Fluorescent conjugated polyfluorene with pendant lactopyranosyl ligands for studies of Ca2+-mediated carbohydrate-carbohydrate interaction

Article abstract of DOI:10.1021/bm901165n

A well-defined fluorescent conjugated polyfluorene with pendant lactopyranosyl ligands was easily prepared through Cu(I)-catalyzed azide/alkyne click ligation and Suzuki coupling polymerization. As a fluorescent multivalent modelsystemof glycoconjugates, thepolymerwasfirstusedfor studies ofmetalion-mediatedcarbohydrate-carbohydrate interaction based on fluorescence spectroscopy. A significant fluorescence quenching of the lactosyl-bearing polyfluorene was observed upon addition of calcium ion, which is attributed to the polymer aggregation derived from Ca²⁺-mediated complex formation. Dynamic light scattering can also prove Ca²⁺-induced aggregation of the polymer based on determination of the corresponding hydrodynamic diameters. The calcium-mediated lactose-lactose interaction was reversible when treated with EDTA. In control studies, Ca²⁺-induced fluorescence quenching can not be observed for cellobiosyl- or galactosyl-functionalized polymer analogues, which show that specific sugar structures are critical for carbohydrate-metal complex formation.

Full text of DOI:10.1021/bm901165n

Biomacromolecules 2010, 11, 13–19
13
Fluorescent Conjugated Polyfluorene with Pendant
Lactopyranosyl Ligands for Studies of Ca2+-Mediated
Carbohydrate-Carbohydrate Interaction
Qi Chen,† Yi Cui,†,‡ Tian-Long Zhang,† Jie Cao,*,‡ and Bao-Hang Han*,†
National Center for Nanoscience and Technology, Beijing 100190, China, and Graduate University of
Chinese Academy of Sciences, Beijing 100049, China
Received May 31, 2009; Revised Manuscript Received November 25, 2009
A well-defined fluorescent conjugated polyfluorene with pendant lactopyranosyl ligands was easily prepared through
Cu(I)-catalyzed azide/alkyne “click” ligation and Suzuki coupling polymerization. As a fluorescent multivalent
modelsystemofglycoconjugates,thepolymerwasfirstusedforstudiesofmetalion-mediatedcarbohydrate-carbohydrate
interaction based on fluorescence spectroscopy. A significant fluorescence quenching of the lactosyl-bearing
polyfluorene was observed upon addition of calcium ion, which is attributed to the polymer aggregation derived
from Ca2+-mediated complex formation. Dynamic light scattering can also prove Ca2+-induced aggregation of
the polymer based on determination of the corresponding hydrodynamic diameters. The calcium-mediated
lactose-lactose interaction was reversible when treated with EDTA. In control studies, Ca2+-induced fluorescence
quenching can not be observed for cellobiosyl- or galactosyl-functionalized polymer analogues, which show that
specific sugar structures are critical for carbohydrate-metal complex formation.
study carbohydrate-carbohydrate interactions based on
fluorescence spectroscopy has not been reported. Our group
have explored the facile prepolymerization and postpoly-
merization functionalization approaches to prepare well-
defined fluorescent conjugated glycopolymers through Cu(I)-
catalyzed azide/alkyne “click” ligation.15 When those methods
were used, conjugated glycopolymers containing tripheny-
lamine or fluorene backbones were prepared for applications
in detecting biomacromolecules.16 In this article, D-lactosyl
bearing polyfluorene has been synthesized (Scheme 1) and
used for investigation of lactose-lactose recognition based
on lactose-metal complexes formation. In the presence of
specific metal ions, this well-defined fluorescent glycopoly-
mer exhibited exceptional fluorescence quenching or energy
transfer efficiency, resulting in amplification of optical signals
for transduction of the carbohydrate-carbohydrate recogniz-
ing event. We believe it will provide some clues for
elucidation of the mechanism underlying in carbohydrate-
carbohydrate interactions.
Introduction
Carbohydrate-carbohydrate interactions between cell surface
glycans play crucial roles in many physiological processes such
as cell adhesion and cellar recognition.1 Systematic investiga-
tions have demonstrated that most of carbohydrate-carbohy-
drate interactions, for example, the LeX-LeX interaction in
compaction in embryogenesis,2 the sulfated disaccharide
GlcpNAc3S(ꢀ1-3)Fucp self-interaction in the cell adhesion of
marine sponges,3 and LacCer-GM3 interaction in the adhesion
of mouse B16 melanoma cells to the endothelial cells,4 are
mediated by calcium ion based on multivalent effect. Various
multivalent model systems such as glyconanoparticles,5 glyco-
polymers,6 glycodendrimers,7 and liposomes8 have been used in
studies of Ca2+-mediated carbohydrate-carbohydrate interactions.
