Analysis of density-dependent binding of glycans by lectins using carbohydrate microarrays

Article abstract of DOI:10.1002/asia.201200202

To investigate the density-dependent binding of glycans by lectins using carbohydrate microarrays, a number of C-terminal hydrazide-conjugated neoglycopeptides with various valences and different spatial arrangements of the sugar ligands were prepared on a solid support. The synthetic strategy includes (1) assembly of alkyne-linked peptides possessing C-terminal hydrazide on a solid support, (2) coupling of azide-linked, unprotected sugars to the alkyne-linked peptides on the solid support utilizing click chemistry, and (3) release of the neoglycopeptides from the solid support. By using this synthetic methodology, sixty five neoglycopeptides with a valency ranging from 1 to 4 and different spatial arrangements of the carbohydrate ligands were generated. Carbohydrate microarrays were constructed by immobilizing the prepared neoglycopeptides on epoxide-derivatized glass slides and were used to analyze the density-dependent binding of glycans by lectins. The results of binding property determinations show that lectin binding is highly dependent on the surface glycan density. Copyright

Full text of DOI:10.1002/asia.201200202

DOI: 10.1002/asia.201200202
Analysis of Density-Dependent Binding of Glycans by Lectins Using
Carbohydrate Microarrays
[
a]
Xizhe Tian, Jaeyoung Pai, and Injae Shin*
th
Dedicated to Professor Hung-wen Liu on the occasion of his 60 birthday
Abstract: To investigate the density-de-
pendent binding of glycans by lectins
using carbohydrate microarrays,
ed sugars to the alkyne-linked peptides
on the solid support utilizing click
chemistry, and (3) release of the neo-
glycopeptides from the solid support.
By using this synthetic methodology,
sixty five neoglycopeptides with a va-
lency ranging from 1 to 4 and different
spatial arrangements of the carbohy-
drate ligands were generated. Carbohy-
drate microarrays were constructed by
immobilizing the prepared neoglyco-
peptides on epoxide-derivatized glass
slides and were used to analyze the
density-dependent binding of glycans
by lectins. The results of binding prop-
erty determinations show that lectin
binding is highly dependent on the sur-
face glycan density.
a number of C-terminal hydrazide-con-
jugated neoglycopeptides with various
valences and different spatial arrange-
ments of the sugar ligands were pre-
pared on a solid support. The synthetic
strategy includes (1) assembly of
alkyne-linked peptides possessing C-
terminal hydrazide on a solid support,
Keywords: carbohydrates
·
click
chemistry · immobilization · lectins ·
microarrays
(
2) coupling of azide-linked, unprotect-
Introduction
ꢀchelate effectꢁ where multivalent glycans interact with
[3b]
a lectin possessing multiple glycan binding sites
and the
The recognition of glycans present in the form of glycocon-
jugates (e.g., glycoproteins, glycosphingolipids, and proteo-
glycans) by proteins in cells is a crucial biological event that
is implicated in various physiological and pathological pro-
ꢀbind and slideꢁ mechanism where lectins diffuse between
[3c]
glycans in the multivalent ligands before dissociation. In
addition, the increased local concentration of glycans con-
tributes to the enhanced binding affinity promoted by multi-
valent interactions.
[1]
cesses. Thus, understanding the role of glycan-mediated
recognition events and blocking glycan–protein interactions
associated with diseases, such as inflammation, cancer, and
bacterial and viral infection, are of great importance for
both biological research and therapeutic applications. In
general, carbohydrate-binding proteins (lectins) interact
with monovalent sugars with weak binding affinity (micro-
Since 2002, a number of research groups including our
own have developed carbohydrate microarrays as high-
throughput analytic tools for functional studies of glycans
because the microarray-based technology has the advantage
of enabling the simultaneous analysis of many glycan–pro-
[4–6]
tein interactions using small amounts of samples.
