125 MHz,
ppm): δ 167.47, 154.17, 151.92, 146.75, 143.69, 141.03, 138.26, 134.97,
132.13, 128.32, 128.10, 127.19, 126.01, 124.73, 123.36, 122.84, 121.01,
85.51, 81.37, 78.65, 73.53, 70.48, 61.65, 56.51, 39.85, 39.69, 39.52, 29.70,
28.58, 19.03. MS (MALDI-TOF, m/z): [M+H]+ cald for C168H12N4O30S9,
3034.1; found, 3034.1.
Cell Culture and Incubation. Hela cells and NIH-3T3 fibroblast
cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium
containing 10% fetal bovine serum and 1% penicillinꢀstreptomycin at
37 οC in a humidified environment containing 5% CO2. Before experi-
ments, the cells were precultured until confluence was reached.
Cell Viability. MTT assays were performed to assess the metabolic
activity of NIH-3T3 fibroblast cells. NIH-3T3 cells were seeded in 96-well
plates (Costar, Vernon Hills, IL, USA) at an intensity of 2 ꢁ 104 cells/mL.
After 48 h of incubation, the medium was replaced by TFBS solutions at
the concentrations of 0.5 and 1 μM, and the cells were then incubated
for 24, 48, and 72 h, respectively. After the designated time intervals,
the wells were washed twice with 1ꢁ PBS buffer and freshly prepared
MTT (100 μL, 0.5 mg/mL) solution in culture medium was added into
each well. The MTT medium solution was carefully removed after 3 h of
incubation in the incubator.
Isopropanol (100 μL) was then added into each well and the plate
was gently shaken for 10 min at room temperature to dissolve all the
precipitate 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 TFBS solutions to
that of the cells incubated with culture medium only.
Two-Photon Fluorescence Imaging. Hela cells were cultured
in chamber (LAB-TEK, Chambered Coverglass System) at 37 °C for
qualitative study. After 80% confluence, the medium was removed and
the adherent cells were washed twice with 1ꢁ PBS buffer. TFBS solution
(0.8 mL, 0.5 μM) was then added to the chamber. After incubation for
2 h, cells were washed three times with 1ꢁ PBS buffer. The cells were
then imaged with multiphoton microscopes (Leica TCS SP5 X) with a
Leica HCX PL APO 63x/1.20 W CORR CS objective lens. The probe
was excited with a mode-locked Ti-Sapphire laser source (Chameleon
Ultra II) at a wavelength of 800 nm with an output power of ∼4 W,
which corresponded to an average power of 10 mW in the focal plane.
Internal PMTs were used to collect the signals at 500ꢀ600 nm in an
8-bit unsigned 1024 ꢁ 1024 pixels at a scan speed of 400 Hz.
Synthesis of 4,40,400-Tris(4-(2-(4-(benzo[d]thiazol-2-yl)-
phenyl)-9,90-bis(6-thiol-β-D-glucose tetraacetate)-hexyl)-
fluoren-7-yl)phenylamine (TFBS-OAc). TFBN (30 mg, 0.013 mmol),
1-thiol-β-D-glucose tetraacetate (45 mg, 0.123 mmol), and potassium
carbonate (130 mg, 1.23 mmol) were placed in a 25-mL round-bottom
flask under nitrogen atmosphere. Degassed THF (7 mL) was added to the
reaction flask, and the mixture was stirred at room temperature for 2 days.