However, the underlying mechanism in these carbohydrate-
carbohydrate interactions has not yet been sufficiently clarified.
Synthetic glycopolymers, as multivalent model systems and
artificial glycoconjugates, have been demonstrated to be im-
portant well-defined tools for investigating carbohydrate-based
biological interactions.9 Moreover, fluorescent conjugated gly-
copolymers with fluorescent backbones and reporting carbohy-
drate moieties are attractively employed in carbohydrate-protein
interaction studies and biosensor applications because of their
intrinsic optical properties, high sensitivities to minor stimuli,
and good biocompatibilities.10 Recently, facile insights into
carbohydrate-lectin and carbohydrate-bacteria bindings using
fluorescent conjugated glycopolymers have been reported by
Bunz’s group11 and Liu’s group,12 respectively.
Experimental Section
Materials and Measurements. All chemical reagents were com-
mercially available and used as received unless otherwise stated.
Deionized water was purified with a Millipore purification system
(Milli-Q water). 9,9-Bis(4-hydroxyphenyl)-2,7-dibromofluorene (1)17
and azido-functionalized sugar derivatives (3-5)18 were prepared
according to reported methods.
The 1H and 13C NMR spectra were recorded on a Bruker DMX300
NMR spectrometer. The optical rotations were measured with a JASCO
DIP-1000 digital polarimeter. Mass spectra were recorded with a VG
PLATFORM mass spectrometer using the ESI(+) technique. The
molecular weights of the polymers were determined by an Agilent 1100
GPC system in THF. The number-average and weight-average molec-
ular weights (Mn and Mw) were estimated by using a calibration curve
of polystyrene standard. IR spectra were recorded using a Perkin-Elmer
Paragon 1000 FTIR spectrometer. Ultraviolet-visible (UV-vis) spectra
were measured using a Perkin-Elmer Lamda 900 UV-vis-NIR
Metal ion-mediated carbohydrate-carbohydrate interac-
tions have been clearly proven by SPR,13 TEM,5 UV-vis
spectrum,5d NMR,14 and π-A isotherms.6b However, as far
as we know, using fluorescent conjugated glycopolymers to
* To whom correspondence should be addressed. Fax: +86-10-82545576.
E-mail: hanbh@nanoctr.cn.
† National Center for Nanoscience and Technology.
‡ Graduate University of Chinese Academy of Sciences.
10.1021/bm901165n
 2010 American Chemical Society
Published on Web 12/09/2009

14 Biomacromolecules, Vol. 11, No. 1, 2010
Chen et al.
Scheme 1. Synthetic Routes to Monomers and Polymers
spectrophotometer and quartz cells with 1 cm path length. The
fluorescence spectra were measured in a conventional cell with 1 cm
path length using a Perkin-Elmer LS55 luminescence spectrometer.
Dynamic light scattering (DLS) measurements were performed on a
ZETASIZER Nano-Series instrument.
Hz, 2H), 7.53 (s, 2H), 7.48 (d, J ) 8.1 Hz, 2H), 7.13 (d, J ) 8.6 Hz,
4H), 6.88 (d, J ) 8.6 Hz, 4H), 4.64 (s, 4H), 2.54 (s, 2H). ESI(+)-MS
calcd for C31H20Br2O2: 584.3 [M]. Found: 607.5 [M + Na]+.
Monomer 6. To a mixture of 2 (584 mg, 1.0 mmol) and 3 (1.66 g,
2.5 mmol) in H2O-THF (1:1, 15 mL) were added freshly prepared
aqueous 1.0 M sodium ascorbate (150 µL, 0.15 mmol) and CuSO4 (12
mg, 0.075 mmol). The heterogeneous mixture was stirred vigorously
in dark room at 50-60 °C until complete consumption of the reactants
based on TLC monitoring. After removal of THF under a reduced
pressure, water (20 mL) was added and the product was extracted with
ethyl acetate (3 × 25 mL). The combined organic layer was dried over
anhydrous Na2SO4 and evaporated in vacuo. The crude product was
subjected to column chromatography (ethyl acetate-petroleum ether,
2:1) to give 6 as a foamy solid (1.86 g, 98%). [R]D25 -50° (c 1, CHCl3).
