Fluorophore-labeled, peptide-based glycoclusters: Synthesis, binding properties for lectins, and detection of carbohydrate-binding proteins in cells

Article abstract of DOI:10.1002/asia.201100319

A facile and efficient solid-phase synthesis of linear peptide-based glycoclusters with various valences and different spatial arrangements of the sugar ligands is described. The synthetic strategy includes 1) solid-phase synthesis of fluorophore-labeled, alkyne-containing peptides, 2) coupling of azide-linked, unprotected mono-, di-, and trisaccharides to the alkyne-conjugated peptides on a solid support by click chemistry, and 3) release of the fluorophore-labeled glycoclusters from the solid support. By using this methodology, 32 fluorescent glycoclusters with a valence ranging from 1 to 4 and different spatial arrangements of the sugar ligands were prepared. Lectin-binding properties of the glycoclusters were initially examined by using microarrays immobilized by various lectins. These glycoclusters were then employed to detect the cell-surface carbohydrate-binding proteins in bacteria. Finally, the uptake of glycoclusters by mammalian cells through receptor-mediated endocytosis was evaluated. The results, obtained from the in vitro and in vivo studies, indicate that the binding affinities toward immobilized and cell-surface proteins are highly dependent on the valence and spatial arrangements of the sugar ligands in glycoclusters.

Full text of DOI:10.1002/asia.201100319

DOI: 10.1002/asia.201100319
Fluorophore-labeled, Peptide-based Glycoclusters: Synthesis, Binding
Properties for Lectins, and Detection of Carbohydrate-Binding Proteins in
Cells
[a]
Xizhe Tian, Jaeyoung Pai, Kyung-Hwa Baek, Sung-Kyun Ko, and Injae Shin*
Dedicated to Professor Eun Lee on the occasion of his retirement and 65th birthday
Abstract: A facile and efficient solid-
phase synthesis of linear peptide-based
glycoclusters with various valences and
different spatial arrangements of the
sugar ligands is described. The synthet-
ic strategy includes 1) solid-phase syn-
thesis of fluorophore-labeled, alkyne-
containing peptides, 2) coupling of
azide-linked, unprotected mono-, di-,
and trisaccharides to the alkyne-conju-
gated peptides on a solid support by
click chemistry, and 3) release of the
fluorophore-labeled glycoclusters from
the solid support. By using this meth-
odology, 32 fluorescent glycoclusters
with a valence ranging from 1 to 4 and
different spatial arrangements of the
sugar ligands were prepared. Lectin-
binding properties of the glycoclusters
were initially examined by using micro-
arrays immobilized by various lectins.
These glycoclusters were then em-
ployed to detect the cell-surface carbo-
hydrate-binding proteins in bacteria.
Finally, the uptake of glycoclusters by
mammalian cells through receptor-
mediated endocytosis was evaluated.
The results, obtained from the in vitro
and in vivo studies, indicate that the
binding affinities toward immobilized
and cell-surface proteins are highly de-
pendent on the valence and spatial ar-
rangements of the sugar ligands in gly-
coclusters.
Keywords: carbohydrates
·
click
chemistry · cluster compounds ·
glycoproteins · oligosaccharides
[
4]
Introduction
take place through these biomolecular interactions. There-
fore, the understanding of the molecular basis of glycan–
protein recognition events is of great importance to eluci-
date the complex biological processes involving glycans.
It is generally accepted that multivalent interactions be-
tween glycans and proteins enhance an otherwise weak
Glycans, which are found mainly in the form of glycoconju-
gates such as glycoproteins, proteoglycans, and glycolipids
inside or on the surface of cells, participate in a wide range
of physiological and pathological processes through interac-
[1,2]
[5]
tions with proteins.
For example, glycan–protein interac-
binding affinity of monomeric sugars with receptors. En-
tions play a pivotal role in cell adhesion, signaling, and traf-
ficking. Intriguingly, glycan-mediated biomolecular interac-
tions are also implicated in the development of various dis-
eases. Bacteria, viruses, and parasites infect hosts by initial
adhesion to host cells through interactions of the pathogenic
hancements in binding affinity by multivalent interactions
(often referred to as the cluster glycoside effect) are attrib-
uted to the chelate effect, clustering of carbohydrate-binding
[5b,c]
proteins, or statistical rebinding.
To achieve a binding en-
hancement through the multivalent glycan–protein interac-
tions, a variety of synthetic glycoclusters with diverse spatial
arrangements and different numbers of glycan ligands have
been prepared. These glycoclusters include linear and cyclic
neoglycopeptides, glyconanoparticles, glyconanotubes, glyco-
dendrimers, glycolipid micelles, glycoproteins, and glycopol-
[3]
proteins with the host cell-surface glycans. In addition,
tumor metastasis and leukocyte-mediated inflammation also
[
a] X. Tian, J. Pai, K.-H. Baek, Dr. S.-K. Ko, Prof. Dr. I. Shin
Center for Biofunctional Molecules
Department of Chemistry
[5,6]
ymers.