After solvent removal, the residue was dissolved in dichloromethane
(15 mL), washed with water, and dried over anhydrous MgSO4. After
solvent removal, the crude product was purified by column chromatography
on silica gel first with hexane/EtOAc (1:1) to remove excess glucose
tetraacetate, which was followed by hexane/EtOAc (1:2) to give the target
compound as yellow solid after precipitation in 20 mL of methanol (35 mg,
67%). 1H NMR (CDCl3, 500 MHz, ppm): δ 8.22 (d, 6H, J = 8 Hz), 8.11
(d, 3H, J= 8 Hz), 7.94 (d, 3H, J= 8 Hz), 7.84 (m, 12H), 7.67 (m, 18H), 7.52
(t, 3H, J = 8 Hz), 7.41 (t, 3H, J = 7.5 Hz), 7.33 (d, 6H, J = 8.5 Hz), 5.17
(m, 6H), 5.03 (t, 6H, J = 10 Hz), 4.97 (t, 6H, J = 9.5 Hz), 4.39 (m, 6H), 4.20
(m, 6H), 4.07 (m, 6H), 3.64 (m, 6H), 2.57 (m, 12H), 2.09 (m, 84H), 1.70 (m,
12H), 1.18 (m, 24H), 0.73 (m, 12H). 13C NMR (CDCl3, 125 MHz, ppm):
δ 170.16, 169.38, 169.32, 167.71, 154.22, 151.51, 146.79, 144.07, 140.91,
139.77, 138.86, 135.07, 132.39, 128.09, 127.99, 127.66, 126.42, 125.25, 124.50,
123.21, 120.24, 83.57, 83.54, 75.80, 73.89, 69.88, 68.33, 62.12, 55.32, 40.52,
32.63, 29.88, 29.57, 29.46, 28.47, 23.78, 20.71, 20.68, 20.61, 20.58.
’ RESULTS AND DISCUSSION
The synthetic route toward conjugated oligomers TFBN, TFBC,
and TFBS is depicted in Scheme 1. 2,7-Dibromo-9,90-bis(6-
bromohexyl)fluorene was synthesized according to our previous
reports.24ꢀ28 Coupling 1-amino-2-thiophenol with 4-bromo-
benzaldehyde in N-methyl-2-pyrrolidone (NMP) at 110 °C for
3 days afforded 2-(4-bromophenyl)benzo[d]thiazole (1) in 80%
yield.29 1 was then converted to boronic ester 1-benzothiazole-4-
phenylpinacolatoborane (2) in 86% yield upon heating with
bis(pinacolato)diborane and KOAc in anhydrous dioxane at
4430
dx.doi.org/10.1021/cm201377u |Chem. Mater. 2011, 23, 4428–4434

Chemistry of Materials
ARTICLE
90 °C.30 Coupling between 1 equiv of 2 and 2,7-dibromo-9,90-
bis(6-bromohexyl)fluorene under Suzuki coupling reaction con-
dition afforded 2-(4-(2-bromo-9,90-bis(6-bromohexyl)-fluoren-
7-yl)phenyl)benzo[d]thiazole (4) in 50% yield. Further Suzuki
coupling reaction between 4 and 4,40,400-tris(pinacolatoborane)-
phenylamine (5) at a molar ratio of 3.6:1 afforded the desired
neutral oligomer (TFBN) in 18% yield. The correct chemical
structure of TFBN was affirmed by NMR spectroscopy (see
Figure S1 in the Supporting Information) and MALDI-TOF
mass spectroscopy. The cationic derivative TFBC was obtained
in 86% yield by quaternization of TFBN, using trimethylamine in
THF/H2O.31 The 1H NMR spectrum of TFBC shows a single
broad peak at 3.02 ppm, which is assigned to the methyl protons
in ꢀCH2CH2N(CH3)3. Integration of the methyl peak area to
that of the other alkyl peaks is 9:12, indicating 100% quaterniza-
tion for TFBN (see Figure S2 in the Supporting Information).
A post-functionalization approach was adopted to synthesize
the glucose-substituted oligomer TFBS.32,33 Coupling TFBN
with 1-thio-β-D-glucose tetraacetate at a molar ratio of 1:9 in THF
at room temperature afforded acetylated precursor TFBS-OAc
in 67% yield. The characteristic single resonance peaks of acetyl
protons34 were observed at 2.01ꢀ1.96 ppm in the 1H NMR spec-
trum of TFBS-OAc (see Figure S3 in the Supporting Information),
indicating successful attachment of acetylated glucose onto TFBN.