1H NMR (300 MHz, CDCl3): δ 7.75 (s, 2H), 7.54 (d, J ) 8.4 Hz, 2H),
7.45-7.42 (m, 4H), 7.02 (d, J ) 8.7 Hz, 4H), 6.82 (d, J ) 8.6 Hz,
Synthesis of Monomers and Polymers. 9,9-Bis(4-propargylox-
yphenyl)-2,7-dibromofluorene 2.16b To a solution of compound 1 (584
mg, 1.15 mmol) and propargyl bromide (330 mg, 2.76 mmol) in acetone
(10 mL) were added anhydrous potassium carbonate (380 mg, 2.76
mmol) and Bu4NBr (37 mg, 0.115 mmol), and the mixture was refluxed
overnight. After removal of acetone, water (15 mL) was added and the
product was extracted with ethyl acetate (3 × 20 mL). The combined
organic layer was dried over anhydrous Na2SO4 and concentrated in
vacuo. The residue was purified with silica gel column chromatography
(ethyl acetate-petroleum ether, 1:6) to give the desired product 2 (544
1
mg, 81%) as a solid. H NMR (300 MHz, CDCl3): δ 7.57 (d, J ) 8.1

Polyfluorene with Pendant Lactopyranosyl Ligands
Biomacromolecules, Vol. 11, No. 1, 2010 15
4H), 5.82 (d, J ) 9.3 Hz, 2H), 5.39-5.34 (m, 6H), 5.12 (s, 4H), 5.08
(d, J ) 8.1 Hz, 2H), 4.95 (dd, J ) 3.3, 10.5 Hz, 2H), 4.52 (d, J ) 7.8
Hz, 2H), 4.45 (d, J ) 11.4 Hz, 2H), 4.15-4.05 (m, 6H), 3.94-3.87
(m, 6H), 2.13 (s, 6H), 2.06 (s, 6H), 2.04 (s, 6H), 2.02 (s, 6H), 2.01 (s,
6H), 1.94 (s, 6H), 1.76 (s, 6H). 13C NMR (75 MHz, CDCl3): δ 169.3,
169.2, 169.1, 169.0, 168.5, 168.1, 156.3, 152.4, 143.8, 136.8, 136.1,
129.9, 128.2, 128.1, 120.8, 120.6, 120.2, 113.7, 100.1, 84.5, 74.9, 74.6,
71.6, 69.9, 69.8, 69.5, 68.0, 65.6, 63.3, 60.9, 60.8, 59.8, 59.4, 20.0,
19.8, 19.7, 19.6, 19.5, 19.3, 19.1. ESI(+)-MS calcd for C83H90Br2N6O36:
1907.4 [M]. Found: 1930.3 [M + Na]+.
99.7, 84.5, 76.4, 74.8, 74.6, 71.7, 71.3, 70.9, 69.4, 67.1, 65.6, 61.1,
60.7, 59.5, 19.8, 19.7, 19.5, 19.2. GPC (THF, polystyrene standard):
Mn ) 27500 g/mol; polydispersity ) 1.78.
Polymer P-1c. Polymer P-1c was obtained from monomer 8 (200
mg, 0.15 mmol) using the same procedure for preparation of polymer
1
P-1a as a solid (152 mg, 81%). H NMR (300 MHz, CDCl3): δ 7.88
(bs, 2H), 7.80-7.60 (m, 6H), 7.49-7.44 (m, 2H), 7.40-7.22 (m, 6H),
6.96-6.83 (m, 4H), 5.82 (br, 2H), 5.68-5.54 (m, 4H), 5.30-5.15 (m,
6H), 4.18 (br, 6H), 2.19 (bs, 6H), 2.05 (bs, 12H), 1.76 (bs, 6H). 13C
NMR (75 MHz, CDCl3): δ 169.3, 169.0, 168.8, 168.0, 156.3, 152.4,
143.8, 136.9, 136.1, 131.2, 129.9, 128.2, 128.1, 126.5, 126.3, 120.8,
120.6, 120.3, 113.8, 113.7, 85.2, 73.1, 69.8, 66.9, 66.8, 65.9, 60.9, 60.2,
19.6, 19.5, 19.1. GPC (THF, polystyrene standard): Mn ) 21600 g/mol;
polydispersity ) 1.81.