In these glycoclusters, the nature of the spacer be-
Yonsei University
Seoul, 120-749 (Korea)
Fax : (+82)2-364-7050
E-mail: injae@yonsei.ac.kr
tween the glycan moieties affects the binding enhancement
achievable by proper complementarities between the clus-
tered glycans and the oligomeric receptors. However, struc-
tural information on multivalent glycan–protein interactions
with atomic resolution is not sufficient for the rational
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100319.
Chem. Asian J. 2011, 6, 2107 – 2113
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2107

FULL PAPERS
design of glycoclusters. Thus, a series of glycoconjugates
with various spacer lengths and a different valence of gly-
cans are prepared for investigating multivalent interactions
with receptors or detecting proteins in cells.
in various solvent systems, and the formed triazole function-
ality is stable under hydrolysis and redox conditions.
[7]
Synthesis of peptides with a different number of alkyne
groups and various spacer lengths was accomplished by a
standard solid-phase peptide synthesis method using propar-
gylamine-containing glutamic acid (Fmoc-Glu(PA)-OH)
prepared according to Scheme 1. To control the spacer
To readily obtain glycoclusters with diverse spatial separa-
tion and various valences of sugars, we have developed an
efficient solid-phase synthetic strategy for fluorophore-la-
beled, peptide-based glycoclusters, which can be utilized to
fluorescently detect proteins in vitro and in vivo. By using
this strategy, 32 fluorescent glycoclusters with a valence (the
number of glycans attached to glycoconjugates) ranging
from 1 to 4 and different spatial arrangements of the glycans
were obtained. Microarray and fluorescence microscopy
analyses using these glycoclusters indicate that the valence
and spatial arrangements of sugar epitopes exert influence
on the binding affinities for immobilized and cell-surface
proteins.
Results and Discussion
Synthesis of Fluorophore-labeled Glycoclusters
Our synthetic procedure includes 1) assembly of fluoro-
phore-labeled, alkyne-conjugated peptides on a solid sup-
port, 2) coupling of azide-linked, unprotected sugars to the
alkyne-conjugated peptides on a solid support through click
chemistry, and 3) release of the glycoclusters from the solid
support. A linear peptide was used as a scaffold of glyco-
clusters because of its facile assembly on the solid support
and the easy control of the spacer length between sugar li-
gands. Azide-containing sugars were attached to alkyne-
linked peptides through click chemistry, as this reaction is
highly compatible with a broad range of functional groups
Scheme 1. Synthesis of Fmoc-Glu(PA)-OH. Abbreviation: HBTU=2-
(
1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,
HOBt=1-hydroxybenzotriazole, DIEA=diisopropylethylamine.
length, glycine, 4-aminobutanoic acid or 6-aminohexanoic
acid was inserted into the alkyne-containing residues (see
Scheme 1 in the Supporting Information). After assembly of
peptides on the solid support (PS-PEG Rink amide linker
resin) under HBTU-HOBt-DIEA conditions (HBTU=2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa-
fluorophosphate, HOBt=1-hydroxybenzotriazole, DIEA=
diisopropylethylamine), a fluorophore of cyanine 3 (Cy3)
was coupled to the peptides to fluorescently detect proteins
in vitro and in vivo. Fluorophore-containing peptides at-
tached to the solid support were then reacted with azidoe-
thylated mono-, di-, and trisaccharides under click chemistry
Abstract in Korean:
conditions (CuSO and sodium ascorbate). Azidoethylated
4
a-Man (a-mannose), a-Fuc (a-fucose), b-GlcNAc (b-N-ace-
tylglucosamine), and b-Lac (b-lactose) were prepared as
[8]
previously reported, and azidoethylated NeuNAca2,6Lac-
NAc (N-acetylneuraminosyl-a-2,6-N-acetyllactosamine) was
synthesized by enzymatic glycosylation of azidoethyl b-
LacNAc by a-2,6-sialyltransferase in the presence of CMP-
NeuNAc (cytidine monophosphate neuraminic acid). After
click chemistry, the assembled fluorescent glycoclusters were
released from the solid support by trifluoroacetic acid
(
TFA) and purified by reversed-phase HPLC. For lectin-
binding studies using protein microarrays and cell experi-
ments, a series of a-Man (1–10) and b-Lac (11–20) clusters
with a valence ranging from 1 to 4 and different spatial ar-
rangements of the sugar ligands were prepared (Figure 1).
In the case of glycoclusters containing a-Fuc (21–24), b-
GlcNAc (25–28), and NeuNAca2,6LacNAc (29–32), mono-
and tetravalent glycoclusters with different spacer lengths
2108
www.chemasianj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2107 – 2113

Fluorophore-labeled, Peptide-based Glycoclusters
Figure 1. Structure of synthesized fluorophore-labeled neoglycopeptides:
Sugar-mn (m=the number of a sugar ligand; n=the number of carbon
between NH and CO groups of the tether).