Moreover, when one compares the NMR spectra between TFBN
and TFBS-OAc, the characteristic triplet for protons in ꢀCH2Br
was not observed in the spectrum of TFBS-OAc, indicating 100%
replacement of Br by acetylated glucose. After deacetylation of
TFBS-OAc in methanol/dichloromethane at room temperature
using NaOCH3, the glucose-substituted oligomer TFBS was obtained
in 93% yield after dialysis against Mill-Q water using a 3KDa
molecular weight cutoff membrane. The disappearance of char-
acteristic peaks of acetyl protons in 1H NMR spectrum of TFBS
(see Figure S4 in the Supporting Information) affirmed complete
deacetylation. In addition, the correct molecular mass of TFBS
was confirmed by MALDI-TOF mass spectroscopy (see Figure S5
in the Supporting Information).
Dynamic light scattering (DLS) was performed to probe the
self-assembly behaviors of TFBC and TFBS in water. Figure 2a
shows the DLS spectrum of 2.5 μM TFBS in water. The mean
hydrodynamic diameter was measured to be 61 nm, with a poly-
dispersity of 0.29. Similar to the previously reported micellization
behaviors of sugar-substituted molecules,35 the formation of nano-
particles for TFBS is due to the glucose substituents. In contrast,
no DLS signal was observed for 2.5 μM TFBC in water, revealing
that the cationic oligomer is well-dissolved at the molecular
level in solution.36 Similarly, there was no DLS signal observed
for 2.5 μM TFBS and TFBC in DMSO.
Scheme 1. Synthetic Route to Oligomers TFBN, TFBC, and
TFBS a
The AFM height image and the corresponding cross section
analysis of TFBS nanoparticles on silicon wafer are shown in
Figures 2b and 2c, respectively. Monolayer spherical nanoparti-
cles are observed with relatively uniform diameter and narrow
height distribution. This indicates ordered self-assembly of TFBS
in pure water, rather than random aggregation. For all the nano-
particles, an average diameter of 80 nm was determined by
statistical analysis and an average central height of 10 nm was
obtained by cross-sectional analysis. The smaller size in vertical
height is caused by collapse upon transforming the nanoparticles
from solution state into dry state, which rationalizes the larger
size of nanoparticles measured by AFM, compared to that by
DLS.37 It is expected that the self-assembly of TFBS in aqueous
solution would enhance the hydrophobicity of local environment
for the star-shaped backbone.
a Reagents and conditions: (i) NMP, 110 °C, 3 days; (ii) Bis(pinacolato)-
diborane, KOAc, Pd(dppf)Cl2, dioxane, 80 °C, overnight; (iii and iv)
Na2CO3, Pd(PPh3)4, toluene/H2O, 100 °C, overnight; (v) THF/H2O,
NMe3, 24 h; (vi) 1-thio-β-D-glucose tetraacetate, THF, K2CO3, RT,
2 days; and (vii) NaOMe, MeOH/DCM, RT, 12 h.
The solvent effect on the optical properties of the neutral
oligomer TFBN was investigated. Figure 3 shows the UVꢀvis
absorption and PL spectra of 2 μM TFBN in toluene, dichloro-
methane (DCM), and dimethylformamide (DMF). With increasing
Figure 2. (a) Hydrodynamic diameter of TFBS in water at [TFBS] = 2.5 μM. (b) AFM height image of TFBS nanoparticles. (c) Cross-sectional analysis
of TFBS nanoparticles.
4431
dx.doi.org/10.1021/cm201377u |Chem. Mater. 2011, 23, 4428–4434

Chemistry of Materials
ARTICLE
Figure 3. Ultraviolet-visible light (UVꢀvis) absorption and photo-
luminescence (PL) spectra of TFBN in toluene, DCM, and DMF at a
concentration of 2 μM (excited at λmax). The inset shows the fluores-
cence from solutions of TFBN in (A) toluene, (B) DCM, and (C) DMF
under a hand-held UV lamp with λmax = 365 nm.