Polymer P-2a. Protected glycoplymer P-1a (150 mg) was added to
a solution of dry CH2Cl2 (5 mL) and MeOH (10 mL) under a nitrogen
atmosphere and followed by 1.0 M CH3ONa/CH3OH solution (0.25
mL). The reaction mixture was stirred overnight at room temperature.
After removal of the solvent under a reduced pressure, 10 mL of water
was added to the residue. The resulting solution was put in a cellulose
dialysis tube (cutoff 3500), dialyzed against water for 2 d, and
lyophilized to give the desired solid polymer P-2a (96.6 mg, 95%). 1H
NMR (300 MHz, DMSO-d6): δ 8.45 (bs, 2H), 8.10-7.76 (m, 6H),
7.69-7.25 (m, 6H), 7.24-7.15 (m, 2H), 7.03-6.94 (m, 4H), 5.67 (br,
2H), 5.11 (s, 4H), 4.28 (br, 2H), 3.87-3.74 (m, 4H), 3.73-3.50 (m,
14H), 3.45-3.30 (m, 6H). 13C NMR (75 MHz, DMSO-d6): δ 157.1,
142.3, 139.2, 139.1, 136.5, 134.3, 131.5, 130.8, 128.9, 128.7, 127.2,
126.5, 125.0, 123.8, 123.7, 114.6, 103.7, 86.9, 79.6, 79.2, 77.8, 75.6,
75.1, 73.3, 71.8, 70.6, 68.1, 60.9, 59.9.
Monomer 7. Monomer 7 was obtained from compounds 2 and 4
using the same procedure for preparation of monomer 6 as a foamy
25
1
solid (1.80 g, 96%). [R]D -38° (c 1, CHCl3). H NMR (300 MHz,
CDCl3): δ 7.70 (s, 2H), 7.49 (d, J ) 8.5 Hz, 2H), 7.38-7.36 (m, 4H),
6.96 (d, J ) 8.7 Hz, 4H), 6.75 (d, J ) 8.5 Hz, 4H), 5.80 (d, J ) 9.1
Hz, 2H), 5.34-5.32 (m, 4H), 5.10 (t, J ) 10.5 Hz, 2H), 5.12 (s, 4H),
5.01 (t, J ) 10.5 Hz, 2H), 4.87 (t, J ) 10.2 Hz, 2H), 4.53 (d, J ) 8.2
Hz, 2H), 4.44 (d, J ) 11.5 Hz, 2H), 4.30 (dd, J ) 3.3, 10.5 Hz, 2H),
4.07 (d, J ) 10.5 Hz, 2H), 3.98 (d, J ) 11.4 Hz, 2H), 3.92-3.89 (m,
4H), 3.68-3.64 (m, 2H), 2.01 (s, 6H), 2.00 (s, 6H), 1.96 (s, 6H), 1.95
(s, 6H), 1.92 (s, 6H), 1.90 (s, 6H), 1.70 (s, 6H). 13C NMR (75 MHz,
CDCl3): δ 169.4, 169.1 (2C), 168.5, 168.3, 168.0, 167.9, 156.3, 152.5,
143.7, 136.9, 136.1, 129.9, 128.2, 128.1, 120.8, 120.7, 120.3, 113.8,
99.8, 84.5, 74.9, 74.8, 71.9, 71.3, 71.1, 70.6, 69.5, 66.9, 63.4, 60.9,
60.7, 60.6, 59.3, 20.0, 19.7, 19.6, 19.5 (2C), 19.4, 19.1. ESI(+)-MS
calcd for C83H90Br2N6O36: 1907.4 [M]. Found: 1930.5 [M + Na]+.
Monomer 8. Monomer 8 was obtained from compounds 2 and 5
using the same procedure for preparation of monomer 6 as a foamy
25
1
solid (1.30 g, 98%). [R]D -30° (c 1, CHCl3). H NMR (300 MHz,
CDCl3): δ 7.89 (s, 2H), 7.55 (d, J ) 8.5 Hz, 2H), 7.46-7.44 (m, 4H),
7.06 (d, J ) 8.7 Hz, 4H), 6.86 (d, J ) 8.6 Hz, 4H), 5.85 (d, J ) 9.3
Hz, 2H), 5.58-5.51 (m, 4H), 5.24 (dd, J ) 3.3, 10.2 Hz, 2H), 5.15 (s,
4H), 4.25-4.21 (m, 2H), 4.17-4.12 (m, 4H), 2.20 (s, 6H), 2.02 (s,
6H), 1.99 (s, 6H), 1.79 (s, 6H). 13C NMR (75 MHz, CDCl3): δ 170.2,
169.9, 169.7, 168.9, 157.3, 153.4, 144.7, 137.8, 137.0, 130.8, 129.2,
129.0, 121.7, 121.5, 121.2, 114.7, 86.2, 74.0, 70.7, 67.7, 66.8, 61.8,
61.1, 60.3, 20.5, 20.4, 20.1. ESI(+)-MS calcd for C59H58Br2N6O20:
1330.9 [M]. Found: 1354.1 [M + Na]+.