Figure 2. Fluorescence images of protein microarrays after probing with
0 mm of mannose-containing clusters (1–10). Graph: quantitative analysis
2
of fluorescence intensity of protein microarrays.
were synthesized for lectin-binding experiments with protein
microarrays.
probes exclusively recognized ConA, which is consistent
with the established specificity of this lectin (Figure 2).
[9d,10]
As expected, monovalent probe 1 rarely bound to ConA
owing to very weak interactions between a monovalent
mannose and the protein. It was also revealed that as the
tether lengthened, fluorescence intensities of mannose clus-
ters associated with ConA increased gradually. The best
mannose ligand among the tested probes for ConA was
found to be tetravalent probe 10 with 5 carbons between the
NH and CO groups of the tether. However, tetravalent
probes 8 and 9, with 1 and 3 carbons between the NH and
CO groups, interacted with ConA more weakly than di- (4)
and trivalent probes (7) with a longer spacer length, thereby
suggesting that the linker length of mannose clusters has a
Studies on Lectin-binding Properties of Glycoclusters Using
Protein Microarrays
Carbohydrate and lectin microarrays have recently received
considerable attention as powerful tools for the rapid analy-
[9]
sis of glycan–protein interactions. Binding properties of
the fluorophore-labeled glycoclusters towards lectins were
initially examined by using microarrays immobilized by vari-
ous carbohydrate-binding proteins. Lectin microarrays used
for this study were fabricated by printing six lectins (3.75,
7.5, and 15 mm) on N-hydroxysuccinimide (NHS) ester-deriv-
atized glass slides, prepared according to the known proce-
[9g]
[6b]
dure,
Maackia amurensis lectin II (MAL II, NeuNAca2,3Gal
binding protein), Ricinus communis agglutinin I (RCA120
with a pin-type microarrayer. The lectins include
significant effect on the ConA binding affinity. The micro-
arrays treated with lactose clusters (11–20, 40 mm) indicated
that these probes solely recognized RCA120 and the probes
with short spacer lengths (12, 15, 18) interacted with the
protein less tightly than those with longer spacers (Figure 3).
In these microarray experiments, tri- and tetravalent lactose
probes, with 3 and 5 carbons between the NH and CO
groups of the tether, bound to RCA120 with similar binding
affinities.
We then analyzed the fluorescence intensities of Fuc (21–
24), GlcNAc (25–28), and NeuNAca2,6LacNAc (29–32)
clusters bound to proteins after incubation of the microar-
rays with 20 mm glycoclusters. Fluorescent signals from Fuc
and GlcNAc clusters were observed for AAL and WGA at-
tached to the surface, respectively (Figure 4). The protein
microarrays incubated with NeuNAca2,6-LacNAc clusters
,
Gal binding protein), Aleuria aurantia lectin (AAL, Fuc
binding protein), Sambucus nigra lectin (SNA, NeuNAca2,6-
Gal binding protein), Concanavalin A (ConA, a-Man/a-Glc
binding protein), and Wheat germ agglutinin (WGA,
[10]
GlcNAc binding protein). After incubation of the printed
slides for 5 hours at room temperature in a humid chamber,
the slides were blocked with bovine serum albumin (BSA)
to suppress nonspecific interactions. The resulting slides
were treated with fluorescent glycoclusters 1–32 for 1 hour
at room temperature to evaluate their lectin-binding proper-
ties.
The results of microarray experiments using mannose-
containing glycoclusters 1–10 (20 mm) showed that these
Chem. Asian J. 2011, 6, 2107 – 2113
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2109

I. Shin et al.
FULL PAPERS
showed fluorescent signals in the regions of SNA and
RCA120. In this case, these glycoclusters interacted with
RCA120 with much weaker binding affinities in comparison
[11]
with SNA, which is consistent with the previous finding.
Analysis of fluorescence intensities of the microarrays also
indicated that monovalent glycan probes (21, 25, 29) very
weakly bound to the lectins under incubation conditions and
tetravalent probes with longer spacers recognized the corre-
sponding lectins more tightly than those with short ones.
Overall, the results obtained from protein microarrays show
that the binding affinities of glycoclusters to proteins are
highly dependent on the valence and spatial arrangements
of sugars.
Detection of Cell-surface Proteins in Bacteria
It has been known that many bacteria express carbohydrate-
[12]
binding proteins on the cell surface. Pathogenic properties
of the bacteria are caused by the initial binding to host cells
through the recognition events of bacterial proteins and
host glycans. To investigate the binding properties of glyco-
clusters toward cell-surface lectins in bacteria, 25 mm of mul-
tivalent mannose clusters 2–10 were incubated for 1 hour
with E. coli ORN178 strain, which produces a mannose-
Figure 3. Fluorescence images of protein microarrays after probing with
4
0 mm of lactose-containing clusters (11–20). Graph: quantitative analysis
of fluorescence intensity of protein microarrays.