solvent polarity, the maximum emission wavelength progres-
sively red-shifts from 440 nm in toluene to 540 nm in DCM and
640 nm in DMF. The solvent-dependent emission maximum
originates from the charge transfer character of the excited state
of TFBN, which is clearly understood by molecular orbital
simulation conducted on a single arm of TFBN. As shown in
Figure S6 in the Supporting Information, the highest occupied
molecular orbital (HOMO) energy level of the arm is mainly
delocalized over the triphenylamine and fluorene units, while the
lowest unoccupied molecular orbital (LUMO) is nearly localized
on the phenylbenzo[d]thiazole unit. The orbital patterns implicate
that the HOMOꢀLUMO transition for TFBN is accompanied
by charge transfer from the electron-donating triphenylamine to
the electron-deficient phenylbenzo[d]thiazole unit in the excited
state. The quantum yield of TFBN decreases from 0.86 in toluene to
0.69 in DCM and 0.10 in DMF, measured using quinine sulfate in
0.1 M H2SO4 as standard. The quenched fluorescence of TFBN
with increasing solvent polarity further proves its charge-transfer
excited state. As a result of the solvent-dependent emission, the
solution fluorescent color of TFBN is blue in toluene, green in
DCM, and orange in DMF (see the inset in Figure 3). On the
other hand, TFBN has the same absorption spectrum in all
solvents, which indicates that the ground state of TFBN is less
sensitive to solvent polarity, as both states are exposed to the
same local environment.37
The UVꢀvis absorption and PL spectra of TFBC and TFBS in
DMSO and pure water are shown in Figure 4. In DMSO, both
the absorption and PL spectra of TFBC and TFBS are almost
the same. The absorption and emission maxima are observed at
388 and 660 nm, respectively. Moreover, they have a similar
quantum yield of 0.09. In water, the absorption maxima for
TFBC and TFBS are observed at 384 and 388 nm, while the
emission maxima for TFBC and TFBS are observed at 575 and
533 nm, respectively. The quantum yields of TFBC and TFBS in
water are 0.03 and 0.10, respectively. Because TFBC and TFBS
share the same conjugated framework, both should show de-
creased fluorescence with increased solvent polarity, because of
the charge transfer character of their excited states. The higher
quantum yield of TFBS in water agrees with the DLS and AFM
results, which show that TFBS self-assembles into nanoparticles
in water. The nanoparticles should increase the hydrophobicity
in the local environment of TFBS, which leads to the higher
quantum yield, relative to that of TFBC.38 As such, the aqueous
Figure 4. (a) UVꢀvis absorption spectra and (b) PL spectra of TFBC
and TFBS in DMSO and water at a concentration of 2 μM (excited at
λmax). The inset in panel (b) shows the fluorescence from solutions of
TFBC (left) and TFBS (right) in water under a hand-held UV lamp with
λmax = 365 nm.
solution of TFBS shows much brighter fluorescence (see the inset
in Figure 4b), making it more suitable for optical applications.
The TPA spectra of TFBN, TFBC, and TFBS were collected
using a standard two photon-excited fluorescence (TPEF) tech-
nique with a femtosecond pulsed laser source.39,40 To avoid
the interference on TPEF by laser excitation, TPA spectra were
measured starting at the wavelength where each individual oligomer
has almost no emission. In addition, limited by the laser availability
in our experiments, TFBN in toluene was investigated in the
range of 640ꢀ810 nm; TFBC and TFBS in water were studied in
the range of 740ꢀ900 nm, and TFBS and TFBC in DMSO were
measured in the range of 800ꢀ970 nm.
The TPA spectrum of TFBN in toluene shows a maximum
TPA cross section (δmax) of 2494 GM at 680 nm (see Figure S7
in the Supporting Information). This value is higher than that for
the linear benzothiazole-containing counterparts (<1600 GM).41ꢀ43
On the other hand, compared to AF-350 reported by Tan et al.,44,45
TFBS has a larger TPA cross section than AF-350 (∼500 GM
vs 132 GM) at ∼790 nm, when both are measured using a
femtosecond pulsed laser. The large δ value of TFBN in toluene
is mainly ascribed to efficient intramolecular charge transfer
(ICT) from the center to peripheries in the octupolar structure.