Polymer P-2b. Polymer P-2b was obtained from polymer P-1b (150
mg) using the same procedure for preparation of polymer P-2a as a
1
solid (93.7 mg, 92%). H NMR (300 MHz, DMSO-d6): δ 8.47 (bs,
2H), 8.12-7.77 (m, 6H), 7.68-7.23 (m, 6H), 7.23-7.13 (m, 2H),
7.03-6.96 (m, 4H), 5.68 (br, 2H), 5.13 (s, 4H), 4.25 (br, 2H), 3.84-3.72
(m, 4H), 3.70-3.49 (m, 14H), 3.46-3.32 (m, 6H). 13C NMR (75 MHz,
DMSO-d6): δ 157.5, 142.5, 139.1, 138.9, 136.5, 134.5, 131.4, 131.0,
129.0, 128.8, 127.2, 126.5, 124.9, 123.8, 123.6, 113.5, 99.5, 87.0, 79.5,
79.2, 77.9, 75.5, 74.9, 73.3, 71.7, 70.8, 68.3, 61.0, 59.4.
Polymer P-2c. Polymer P-2c was obtained from polymer P-1c (130
mg) using the same procedure for preparation of polymer P-2a as a
Polymer P-1a. Under a nitrogen atmosphere, sugar-carrying mono-
mer 6 (280 mg, 0.15 mmol), 1,4-phenyldiboronic acid (30 mg, 0.18
mmol), Pd(PPh3)4 (10 mg), and potassium carbonate (250 mg, 1.8
mmol) were placed in a 50 mL round-bottomed flask, and then THF
(15 mL) was added. The mixture was stirred at 70 °C for 36 h under
a nitrogen atmosphere. The resulting polymer was purified by precipita-
tion in methanol and washed with methanol-acetone in a Soxhlet
apparatus for 48 h. P-1a was obtained as a gray powder (214 mg, 78%).
1H NMR (300 MHz, CDCl3): δ 7.76 (bs, 2H), 7.62-7.55 (m, 6H),
7.52-7.44 (m, 4H), 7.24-7.04 (m, 4H), 6.86-6.84 (m, 4H), 5.82 (br,
2H), 5.40-5.33 (m, 6H), 5.15-5.03 (m, 6H), 4.99-4.95 (m, 2H),
4.53-4.45 (m, 4H), 4.11 (br, 6H), 3.92 (br, 6H), 2.15 (bs, 6H), 2.05
(bs, 24H), 1.96 (bs, 6H), 1.76 (bs, 6H). 13C NMR (75 MHz, CDCl3):
δ 169.3, 169.2, 169.1, 169.0, 168.5, 168.4, 168.0, 156.3, 152.4, 143.9,
137.6, 136.9, 130.9, 129.9, 128.3, 128.1, 126.4, 126.1, 120.8, 120.2,
113.7, 113.4, 100.1, 84.5, 76.2, 74.9, 74.6, 71.6, 69.9, 69.8, 69.4, 68.0,
65.6, 60.9, 60.7, 59.8, 19.8, 19.6, 19.5, 19.1. GPC (THF, polystyrene
standard): Mn ) 28300 g/mol; polydispersity ) 1.73.
1
solid (91.1 mg, 96%). H NMR (300 MHz, DMSO-d6): δ 8.39 (bs,
2H), 8.14-7.70 (m, 6H), 7.68-7.40 (m, 6H), 7.28-7.20 (m, 2H),
7.10-6.98 (m, 4H), 5.54 (br, 2H), 5.15 (s, 4H), 4.10 (br, 2H), 3.82-3.70
(m, 4H), 3.65-3.50 (m, 6H). 13C NMR (75 MHz, DMSO-d6): δ 157.2,
142.6, 139.1, 139.0, 136.3, 134.2, 131.5, 128.9, 128.7, 127.3, 126.5,
124.8, 123.8, 123.4, 120.5, 114.6, 88.1, 78.4, 73.7, 69.3, 68.4, 65.3,
60.9, 60.4.