[13]
binding protein, a gene product of fimH, on pili.
After
washing to remove unbound mannose clusters, bacterial
cells were imaged by using confocal fluorescence microsco-
py. It was found that a valence of a sugar ligand in glycoclus-
ters had influence on the binding to the cells. Tri- and tetra-
valent glycoclusters (5–10) recognized the cells more tightly
than divalent ones (2–4) (Figure 5a and Figure 1a in the
Supporting Information). Trivalent mannose probes (5–7)
showed a slightly lower binding affinity with the cells than
those with tetravalent probes (8–10). We also found that gly-
coclusters 2, 5, and 8, with short spacer lengths, weakly in-
teracted with proteins in bacteria, in comparison with those
with a longer tether. These findings indicate the importance
of the valence of a sugar ligand and the nature of the tether
in glycoclusters for the strong binding to bacterial cells. Fi-
nally, the specific binding of mannose clusters to bacterial
proteins was investigated by competition experiments. Bind-
ing of tetravalent mannose probes (8, 9, and 10, 25 mm) to
bacterial proteins was inhibited by pre-incubation of the
bacteria with 20 mm mannose for 1 hour (IC50 of free man-
nose to inhibit 10 binding to mannose-binding proteins on
E. coli ORN178 was measured to be 400 mm, see Figure 1b,
in the Supporting Information), thereby demonstrating that
the mannose probes selectively interact with mannose-bind-
ing proteins on the bacterial cell surface (Figure 5b).
Evaluation of Uptake of Lactose-containing Clusters by
Mammalian Cells
Hepatocytes abundantly express an asialoglycoprotein re-
ceptor (ASGP-R) on the cell surface, which binds to termi-
nal galactose or N-acetylgalactosamine on glycoproteins
Figure 4. Fluorescence images of protein microarrays after probing with
2
0 mm of glycoclusters 21–32. Graph: quantitative analysis of fluorescence
[14]
intensity of protein microarrays.
through multivalent interactions. This binding event trig-
2110
www.chemasianj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2107 – 2113

Fluorophore-labeled, Peptide-based Glycoclusters
Figure 5. a) Confocal fluorescence microscopy images of E. coli ORN178 incubated with 25 mm mannose-containing clusters 2–10 for 1 h. b) Confocal
fluorescence microscopy images of E. coli ORN178 pre-treated with 20 mm mannose for 1 h followed by further incubation with 25 mm mannose-contain-
ing clusters 8–10 for 1 h (bar=10 mm).
gers the receptor-mediated endocytotic uptake of the glyco-
proteins into cells, which are then transported to the lyso-
some where they are degraded. Thus, ASGP-R is involved
in the clearance of Gal/GalNAc-terminated glycoproteins
from circulation into hepatocytes.
treated cells revealed that the number of lactose ligands in
the glycoclusters was important for uptake by HepG2 cells
(Figure 6). However, lactose ligands having the same va-
lence with different lengths of the tether were internalized
into cells to a similar degree, suggesting that the nature of
the tether plays a very small role in the uptake of lactose-
containing probes by cells.
To analyze the uptake of lactose-containing probes by
cells through receptor-mediated endocytosis, we used the
hepatocellular carcinoma cell line HepG2 that produces
To further examine whether lactose-containing glycoclus-
ters enter cells through ASGP-R-mediated endocytosis,
HepG2 cells were pre-treated with galactose (2 or 20 mm)
or lactose (2 or 20 mm) as a competitor for 1 hour at 378C,
and then incubated with 20 (20 mm) for an additional 3 hours
at 378C. The uptake of 20 by cells was inhibited by pre-incu-
bation with galactose and lactose in a competitor concentra-
tion-dependent manner (Figure 7a and b), suggesting that
galactose, lactose, and 20 share a common cellular receptor,
ASGP-R. The results also showed that lactose was a more
effective competitor than galactose, which is consistent with
the previous finding that galactose has a lower binding affin-
[14]
abundant ASGP-R but not the mannose receptor.
The
HepG2 cells were incubated with 20 mm of multivalent lac-
tose probes 12–20 for 3 hours at 378C. After washing to
remove the unbound glycoclusters, fluorescence intensity of
cells was measured by using an ArrayScan Reader (Cello-
mics). Quantitative analysis of fluorescence intensity of the
[15]
ity for ASGP-R than lactose.
To ascertain the uptake of lactose probes by cells through
receptor-mediated endocytosis, HepG2 cells were incubated
with 20 (20 mm) for 4 hours at 48C, as the receptor-mediated
[16]
endocytosis is suppressed at low temperature. A remarka-
bly reduced fluorescence intensity was observed, thus indi-
cating that the uptake of 20 by cells was inhibited by low
temperature incubation (Figure 7c). These results, together
with competition results, suggest that lactose-containing gly-
coclusters are taken up by the HepG2 cells through ASGP-
R-mediated endocytosis.