In DMSO, the δmax values of TFBC and TFBS are observed at
800 nm (see Figure S8 in the Supporting Information), which are
435 and 444 GM, respectively. In water, TFBS shows a δmax value
of 1195 GM at 740 nm (see Figure 5), while the TPEF spectra
for TFBC are barely detectable under the same experimental
conditions. Note that the δ value of TFBS in water at 800 nm is
574 GM, which is also larger than that in DMSO (444 GM).
4432
dx.doi.org/10.1021/cm201377u |Chem. Mater. 2011, 23, 4428–4434

Chemistry of Materials
ARTICLE
Figure 6. (A) TPEF, (B) transmission, and (C) TPEF/transmission
overlapped images of live Hela cells upon incubation with TFBS for 2 h
at a concentration of 0.5 μM. Images shown in panels AꢀC have the
same scale bar.
that TFBS is mainly located in the cytoplasm area. To identify its
intracellular localization, we costained Hela cells with TFBS and
an endosomal tracker (LysoTracker Red).50 However, the lack of
overlap between LysoTracker Red and TFBS indicates that
TFBS randomly accumulates throughout cytosol (see Figure S9
in the Supporting Information). Further work on costaining cells
with TFBS and other vesicular specific antibodies would be
needed to identify the accurate TFBS localization.
The cytotoxicity of TFBS was evaluated for mouse embryonic
fibroblast normal cells (NIH-3T3), using methylthiazolyldiphenyl-
tetrazolium (MTT) cell viability assay. Figure S10 in the Supporting
Information shows the in vitro NIH-3T3 cell viabilities after
being cultured with aqueous TFBS solution at the concentrations
of 0.5 and 1 μM for 24, 48, and 72 h, respectively. The cell
viabilities are close to 100% within the tested period of time,
which indicates low cytotoxicity of TFBS. This low cytotoxicity
would benefit TFBS for in vitro and in vivo cellular imaging and
other clinical applications.
Figure 5. TPA cross sections of TFBS in water.
Because TFBN, TFBC, and TFBS have the same conjugated
framework, their TPA values in different solvents should be
associated with the solvent effect and molecular assembly. Wang
et al.46 suggested that the enhancement of δ from a solvent of
low polarity to that of intermediate polarity is associated with
the charge separation assisted by the solvent, and the resulting
change in the electronic structure provides a main perturbation
on δ. On the other hand, Woo et al. reported that water could
influence the electronic structure of D-π-A-π-D chromophores
via hydrogen bonding, which led to decreased ICT.47 As a con-
sequence, a substantial drop in δ was observed in water,
compared to that in organic solvents. A similar trend is observed
for TFBC, which is well-dissolved at the molecular level in both
DMSO and water. However, as TFBS forms self-assembled
nanoparticles in water, the star-shaped backbone is isolated within
a relatively hydrophobic environment. As such, TFBS shows a
significantly higher δmax in water, compared to that for TFBC.
This argument is further supported by the comparable δmax
values in DMSO for TFBC and TFBS (see Figure S8 in the
Supporting Information), because both are well-dissolved at the
molecular level. However, the δmax value of TFBS in water is still
lower than that for TFBN in toluene, indicating the inevitable
loss of TPA when converting TPA molecules from low-polar
organic solvents into high-polar aqueous media.
To demonstrate the ability of TFBS as a probe for live cell
imaging in vitro, the human cervical cancer cell (Hela cell) line
was used as an example. Hela cells were incubated with an
aqueous solution of TFBS at a concentration of 0.5 μM for 2 h.