Studies of Metal Ions-Mediated Carbohydrate-Carbohydrate
Interaction Based on Spectrofluorometric Titration. To a solution
of glycopolymer (P-2a, P-2b, or P-2c) in water or H2O-DMSO(9:1,
v/v) was added different metal chloride solutions (in the same solvents)
individually. The final concentration of glycopolymer is 1.5 × 10-6
M, corresponding to the repeating unit. The final concentration for the
metal ion is 20 mM. After addition of the metal ion, the sample was
allowed to incubate at room temperature overnight prior to recording
a spectrum. The excitation wavelength was 370 nm and the emission
scan ranged from 370-650 nm.
Polymer P-1b. Polymer P-1b was obtained from monomer 7 (280
mg, 0.15 mmol) using the same procedure for preparation of polymer
1
P-1a as a gray powder (198 mg, 75%). H NMR (300 MHz, CDCl3):
Results and Discussion
δ 7.79 (bs, 2H), 7.65-7.53 (m, 6H), 7.47-7.40 (m, 4H), 7.23-7.07
(m, 4H), 6.86-6.79 (m, 4H), 5.82 (br, 2H), 5.41-5.37 (m, 4H),
5.16-5.08 (m, 8H), 4.99-4.95 (m, 2H), 4.58-4.49 (m, 4H), 4.39-4.37
(m, 2H), 4.13 (br, 2H), 4.05-4.04 (m, 2H), 3.94-3.92 (m, 4H), 3.69
(br, 2H), 2.15 (bs, 6H), 2.09 (bs, 12H), 2.04 (bs, 12H), 2.01 (bs, 6H),
1.99 (br, 6H), 1.76 (bs, 6H). 13C NMR (75 MHz, CDCl3): δ 169.4,
169.2, 169.1, 169.0, 168.8, 168.5, 168.1, 156.5, 152.4, 143.6, 137.7,
136.9, 130.7, 123.0, 128.3, 128.0, 126.3, 120.7, 120.4, 113.8, 113.6,
The synthetic routes to the monomers and glycopolymers are
outlined in Scheme 1. Propargyl-attached fluorene derivative 2
was easily prepared from 9,9-bis(4-hydroxyphenyl)-2,7-dibro-
mofluorene 1 by treating with propargyl bromide under a basic
condition. The Cu(I)-catalyzed “click reaction”19 facilitated
selective ligation between azido-attached lactose 3 and 2 to
furnish lactosyl-bearing monomer 6 smoothly in 98% yield.

16 Biomacromolecules, Vol. 11, No. 1, 2010
Chen et al.
1
Figure 1. H NMR spectra of monomer 6 and polymers P-1a and P-2a.
Formation of triazole ring is confirmed by chemical shift at 7.75
dant lactosyl derivative. After removing all the acetyl groups
with CH3ONa/CH3OH, the desired glycopolymer (P-2a) was
obtained in an excellent yield (95%). According to the same
procedures mentioned above, cellobiosyl-functionalized and
galactosyl-functionalized polyfluorenes (P-2b and P-2c) were
obtained from monomers 7 and 8, respectively. Employing an
1
ppm (single peak, 2H) on H NMR spectrum (Figure 1) and
two peaks at 120.8 and 143.8 ppm on 13C NMR spectrum. A
palladium-catalyzed Suzuki coupling polymerization between
monomer 6 and 1,4-phenyldiboronic acid gave a well-defined
copolymer P-1a, poly(fluorene-alt-phenylene) containing pen-

Polyfluorene with Pendant Lactopyranosyl Ligands
Biomacromolecules, Vol. 11, No. 1, 2010 17
Figure 2. UV-vis absorption and fluorescence spectra of polymers
P-1a and P-2a.
Figure 3. Fluorescence spectra of polymer P-2a in the absence and
presence of various metal ions in water at room temperature. [P-2a]
) 1.5 × 10-6 M, [metal ion] ) 20 mM.
extended 9,9-bis(4-hydroxyphenyl)fluorenyl core as the polym-
erization monomer is advantageous for providing a more
efficient shielding effect on the polyfluorene main chain after
insertion of a rigid phenylene spacer between the sugar side
chain and the polymer backbone, which would suppress the
formation of aggregates or excimers.20 Figure 1 shows the H
1
NMR spectra of monomer 6 and polymers P-1a and P-2a. These
data also apparently indicate that polymerization of sugar-
bearing monomers is a good approach to prepare well-defined
fluorescent conjugated glycopolymers.