Figure 6. Fluorescence microscopy images of HepG2 cells incubated with
2
0 mm lactose-containing clusters 12–20 for 3 h. Graph: quantitative anal-
ysis of fluorescence intensity of the treated HepG2 cells (bar=50 mm).
Chem. Asian J. 2011, 6, 2107 – 2113
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2111

I. Shin et al.
FULL PAPERS
click chemistry, azide-linked mono-, di-, and trisaccharides (2.0 equiv per
triple bond) dissolved in DMF (300 mL) were added to the fluorophore-
labeled peptide resin and then sodium ascorbate (1.0 equiv per triple
bond) and CuSO (1.0 equiv per triple bond) in water (50 mL) was added
4
to the reaction mixture. After 24 h, fluorophore-labeled glycopeptides
were cleaved from the solid support by treatment with TFA-triethylsilane
(
TES; 98:2) for 2 h. The fluorescent neoglycopeptides were analyzed by
analytical RP-HPLC with a gradient of 5–100% CH CN (0.1% TFA) in
3
water (0.1% TFA) over 45 min and the purified products were character-
ized by MALDI-TOF MS (Table 1, see the Supporting Information).
Lectin-binding Properties of Glycoclusters using Protein Microarrays
Proteins (ConA, WGA, AAL, SNA, MAL II, RCA120) used for this
study were dissolved in sodium phosphate buffer (pH 8.2) containing
4
0% glycerol. Solutions of proteins (1 nL, 15 mm) from a 384-well plate
were printed in a predetermined place on a NHS-derivatized glass slide
[
9g]
prepared according to the procedure reported previously (a distance of
40 mm between the centers of adjacent spots) by using a pin-type micro-
2
TM
arrayer (Cartesian MicroSys 5100 PA). After completion of printing,
the slide was placed into a humid chamber (60%) at room temperature
for 5 h. Then, a compartmentalized plastic film, which is coated by adhe-
sive on one side (thickness: 0.2 mm), was attached to the glass slide. The
slide was inversely immersed into PBS (pH 7.4) containing 0.1%
Tween 20 to prevent spot spreading and washed with gentle shaking by
hand. The solution (15–20 mL) of PBS (pH 7.4) containing 0.1%
Tween 20 and 1% BSA was dropped onto each block compartmented by
a plastic film and then incubated for 0.5–1 h. The solution was removed
by washing the slide with PBS containing 0.1% Tween 20 (30 mL, 3ꢁ
Figure 7. a) Fluorescence microscopy images of HepG2 cells incubated
with 20 mm galactose or 20 mm lactose for 1 h at 378C, and then incubat-
ed with 20 mm 20 for additional 3 h at 378C (bar=50 mm). b) Quantitative
analysis of fluorescence intensity of HepG2 cells after incubation with a
competitor and 20. c) Quantitative analysis of fluorescence intensity of
HepG2 cells incubated with 20 at 378C or 48C for 4 h.
1
0 min). The fabricated protein microarrays were used immediately to
Conclusions
get reproducible results.
Solutions (15–20 mL) of fluorophore-labeled neoglycopeptides (20–40 mm)
in PBS (pH 7.4) containing 0.1% Tween 20 (in the case of ConA, 0.5 mm
MnCl and 0.5 mm CaCl were added to the solution) were dropped into
2 2
each block compartment of the plastic film and then incubated for 1 h.
The unbound glycoclusters were removed by washing the slide with PBS
containing 0.1% Tween 20 (30 mL, 3ꢁ10 min). The slide was scanned
using an ArrayWoRx scanner.
We have developed an efficient method for the preparation
of fluorophore-labeled glycoclusters with various valences
and different spatial arrangements of the sugars. The syn-
thetic fluorescent glycoclusters were employed for probing
lectin-binding properties using protein microarrays, detec-
tion of proteins on the bacterial cell surface, and evaluation
of uptake of glycoclusters by mammalian cells through re-
ceptor-mediated endocytosis. The results obtained from in
vitro and in vivo experiments indicate that the binding affin-
ities of free proteins and cell-surface proteins are highly de-
pendent on the valence and spatial arrangements of sugar li-
gands in glycoclusters. We believe that this synthetic strategy
can be expanded in the future for preparation of more di-
verse glycoclusters for basic biological research to under-
stand glycan–protein interactions as well as for development
of effective inhibitors to block these biomolecular interac-
tions.
Detection of Bacterial Cells
E. coli ORN178 cells were grown overnight at 378C in LB medium in
8
À1
order to attain an optical density of approximately 1.0 (~10 cellsmL
)
at 600 nm (OD600). The cell culture was centrifuged at 13000 rpm for
30 sec, washed with PBS buffer and spun down twice, and finally sus-
pended in PBS buffer. E. coli ORN178 cells were incubated with 25 mm
mannose-containing clusters (1–10) in PBS for 1 h at room temperature
with gentle shaking and then centrifuged to collect the cell pellet. The su-
pernatant was discarded and the pellet was resuspended in the same
buffer. The process was repeated three times to remove unbound glyco-
clusters. The E. coli cells were imaged by using confocal fluorescence mi-
croscopy (LSM510 META, Carl Zeiss, Berlin, Germany). Fluorescence
intensity was quantitatively measured by using NIS-Elements D 3.2 soft-
ware (Nikon, Japan).