The
[2]
to millimolar range) and low selectivity.
most extensive use of carbohydrate microarrays is to rapidly
analyze the specificities of carbohydrate-binding proteins
(lectins, antibodies, cytokines, chemokines, and growth fac-
To enhance the binding affinity and selectivity in natural
environments, lectins generally form oligomers and/or pos-
sess multiple binding sites per monomer (see below). These
phenomena lead to multivalent interactions between glycan
ligands and proteins. Several mechanisms have been pro-
posed to explain the enhanced binding affinity promoted by
[6]
tors). The microarray technology has been also utilized to
[5]
examine mammalian cell–glycan interactions.
Further-
more, carbohydrate microarrays have been employed to
detect pathogens and disease-related anti-glycan antibodies
[3]
[6l]
multivalent interactions. These mechanisms include the
for diagnosis. The microarray technology has been further-
more used for the fast assessment of acceptor specificities of
glycosyltransferases and for measurement of the relative
rates of glycosylation of substrates by these enzymes. An-
other interesting application of carbohydrate microarrays in-
volves the quantitative analysis of protein-binding affinities
+
+
[
a] X. Tian, J. Pai, Prof. Dr. I. Shin
Center for Biofunctional Molecules
Department of Chemistry
Yonsei University
[6j]
Seoul 120-749 (Korea)
Fax : (+82)2-364-7050
E-mail: injae@yonsei.ac.kr
such as IC50 values of soluble inhibitors and K values (dis-
d
[6a,j,k]
sociation constants) for glycan–protein interactions.
+
It has been suggested that glycans on the solid surface can
interact with proteins in a similar way as when they are lo-
cated on the cell surface and that carbohydrate microarrays
[
] These two authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201200202.
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are therefore suitable for the investigation of features of
[5g]
binding events in vivo. In the microarray format, glycans
are densely immobilized on the solid surface and thus inter-
and intramolecular binding to lectins both take place unlike
glycan binding to lectins in solution, where both intra- and
intermolecular interactions do not occur so readily. Al-
though it has been shown that glycans on the microarrays
act multivalently in glycan–protein interactions, a systematic
analysis of the density-dependence of binding of glycans by
lectins using carbohydrate microarrays is needed to better
understand the nature and consequence of glycan–protein
interactions. Thus, we have constructed carbohydrate micro-
arrays containing various neoglycopeptides that have a va-
lency ranging from 1 to 4 and different spatial arrangements
of the glycan ligands. By using a single carbohydrate micro-
array, a total of 260 glycan–protein interactions were simul-
taneously investigated. The observations made in this effort
are described below.
Scheme 1. Synthesis of Fmoc-Glu(PA)-OH.
(HBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroni-
um hexafluorophosphate, HOBt=1-hydroxybenzotriazole,
DIEA=diisopropylethylamine), the alkyne-conjugated pep-
tides were reacted with several azidoethylated sugars under
Results and Discussion
click conditions (CuSO and sodium ascorbate). Azidoethy-
4
lated sugars (Fuc-a, GlcNAc-b, Man-a, a-1,6-mannobiose-a,
[8]
Recently, different carbohydrate microarrays, that is, immo-
bilized peptide nucleic acid-conjugated glycans, DNA-linked
glycans, glycodendrimers, and neoglycoproteins with varying
glycan density, have been fabricated to study the density-de-
and LacNAc-b) were prepared as previously reported.
After click chemistry, the assembled neoglycopeptides were
released from the solid support by trifluoroacetic acid
(TFA) and purified by reversed-phase HPLC. Based on this
synthetic methodology, sixty five neoglycopeptides with a va-
lency ranging from 1 to 4 and different spatial arrangements
of the carbohydrate ligands were obtained (Table 1 and
Table S1 in the Supporting Information).