Gel-permeation chromatography (GPC) analysis with poly-
styrene standards shows number-averaged molecular masses
(Mn) ranged from 28300 to 21600 g/mol for all protected
polymers, with polydispersities from 1.73 to 1.81. The protected
polymers are readily soluble in common solvents such as
methylene chloride, chloroform, and THF, but are insoluble in
methanol, ethanol, acetone, and water. The solubility of the
resulting polymers is different from their precursor polymers,
showing good solubility in DMF, DMSO, and H2O-DMSO
(9:1, v/v) as well. The deprotected polymers are not soluble in
water freely, only P-2a and P-2b are slightly soluble in water.
Similar results were reported by Takasu for sugar-carrying
poly(phenylenevinylene).10f
The precursor polymer P-1a exhibits an absorption maximum
peak at 356 nm and an emission maximum peak at 397 nm in
THF solution (Figure 2), which are assigned to the π-π*
transition of the conjugated polymer backbone. As for the
resulting polymer P-2a, it exhibits an absorption maximum peak
at 365 nm and an emission maximum peak at 409 nm with a
vibronic shoulder peak at 431 nm in DMSO solution. Whereas,
in H2O-DMSO (9:1, v/v), P-2a displays an absorption maxi-
mum peak at 370 nm and an emission maximum peak at 420
nm with a vibronic shoulder peak at 436 nm (Figure 2). The
obvious bathochromic/red shift in both absorption and emission
spectra for polymers P-2a could be attributed to the enhanced
planar conformation or aggregation of polymer main chain in
the solvent with increased polarity.10h Fluorescence quantum
yields of the polymer were measured in dilute DMSO or aqueous
solution, and calculated by using quinine sulfate in 0.1 M
sulfuric acid as the reference absolute quantum efficiency (55%).
The fluorescence quantum yields for P-2a are 68% in DMSO
solution and 30% in solution of H2O-DMSO (9:1, v/v),
respectively.
Figure 4. Fluorescence spectra of polymer P-2a (1.5 × 10-6 M) in
the absence and presence of various concentrations of Ca2+ in water
at room temperature. Ca2+ concentrations (mM) from the top
downward are 0, 2.0, 5.0, 10.0, 15.0, 17.5, 20.0, and 30.0.
almost no change in the fluorescence intensity after addition of
Na+ or K+. Addition of Mg2+ or Ba2+ only caused a slight
fluorescence quenching of P-2a. However, a significant fluo-
rescence quenching of this glycopolymer was observed when
Ca2+ was added. It can be inferred that the size of the cation
used is important to form a complex with lactose. Previous
studies indicated that carbohydrate-metal complexes formed
only with cations of ionic radii larger than 0.8 Å, the optimum
size being 1.0 Å.5d,21 Thus, calcium ions with ionic radii of
0.99 Å are ideal for complex formation. Russell’s group has
reported that, based on SPR measurement, an increased response
in aggregation of lactose-coated gold nanoparticles for group 2
metal ions occurred as the ionic radii increased (i.e., Mg < Ca
< Ba),5d which is different from our results (Mg ≈ Ba < Ca).
The discrepancy may be attributed to the different artificial
multivalent model systems used. For example, using liposome
as the model system, the binding data showing that binding of
(KDN)GM3 liposome to Gg3 epitope was much stronger than
that of GM3.22 When glycopolymer was employed as the
multivalent carrier, an opposite result was observed.23
The preliminary studies of Ca2+-mediated lactose-lactose
interaction were performed by probing the changes in fluores-
cence intensity of P-2a when titrated with calcium ion. As
shown in Figure 4, a substantial fluorescence quenching for P-2a
([P-2a] ) 1.5 × 10-6 M) was observed upon adding calcium
ion ([Ca2+] ) 0-30 mM) in water. A possible mechanism for
the fluorescence quenching of polymer P-2a is attributed to its
Metal ion-mediated interaction of the lactose-bearing poly-
fluorene P-2a was investigated based on the change in fluores-
cence intensity of P-2a in the solution of water upon gradual
addition of metal ions. As shown in Figure 3, P-2a exhibits

18 Biomacromolecules, Vol. 11, No. 1, 2010
Chen et al.