After the cell uptake, excess TFBS was washed away and the live
cells were imaged using TPM. The excitation wavelength was
fixed at 800 nm with a laser power of ∼4 W, which corresponded
to an average power of 10 mW in the focal plane. The fluorescent
signals were collected over the range of 500ꢀ600 nm. Note that
TFBS has a δ value of ∼570 GM at 800 nm in water, which is
sufficient to obtain bright TPM images. Moreover, the δ values of
TFBS reach up to 1200 GM in the range of 730ꢀ800 nm, which are
larger than that for many recently reported two-photon fluores-
cence probes designed for cellular imaging applications.19ꢀ22,48,49
As shown in Figure 6A, the bright green fluorescence indicates
successful internalization of TFBS by live Hela cells. As a glucose
analogue, TFBS is possibly internalized by Hela cells through a
glucose-specific transport system rather than by passive diffusion.49
The TPEF/transmission overlapped image (Figure 6C) suggests
’ CONCLUSION
In conclusion, we have synthesized a glycosylated star-shaped
neutral conjugated oligomer (4,40,400-tris(4-(2-(4-(benzo[d]thiazol-2-
yl)phenyl)-9,90-bis(6-thiol-β-D-glucose)-hexyl)-fluoren-7-yl)-
phenylamine (TFBS)), using a post-functionalization strategy
for two-photon fluorescence imaging of live cells. Because of the
presence of hydrophilic glucose substituents, TFBS is able to self-
assemble into nanoparticles in aqueous media. The nanoparticles
show a high two-photon absorption (TPA) cross section of
∼1200 GM at 740 nm in water, which is higher than that for
recently reported water-soluble TPA chromophores for two-
photon microscopy (TPM) applications. In contrast, the TPA
spectrum for its cationic counterpart (4,40,400-tris(4-(2-(4-(benzo-
[d]thiazol-2-yl)phenyl)-9,90-bis(6-N,N,N-trimethylammonium)-
hexyl)-fluoren-7-yl)phenylamine (TFBC)) is not measurable
using TPEF method, because of its extremely low fluorescence
in water, which highlights the importance of glucose substitution
in achieving high TPA action cross-section in water. Moreover,
TFBS has low cytotoxicity and efficient permeability to live cells,
which makes it an ideal probe for two-photon fluorescence
imaging of live cells in a high-contrast and nonviral manner.
This study thus demonstrates an effective molecular engineering
strategy to develop robust water-soluble TPA materials for TPM
applications.
’ ASSOCIATED CONTENT
1
S
Supporting Information. TPA measurement, H NMR
b
of TFBN, TFBC, TFBS-OAc and TFBS, MALDI-TOF mass
4433
dx.doi.org/10.1021/cm201377u |Chem. Mater. 2011, 23, 4428–4434

Chemistry of Materials
ARTICLE
spectrum of TFBS, density functional theory (DFT) calculation,
TPA cross sections of TFBN in toluene as well as TFBS and
TFBC in DMSO, and cell viability of NIH-3T3 fibroblast cells
after incubation with TFBS. This material is available free of
charge via the Internet at http://pubs.acs.org.
(21) McRae, R. L.; Phillips, R. L.; Kim, I.-B.; Bunz, U. H. F.; Fahrni,
C. J. J. Am. Chem. Soc. 2008, 130, 7851.
(22) Warther, D.; Bolze, F.; Lꢀeonard, J.; Gug, S.; Specht, A.; Puliti,
D.; Sun, X.-H.; Kessler, P.; Lutz, Y.; Vonesch, J.-L.; Winsor, B.; Nicoud,
J.-F.; Goeldner, M. J. Am. Chem. Soc. 2010, 132, 2585.
(23) Jiang, Y.; Wang, Y.; Wang, B.; Yang, J.; He, N.; Qian, S.; Hua, J.
Chem. Asian J. 2011, 6, 157.
(24) Liu, B.; Gaylord, B. S.; Wang, S.; Bazan, G. C. J. Am. Chem. Soc.
2003, 125, 6705.
(25) Liu, B.; Wang, S.; Bazan, G. C.; Mikhailovsky, A. J. Am. Chem.
Soc. 2003, 125, 13306.
(26) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 1188.
(27) Liu, B.; Bazan, G. C. Chem. Asian J. 2007, 2, 499.
(28) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942.
(29) Lin, T.-C.; He, G. S.; Prasad, P. N.; Tan, L.-S. J. Mater. Chem.
2004, 14, 982.
(30) Pu, K.-Y.; Fang, Z.; Liu, B. Adv. Funct. Mater. 2008, 18, 1321.
(31) Pu, K. Y.; Pan, S. Y. H.; Liu, B. J. Phys. Chem. B 2008, 112, 9295.
(32) Xue, C.; Jog, S. P.; Murthy, P.; Liu, H. Biomacromolecules 2006,
7, 2470.
(33) Xue, C.; Donuru, V. R. R.; Liu, H. Macromolecules 2006,
39, 5747.
(34) Pu, K.-Y.; Shi, J.; Wang, L.; Cai, L.; Wang, G.; Liu, B. Macro-
molecules 2010, 43, 9690.