For determination of IC50, E. coli ORN178 strain was incubated with var-
ious concentrations (0, 0.1, 0.3, 0.5, 0.7, 1, 5, 10 mm) of free mannose in
PBS for 1 h at 378C and then treated with 25 mm 10 in PBS for 1 h. After
washing twice with PBS, fluorescence intensity was quantitatively mea-
sured by using a Typhoon 9410 scanner (GE, Germany) and ImageQuant
software.
Experimental Section
Synthesis of Fluorophore-labeled Neoglycopeptides
Fluorophore-labeled neoglycopeptides were synthesized by Fmoc/tBu
strategy on PS-PEG Rink amide linker resin (0.25 mmolg ): Fmoc
amino acid (3 equiv) was manually coupled on the resin (5.0 mmol) in the
presence of HBTU (3 equiv), HOBt (3 equiv), and DIEA (6 equiv).
Fmoc group was removed by treatment with 20% piperidine in N,N-di-
methylformamide (DMF) and the resin was washed with DMF and
Cl several times. For coupling of Cy3 to the peptides, Cy3
2 2
3.0 equiv) was preactivated with HOAt (3.0 equiv), HATU (2.9 equiv),
and DIEA (6.0 equiv) in DMF (400 mL) for 10 min. The activated Cy3
was then added to the N-terminal amino-containing resin and shaken for
À1
Evaluation of Uptake of Lactose-containing Clusters by Mammalian Cells
The hepatocellular carcinoma cell line HepG2 that expresses ASGP-R
5
was seeded in a 24-well plate at a density of ~10 cells per well and cul-
tured in DMEM supplemented with 10% fetal bovine serum (FBS).
After 24 h, the cells were incubated with 20 mm lactose-containing glyco-
clusters (12–20) in culture media for 3 h at 378C. After washing twice
with Dulbeccoꢂs Phosphate Buffered Saline (DPBS, without calcium and
magnesium) to remove unbound glycoclusters, the cells were fixed with
3.7% formaldehyde containing 4,6-diamidino-2-phenyllindole (DAPI,
CH
(
6
h. For coupling of sugars to the fluorophore-conjugated peptides by
2112
www.chemasianj.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 2011, 6, 2107 – 2113

Fluorophore-labeled, Peptide-based Glycoclusters
À1
1
mgmL ) as the internal control for the ArrayScan VTI HCS Reader.
i) A. H. Courtney, E. B. Puffer, J. K. Pontrello, Z.-Q. Yang, L. L.
Kiessling, Proc. Natl. Acad. Sci. USA 2009, 106, 2500–2505; j) C.-C.
Lin, Y.-C. Yeh, C.-Y. Yang, C.-L. Chen, G.-F. Chen, C.-C. Chen, Y.-
C. Wu, J. Am. Chem. Soc. 2002, 124, 3508–3509; k) K. El-Boubbou,
C. Gruden, X. Huang, J. Am. Chem. Soc. 2007, 129, 13392–13393;
l) R. Kikkeri, B. Lepenies, A. Adibekian, P. Laurino, P. H. Seeberg-
er, J. Am. Chem. Soc. 2009, 131, 2110–2112; m) J. M. de La Fuente,
A. G. Barrientos, T. C. Rojas, J. Rojo, J. CaÇada, A. Fernꢃndez, S.
Penadꢄs, Angew. Chem. 2001, 113, 2317–2321; Angew. Chem. Int.
Ed. 2001, 40, 2257–2261; n) S. I. van Kasteren, S. J. Campbell, S.
Serres, D. C. Anthony, N. Sibson, B. G. Davis, Proc. Natl. Acad. Sci.
USA 2009, 106, 18–23; o) D. H. Charych, J. O. Nagy, W. Spevak,
M. D. Bednarski, Science 1993, 261, 585–588; p) C.-H. Lai, C.-Y.
Lin, H.-T. Wu, H.-S. Chan, Y.-J. Chuang, C.-T. Chen, C.-C. Lin, Adv.
Funct. Mater. 2010, 20, 3948–3958.
After washing twice with DPBS, the cells were imaged by fluorescence
microscopy (Eclipse TE2000-S, Nikon, Japan) or ArrayScan VTI HCS
Reader with a 20X objective (Cellomics, USA). Fluorescence microsco-
py: the excitation wavelength range was from 510 to 560 nm for Cy3 and
from 330 to 380 nm for DAPI. ArrayScan VTI HCS Reader: Target Acti-
vation BioApplication was used to acquire and analyze the images.
Images of 1000 cells for each probe were analyzed to obtain the average
cell number per field, fluorescence area and intensity per cell.