[
7]
pendent glycan binding properties of lectins. In these gly-
coconjugates, the number of glycans was varied but distan-
ces between glycans in the glycoconjugates were not system-
atically varied. In addition, the preparation of the glycocon-
jugates is often a difficult process and their structures are in
some cases not well defined, both of which makes the analy-
sis of glycan–protein interactions difficult. To easily prepare
glycocluster probes in which the number of glycans and the
distance between sugar ligands are readily controlled, we
used a linear peptide as a scaffold for multivalent glycocon-
jugates.
Our synthetic strategy includes assembly of alkyne-linked
peptides possessing a C-terminal hydrazide on a solid sup-
port, coupling of azide-conjugated sugars to the alkyne-
linked peptides on the solid support utilizing click chemistry,
and release of the neoglycopeptides from the solid support
The carbohydrate microarrays used in this study were fab-
ricated by printing various concentrations (0.25, 0.5, 1.0, and
2.0 mm) of 65 neoglycopeptides bearing C-terminal hydra-
zide groups on an epoxide-derivatized glass slide prepared
[6d,j,l]
as described previously.
The resulting microarrays were
treated with Cy3-labeled lectins (concanavalin A, ConA; Ri-
cinus communis agglutinin I, RCA120; Aleuria aurantia
lectin, AAL; wheat germ agglutinin, WGA) to evaluate
density-dependent glycan binding properties.
ConA is an a-Man/a-Glc binding protein composed of
four identical subunits at pH 7 (ca. 26 kDa per monomer).
This lectin has one sugar-binding site per monomer with the
binding sites in the tetramer being separated by 65–70 ꢃ
[
8]
(
Scheme S1 in the Supporting Information). Since the hy-
[9]
drazide group is known to react selectively and efficiently
(Figure 1a). The results of the microarray experiments
using ConA show that this protein selectively recognizes
sugars possessing a-mannose units (a-mannose and a-1,6-
mannobiose) with similar binding affinities (Figure 2a and
[6d,j,l]
with an epoxide on the solid surface,
this functional
group was conjugated to the C-terminus of neoglycopeptides
for the construction of carbohydrate microarrays.
[6l,10]
The preparation of peptides with a different number of
alkyne groups and various spacer lengths was accomplished
by using propargylamine-containing glutamic acid (Fmoc-
Glu(PA)-OH), which was prepared according to Scheme 1,
in a standard solid-phase peptide synthesis protocol. To vary
the spacer length, glycine, 4-aminobutanoic acid, 6-amino-
hexanoic acid, or 8-aminooctanoic acid was inserted into the
alkyne-containing residues (Scheme S1 in the Supporting In-
formation). After assembly of peptides on the solid support
2e).
Interestingly, the length of the tether between the
glycans and the number of sugars in the neoglycopeptide
significantly affects the binding affinity of ConA to the gly-
cans. Specifically, the binding gradually increases with in-
creasing valency and tether length. Because the maximal
distance between glycans on the neoglycopeptide is less
than 70 ꢃ (Figure S1a in the Supporting Information) and
sugar-binding sites in the tetrameric Con A are located re-
motely, intramolecular multivalent interactions between
binding sites of the protein and glycans in the same neogly-
(
Wang resin) under HBTU–HOBt–DIEA conditions
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Density-Dependent Binding of Glycans by Lectins
Table 1. Structure of neoglycopeptides used for the construction of carbohydrate microarrays. Symbol: Sugar-mn (m: the number of sugar residues, n:
the number of carbons between NH and CO groups of the tether).