Figure 7. Schematic representation of the reversible calcium-lactose
polymer aggregation.
glucose moiety of another lactose.5d,24 Figure 7 shows a
schematic representation of the reversible calcium-lactose
polymer aggregate formation in which water molecules coor-
dinated with calcium ions are omitted. A control experiment
was performed to confirm the importance of the disaccharide
component in effecting Ca2+ complexation. Glycopolymers P-2b
or P-2c, cellobiosyl- or glactose-bearing polyfluorenes, were
designed and prepared for control experiments. Upon addition
of 30 mM calcium ions to a solution of the polymer P-2b in
water or the polymer P-2c in H2O-DMSO(9:1, v/v), no
significant changes in the fluorescence intensity were observed
(Figures S1 and S2), which suggested that specific sugar
structures are critical for the formation of carbohydrate-metal
complex. These results revealed that conjugated glycopolymer,
as a fluorescent multivalent model system of carbohydrate, was
expected to have potential applications in detecting specific
carbohydrate-carbohydrate interactions.
Figure 5. Hydrodynamic diameters of polymer P-2a in the absence
and presence of calcium ion in water determined by DLS at room
temperature.
Conclusions
In conclusion, a fluorescent conjugated polyfluorene with
pendant lactopyranosyl ligands was easily prepared through
Cu(I)-catalyzed azide/alkyne “click” ligation and Suzuki cou-
pling polymerization. With fluorescent scaffolding and reporting
carbohydrate ligands, this well-defined fluorescent conjugated
glycopolymer was first used for studies of metal ion-mediated
carbohydrate-carbohydrate interaction through spectrofluoro-
metric titration. Upon addition of calcium ion, a significant
fluorescence quenching of the lactosyl-bearing polyfluorene was
observed, which is attributed to its aggregation derived from
Ca2+-mediated complex formation. Additionally, calcium-
induced aggregation of the polymer can be confirmed using
dynamic light scattering by determination of the corresponding
hydrodynamic diameters. It has been shown that not only the
cation size but also the sugar structures are critical for
carbohydrate-metal complex formation. We believe this kind
of fluorescent conjugated glycopolymer can provide an excellent
platform for studies of carbohydrate-carbohydrate interactions.
Figure 6. Fluorescence spectra of polymer P-2a, Ca2+ (20 mM)-
induced aggregation of polymer P-2a, and polymer P-2a following
addition of Ca2+ and subsequent addition of 20 mM EDTA at room
temperature.
aggregation derived from P-2a-Ca2+ complex formation,
resulting in self-quenching. Dynamic light scattering can also
prove Ca2+-induced aggregation of the polymer by determining
the corresponding hydrodynamic diameters (Figure 5). It can
be seen that the hydrodynamic diameter of the glycopolymer
P-2a increases from 105 to 1030 nm after addition of calcium
ion (0-30 mM). The increment in hydrodynamic diameter is
consistent with the spectrofluorometric titration data, which
clearly indicates that continuous aggregation occurred during
the P-2a-Ca2+ complex formation. In addition, the hydrody-
namic diameter obtained by DLS is related to the solvent. In
DMSO solution, polymer P-2a shows a good solubility and its
diameter in the absence of calcium ion is about 35 nm, which
is reasonable, according to the contour length of the polymer
(nearly 25 nm). Furthermore, the hydrodynamic diameter is also
related to the concentration of the polymer. When the concen-
tration of the polymer P-2a is 1.0 × 10-6 M, the hydrodynamic
diameter of aggregated ensemble in the presence of 30 mM
calcium ion decreases to 800 nm, as compared with the 1030
nm at the concentration (1.5 × 10-6 M) of polymer P-2a.
The calcium-mediated lactose-lactose interaction is reversible
when an EDTA solution (20 mM) was added to the P-2a-Ca2+
complex aggregate. As shown in Figure 6, the associated
fluorescence spectrum almost recovers to the original spectrum
of glycopolymer P-2a after treated with EDTA. It has been
proved that calcium binds with two lactose molecules and four
water molecules in an octa-coordinate fashion, in which calcium
ion is coordinated to a galactose moiety of one lactose and a
Acknowledgment. The financial support of the Ministry of
Science and Technology of China (National Basic Research
Program, Grant No. 2007CB808000) and National Science
Foundation of China (Grant Nos. 20672025 and 20972035) is
acknowledged.
Supporting Information Available. Fluorescence spectra of
polymers P-2b and P-2c in the absence and presence of calcium
ion; 13C NMR spectra of monomer 6, polymer P-1a, and
polymer P-2a. This material is available free of charge via the
Internet at http://pubs.acs.org.
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