For competition experiments, HepG2 cells were seeded in a 24-well plate
at a density of ~10 cells per well and cultured in DMEM supplemented
with 10% FBS. After 24 h, the cells were incubated with galactose (2 or
0 mm) or lactose (2 or 20 mm) for 1 h at 378C. The cells were then treat-
ed with 20 (20 mm) in culture media and incubated for 3 h at 378C. After
washing twice with DPBS, the cells were fixed with 3.7% formaldehyde
containing 1 mgmL DAPI. After washing twice with DPBS, the cells
were imaged and evaluated by fluorescence microscopy or ArrayScan
VTI HCS Reader with a 20X objective.
5
2
À1
[7] a) M. Meldal, C. W. Tornøe, Chem. Rev. 2008, 108, 2952–3015; b) A.
Dondoni, Chem. Asian J. 2007, 2, 700–708.
8] a) S. Park, I. Shin, Org. Lett. 2007, 9, 1675–1678; b) S. Park, M.-R.
Lee, I. Shin, Nat. Protoc. 2007, 2, 2747–2758.
9] a) S. Park, M.-R. Lee, I. Shin, Chem. Commun. 2008, 4389–4399;
b) T. Horlacher, P. H. Seeberger, Chem. Soc. Rev. 2008, 37, 1414–
1422; c) J. C. Paulson, O. Blixt, B. E. Collins, Nat. Chem. Biol. 2006,
[
For confirmation of the receptor-mediated uptake mechanism, HepG2
cells were incubated with 20 (20 mm) for 4 h at 48C to block receptor-
mediated endocytosis. After washing twice with DPBS, the cells were
fixed with 3.7% formaldehyde containing 1 mgmL DAPI. After wash-
ing twice with DPBS, the cells were imaged and evaluated by ArrayScan
VTI HCS Reader with a 20X objective.
[
À1
2
, 238–248; d) S. Park, M.-R. Lee, S.-J. Pyo, I. Shin, J. Am. Chem.
Soc. 2004, 126, 4812–4819; e) A. Kuno, N. Uchiyama, S. Koseki-
Kuno, Y. Ebe, S. Takashima, M. Yamada. J. Hirabayashi, Nat. Meth-
ods 2005, 2, 851–856; f) K. L. Hsu, K. T. Pilobello, L. K. Mahal, Nat.
Chem. Biol. 2006, 2, 153–157; g) M.-R. Lee, S. Park, I. Shin, Bioorg.
Med. Chem. Lett. 2006, 16, 5132–5135; h) X. Z. Song, B. Y. Xia, Y.
Lasanajak, D. F. Smith, R. D. Cummings, Glycoconjugate J. 2008, 25,
Acknowledgements
1
5–25; i) T. Feizi, F. Fazio, W. C. Chai, C. H. Wong, Curr. Opin.
Struct. Biol. 2003, 13, 637–645; j) D. N. Wang, S. Y. Liu, B. J.
Trummer, C. Deng, A. L. Wang, Nat. Biotechnol. 2002, 20, 275–281.
This work was supported by grants from the National Creative Research
Initiative and WCU programs (R32-2008-000-10217-0). Escherichia coli
ORN 178 and ORN 208 strains were kindly provided by Professor P. E.
Orndorff (North Carolina State Univ.).
[
10] a) I. E. Liener, N. Sharon, I. J. Goldstein, The Lectins: Properties,
Functions, and Applications in Biology and Medicine, Academic
Press, Orlando, 1986, pp. 35–244; b) E. J. M. Van Damme, W. J.
Psumans, A. Pusztai, S. Bardocz, Handbook of Plant Lectins: Prop-
erties and Biomedical Applications, Wiley, Chichester, UK, 1998.
11] S. S. Spicer, S. L. Erlandsen, A. C. Wilson, M. F. Hammer, R. A.
Hennigar, B. A. Schulte, J. Histochem. Cytochem. 1987, 35, 1231–
[
1] a) C. R. Bertozzi, L. L. Kiessling, Science 2001, 291, 2357–2364;
b) A. Varki, Glycobiology 1993, 3, 97–130; c) S. Park, M.-R. Lee, I.
Shin, Chem. Soc. Rev. 2008, 37, 1579–1591.
[
[
[
1
244.
[2] P. R. Crocker, Curr. Opin. Pharmacol. 2005, 5, 431–437.
[3] A. E. Smith, A. Helenius, Science 2004, 304, 237–242.
[4] a) M. M. Fuster, J. D. Esko, Nat. Rev. Cancer 2005, 5, 526–542;
b) E. E. Simanek, G. J. McGarvey, J. A. Jablonowski, C. H. Wong,
Chem. Rev. 1998, 98, 833–862.
12] a) K.-A. Karlsson, Curr. Opin. Struct. Biol. 1995, 5, 622–635;
b) M. D. Disney, J. Zheng, T. T. Swager, P. H. Seeberger, J. Am.
Chem. Soc. 2004, 126, 13343–13346.