No
Symbol
No
Symbol
No
Symbol
No
Symbol
No
Symbol
1
2
3
4
5
6
7
8
9
Fuc-10
Fuc-21
Fuc-23
Fuc-25
Fuc-27
Fuc-31
Fuc-33
Fuc-35
Fuc-37
Fuc-41
Fuc-43
Fuc-45
Fuc-47
14
15
16
17
18
19
20
21
22
23
24
25
26
Mannobiose-10
Mannobiose-21
Mannobiose-23
Mannobiose-25
Mannobiose-27
Mannobiose-31
Mannobiose-33
Mannobiose-35
Mannobiose-37
Mannobiose-41
Mannobiose-43
Mannobiose-45
Mannobiose-47
27
28
29
30
31
32
33
34
35
36
37
38
39
Mannose-10
Mannose-21
Mannose-23
Mannose-25
Mannose-27
Mannose-31
Mannose-33
Mannose-35
Mannose-37
Mannose-41
Mannose-43
Mannose-45
Mannose-47
40
41
42
43
44
45
46
47
48
49
50
51
52
GlcNAc-10
GlcNAc-21
GlcNAc-23
GlcNAc-25
GlcNAc-27
GlcNAc-31
GlcNAc-33
GlcNAc-35
GlcNAc-37
GlcNAc-41
GlcNAc-43
GlcNAc-45
GlcNAc-47
53
54
55
56
57
58
59
60
61
62
63
64
65
LacNAc-10
LacNAc-21
LacNAc-23
LacNAc-25
LacNAc-27
LacNAc-31
LacNAc-33
LacNAc-35
LacNAc-37
LacNAc-41
LacNAc-43
LacNAc-45
LacNAc-47
10
11
12
13
copeptide should be geometrically difficult. It is likely that
intermolecular interactions between the tetrameric protein
and glycans in different neoglycopeptides are the major
factor leading to the enhancement in binding affinities. Pre-
sumably, glycans linked by a short tether on neoglycopepti-
des exert steric hindrance to protein binding and, according-
ly, neoglycopeptides with long tethers interact with the pro-
Next, the binding properties of lectins that have relatively
short distances between sugar-binding sites (AAL and
WGA) were investigated using the glycan microarrays.
AAL is a fucose-binding protein possessing two identical
subunits (36 kDa per monomer) and each monomer has five
sugar-binding sites with different binding affinity toward
fucose. The sugar-binding sites in the AAL monomer are
estimated to be separated by about 16 ꢃ (Figure 1c). The
findings show that this lectin selectively recognizes neogly-
copeptides containing fucose and that the tether length be-
tween fucose ligands influences the binding affinity of AAL
to the sugar. The fluorescence intensity of AAL bound to
fucose was observed to increase gradually with increasing
tether length (Figure 2c and 2g). Interestingly, AAL inter-
acts more strongly with the multivalent than the monovalent
fucose form and it has similar binding affinities to tri- and
tetravalent fucose. To validate these results, dissociation
[14]
[11]
tein more strongly than those with shorter ones.
Similar results were obtained in studies with RCA120
,
which is a galactose-binding protein. This lectin exists as
a 120 kDa tetramer (A B ) composed of two ricin-like
2
2
[
12]
dimers that are held together by noncovalent interactions.
The distance between the two sugar-binding sites in RCA
1
20
[13]
is approximately 110 ꢃ (Figure 1b). This lectin selectively
binds to immobilized LacNAc and the fluorescence intensity
of RCA120 associated with LacNAc increases gradually as
the tether length between glycan ligands and the number of
glycans in the neoglycopeptide is increased (Figure 2b and
constants (K ) were measured for complexes between AAL
d
2
f).
and di-, tri-, or tetravalent neoglycopeptides (5, 9, and 13,
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bilization density of neoglyco-
peptides on the surface, solu-
tions containing different ratios
of neoglycopeptides and an un-
liganded molecule, 6-(hydroxya-
cetamido)hexanoyl hydrazide,
[11]
were used. Solutions contain-
ing various ratios (1:0, 1:1, 1:5,
1
:10, 1:20) of neoglycopeptides
(LacNAc-47 (65), GlcNAc-47
(52), Man-47 (39), Manbio-47
(26), and Fuc-47 (13)) to the
unliganded
molecule
were
printed on the epoxide surface,
and the resulting microarrays
were incubated with Cy3-labled
AAL and WGA (Figure 4).