(35) Eastoe, J.; Rogueda, P.; Harrison, B. J.; Howe, A. M.; Pitt, A. R.
Langmuir 1994, 12, 4429.
(36) Chu, B. Laser Light Scattering: Basic Principles and Practice, 2nd ed.;
Academic Press: San Diego, CA, 1991.
(37) Pu, K.-Y.; Li, K.; Liu, B. Adv. Funct. Mater. 2010, 20, 2770.
(38) Pu, K.-Y.; Liu, B. Adv. Funct. Mater. 2009, 19, 277.
(39) Xu, C.; Webb, W. W. J. Opt. Soc. Am. B 1996, 13, 481.
(40) Makarov, N. S.; Drobizhev, M.; Rebane, A. Opt. Express 2008,
16, 4029.
’ AUTHOR INFORMATION
Corresponding Author
*Fax: (+65) 6779-1936 (B.L.). E-mails: cheliub@nus.edu.sg,
(B.L.), chmlaiyh@nus.edu.sg (Y.-H.L.).
’ ACKNOWLEDGMENT
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 Engineering (IMRE/11-1C0213) for financial
support.
’ REFERENCES
(1) Albota, M.; Beljonne, D.; Brꢀedas, J.; Ehrlich, J. E.; Fu, J.; Heikal,
A. A.; Hess, S.; Kogej, T.; Levin, M. D.; Marder, S. R.; McCord-Maughon,
D.; Perry, J. W.; R€ockel, H.; Rumi, M.; Subramaniam, G.; Webb, W.;
Wu, X.; Xu, C. Science 1998, 281, 1653.
(2) Cumpston, B. H.; Ananthavel, S. P.; Barlow, S.; Dyer, D. L.;
Ehrlich, J. E.; Erskine, L. L.; Heikal, A. A.; Kuebler, S. M.; Lee, I. L. S.;
McCord-Maughon, D.; Qin, J.; R€ockel, H.; Rumi, M.; Wu, X.; Marder,
S. R.; Perry, J. W. Nature 1999, 389, 51.
(3) Pawlicki, M.; Collins, H. A.; Denning, R. G.; Anderson, H. L.
Angew. Chem., Int. Ed. 2009, 48, 3244.
(41) Hrobarikova, V.; Hrobarik, P.; Gajdos, P.; Fitilis, I.; Fakis, M.;
Persephonis, P.; Zahradnik, P. J. Org. Chem. 2010, 75, 3053.
(42) Yao, S.; Ahn, H.-Y.; Wang, X.; Fu, J.; Van Stryland, E. W.;
Hagan, D. J.; Belfield, K. D. J. Org. Chem. 2010, 75, 3965.
(43) Andrade, C. D.; Yanez, C. O.; Rodriguez, L.; Belfield, K. D.
J. Org. Chem. 2010, 75, 3975.
(44) He, G. S.; Swiatkiewicz, J.; Jiang, J.; Prasad, P. N.; Reinhardt,
B. A.; Tan, L.-S.; Kannan, R. J. Phys. Chem. A 2000, 104, 4805.
(45) Kannan, R.; He, G. S.; Lin, T.-C.; Prasad, P. N.; Vaia, R. A.; Tan,
L.-S. Chem. Mater. 2004, 16, 185.
(4) Denk, W.; Strickler, J. H.; Webb, W. W. Science 1990, 248, 73.
(5) Xu, C.; Zipfel, W.; Shear, J. B.; Williams, R. M.; Webb, W. W.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10763.
(6) So, P. T. C.; Dong, C. Y.; Masters, B. R.; Berland, K. M. Annu.
Rev. Biomed. Eng. 2000, 2, 399.
(7) Le Droumaguet, C.; Mongin, O.; Werts, M. H. V.; Blanchard-
Desce, M. Chem. Commun. 2005, 2802.
(8) Mongin, O.; Porrꢁes, L.; Katan, C.; Mertzb, T. P. J.; Blanchard-
Descea, M. Tetra. Lett. 2003, 44, 8121.