13] a) H. Connell, W. Agace, P. Klemm, M. Schembri, S. Marild, C.
Svanborg, Proc. Natl. Acad. Sci. USA 1996, 93, 9827–9832; b) S. L.
Harris, P. A. Spears, E. A. Havell, T. S. Hamrick, J. R. Horton, P. E.
Orndorff, J. Bacteriol. 2001, 183, 4099–4102; c) N. Nagahori, R. T.
Lee, S.-I. Nishimura, D. Pagꢄ, R. Roy, Y. C. Lee, ChemBioChem
[
5] a) M. Mammen, S.-K. Choi, G. M. Whitesides, Angew. Chem. 1998,
1
10, 2908–2953; Angew. Chem. Int. Ed. 1998, 37, 2754–2794; b) J. J.
Lundquist, E. J. Toone, Chem. Rev. 2002, 102, 555–578; c) L. L.
Kiessling, J. E. Gestwicki, L. E. Strong, Angew. Chem. 2006, 118,
2
002, 3, 836–844; d) S. Park, M.-R. Lee, I. Shin, Bioconjugate
2
408–2429; Angew. Chem. Int. Ed. 2006, 45, 2348–2368; d) R. J. Pi-
Chem. 2009, 20, 155–162.
eters, Org. Biomol. Chem. 2009, 7, 2013–2025; e) M. M. Lee, J. L.
Childs-Disney, A. Pushechnikov, J. M. French, K. Sobczak, C. A.
Thornton, M. D. Disney, J. Am. Chem. Soc. 2009, 131, 17464–17472;
f) M. M. Lee, A. Pushechnikov, M. D. Disney, ACS Chem. Biol.
[
14] a) Y. Higuchi, M. Oka, S. Kawakami, M. Hashida, J. Controlled Re-
lease 2008, 125, 131–136; b) K. Drickamer, M. E. Taylor, Annu. Rev.
Cell Biol. 1993, 9, 237–264; c) J. Stockert, A. G. Morell, I. H.
Scheinberg, Science 1974, 186, 365–366; d) D. T. Connolly, R. R.
Townsend, K. Kawaguchi, W. R. Bell, Y. C. Lee, J. Biol. Chem. 1982,
2
009, 4, 345–355.
[
6] a) V. Wittmann, S. Seeberger, Angew. Chem. 2000, 112, 4508–4512;
Angew. Chem. Int. Ed. 2000, 39, 4348–4352; b) D. Schwefel, C.
Maierhofer, J. G. Beck, S. Seeberger, K. Diederichs, H. M. Mçller,
W. Welte, V, Wittmann, J. Am. Chem. Soc. 2010, 132, 8704–8719;
c) Y. Koshi, E. Nakata, M. Miyagawa, S. Tsukiji, T. Ogawa, I. Hama-
chi, J. Am. Chem. Soc. 2008, 130, 245–251; d) M.-R. Lee, D.-W.
Jung, D. Williams, I. Shin, Org. Lett. 2005, 7, 5477–5480; e) B. N.
Murthy, N. H. Voelcker, N. Jayaraman, Glycobiology 2006, 16, 822–
2
57, 939–945; e) A. L. Schwartz, S. E. Fridovich, H. F. Lodish, J.
Biol. Chem. 1982, 257, 4230–4237; f) A. L. Schwartz, A. Bolognesi,
S. E. Fridovich, J. Cell Biol. 1984, 98, 732–738.
[
15] a) M. Mammen, S.-K. Choi, G. M. Whitesides, Angew. Chem. 1998,
1
10, 2908–2953; Angew. Chem. Int. Ed. 1998, 37, 2754–2794; b) K.
Ozaki, R. T. Lee, Y. C. Lee, T. Kawasaki, Glycoconjugate J. 1995, 12,
68–274; c) A. David, P. Kope cˇ kovꢃ, A. Rubinstein, J. Kope cˇ cek,
2
Bioconjugate Chem. 2001, 12, 890–899.
8
32; f) N. Frison, M. E. Taylor, E. Soilleux, M. T. Bousser, R. Mayer,
M. Monsigny, K. Drickamer, A. C. Roche, J. Biol. Chem. 2003, 278,
3922–23929; g) X. Chen, U. C. Tam, J. L. Czlapinski, G. S. Lee, D.
Rabuka, A. Zettl, C. R. Bertozzi, J. Am. Chem. Soc. 2006, 128,
[
16] a) W. A. Dunn, A. L. Hubbard, N. N. Aronson, Jr., J. Biol. Chem.
1
980, 255, 5971–5978; b) S. C. Mueller, A. L. Hubbard, J. Biol.
2
Chem. 1986, 261, 932–942.
6
1
292–6293; h) G. Pasparakis, C. Alexander, Angew. Chem. 2008,
20, 4925–4928; Angew. Chem. Int. Ed. 2008, 47, 4847–4850;
Received: March 31, 2011
Published online: June 1, 2011
Chem. Asian J. 2011, 6, 2107 – 2113
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2113