(9) Yoo, J.; Yang, S. K.; Jeong, M.-Y.; Ahn, H. C.; Jeon, S.-J.; Cho,
B. R. Org. Lett. 2003, 5, 645.
(10) Cui, Y.-Z.; Fang, Q.; Xue, G.; Xu, G.-B.; Yin, L.; Yu, W.-T.
Chem. Lett. 2005, 34, 644.
(11) Cho, B. R.; Son, K. H.; Lee, S. H.; Song, Y.-S.; Lee, Y.-K.; Jeon,
S.-J.; Choi, J. H.; Lee, H.; Cho, M. J. Am. Chem. Soc. 2001, 123, 10039.
(12) Tian, Y.; Chen, C.-Y.; Cheng, Y.-J.; Young, A. C.; Tucker,
N. M.; Jen, A. K.-Y. Adv. Funct. Mater. 2007, 17, 1691.
(13) Woo, H. Y.; Korystov, D.; Mikhailovsky, A.; Nguyen, T.-Q.;
Bazan, G. C. J. Am. Chem. Soc. 2005, 127, 13794.
(46) Wang, C.-K.; Zhao, K.; Su, Y.; Ren, Y.; Zhao, X.; Luo, Y. J. Chem.
Phys. 2003, 119, 1208.
(47) Woo, H. Y.; Liu, B.; Kohler, B.; Korystov, D.; Mikhailovsky, A.;
Bazan, G. C. J. Am. Chem. Soc. 2005, 127, 14721.
(48) Morales, A. R.; Luchita, G.; Yanez, C. O.; Bondar, M. V.;
Przhonskab, O. V.; Belfield, K. D. Org. Biomol. Chem. 2010, 8, 2600.
(49) Tian, Y. S.; Lee, H. Y.; Lim, C. S.; Park, J.; Kim, H. M.; Shin,
Y. N.; Kim, E. S.; Jeon, H. J.; Park, S. B.; Cho, B. R. Angew. Chem., Int. Ed.
2009, 48, 8027.
(50) Khan, S.; Bijker, M. S.; Weterings, J. J.; Tanke, H. J.; Adema,
G. J.; van Hall, T.; Drijfhout, J. W.; Melief, C. J. M.; Overkleeft, H. S.; van
der Marel, G. A.; Filippov, D. V.; van der Burg, S. H.; Ossend, F. J. Biol.
Chem. 2007, 282, 21145.
(14) Jager, W. F.; Volkers, A. A.; Neckers, D. C. Macromolecules
1995, 28, 8153.
(15) Stubenrauch, C. Curr. Opin. Colloid Interface Sci. 2001, 6, 160.
(16) Koo, C.-K.; Wong, K.-L.; Man, C. W.-Y.; Lam, Y.-W.; So, L. K.-Y.;
Tam, H.-L.; Tsao, S.-W.; Cheah, K.-W.; Lau, K.-C.; Yang, Y.-Y.; Chen, J.-C.;
Lam, M. H.-W. Inorg. Chem. 2009, 48, 878.
(17) Koo, C.-K.; Wong, K.-L.; Man, C. W.-Y.; Tam, H.-L.; Tsao,
S.-W.; Cheah, K.-W.; Lam, M. H.-W. Inorg. Chem. 2009, 48, 7501.
(18) Koo, C.-K.; So, L. K.-Y.; Wong, K.-L.; Ho, Y.-M.; Lam, Y.-W.;
Lam, M. H.-W.; Cheah, K.-W.; Cheng, C. C.-W.; Kwok, W.-M. Chem.—
Eur. J. 2010, 16, 3942.
(19) Ohulchanskyy, T. Y.; Pudavar1, H. E.; Yarmoluk, S. M.; Yashchuk,
V. M.; Bergey, E. J.; Prasad, P. N. Photochem. Photobiol. 2003, 77, 138.
(20) Dyrager, C.; Friberg, A.; Dahlꢀen, K.; Fridꢀen-Saxin, M.;
B€orjesson, K.; Wilhelmsson, L. M.; Smedh, M.; Grøtli, M.; Luthman,
K. Chem.—Eur. J. 2009, 15, 9417.
4434
dx.doi.org/10.1021/cm201377u |Chem. Mater. 2011, 23, 4428–4434