The findings show that both lec-
tins bind more tightly to the
sugars in microspots that con-
tain a high proportion of neo-
glycopeptides (short distances
between
neoglycopeptides)
than to those containing a low
proportion of neoglycopeptides
(
long distances). These results
indicate that immobilization
density of sugars influences
lectin binding to glycans on the
surface because a lower density
of a printed sugar leads to
Figure 1. Estimated distances between the sugar-binding sites in four lectins: a) ConA (PDB ID: 1QDO),
b) RCA120 (PDB ID: 1RZO), c) AAL (PDB ID: 1OFZ) and d) WGA (PDB ID: 2X52).
respectively, in Table 1) with the longest tether using carbo-
a lower binding affinity towards lectins.
[6j,k]
hydrate microarrays.
For this study, carbohydrate micro-
To further adjust the distance between neoglycopeptides
immobilized on the surface, cone-shaped dendron-modified
arrays with three neoglycopeptides immobilized were incu-
bated with various concentrations (0.61 nm–5 mm) of Cy3-la-
beled AAL. The Kd values for complexes between AAL
and di-, tri-, or tetravalent neoglycopeptides were deter-
[15]
glass slides were used (Figure 5a). It has been estimated
that the lateral distance between immobilized molecules is
about 0.5 nm on a conventionally modified glass slide used
À8
À8
À8
[6k,6 m,15b]
mined to be 9.1ꢄ10 m, 3.1ꢄ10 , and 2.7ꢄ10 m, respec-
tively (Figure 3), in agreement with the above results.
for microarray construction .
By contrast, the lateral
spacing between surface functional groups on second- and
third-generation dendron-modified glass slides was mea-
sured to be 3–4 nm and 6–7 nm, respectively.
WGA is a dimeric GlcNAc-binding protein with a molecu-
lar weight of 36 kDa that consists of two identical subunits.
This lectin contains eight sugar-binding sites (four binding
sites per monomer), and the sugar-binding sites are separat-
[15]
Fuc- or
GlcNAc-containing neoglycopeptides were printed on epox-
ide-modified surfaces of a typically modified glass slide and
on second- and third-generation dendron-modified surfaces.
The resulting microarrays were incubated with Cy3-AAL
and Cy3-WGA, respectively. The results show that glycans
immobilized on a normally modified surface are strongly
recognized by lectins, while those on the third-generation
dendron-modified surface interact very weakly with lectins
due to the long distance between the immobilized neoglyco-
peptides (Figure 5). The binding affinities of lectins to gly-
cans are weaker on the second-generation dendron-modified
surface than those on the normally modified surface, al-
though the valency- and tether length-dependent glycan-
binding properties of lectins are similar with both surfaces.
These results support the notion that multivalent interac-
tions between lectins and glycans in different neoglyopep-
[2d]
ed by about 14 ꢃ (Figure 1d). WGA recognizes GlcNAc
and LacNAc with slightly different binding affinities
[6j]
(
GlcNAc>LacNAc).
Similar to the observations for
AAL, the binding affinities of WGA to the sugars increases
gradually with increasing length of the tether (Figure 2d and
2
h). In addition, this protein binds more strongly to multiva-
lent sugars, with similar binding affinities for tri- and tetra-
valent sugars, than to the monovalent one. The results ob-
tained from the microarray experiments show that the acces-
sibility of a glycan ligand attached to the surface affects
lectin binding.
Experiments were performed to examine whether distan-
ces between neoglycopeptides immobilized on the surface
affect the binding properties of lectins. To control the immo-
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Density-Dependent Binding of Glycans by Lectins
Figure 2. Fluorescence images (a–d) and the corresponding quantitative analyses (e–h) of glycan microarrays after probing with Cy3-labeled lectins:
a,e) ConA, b,f) RCA120, c,g) AAL, and d,h) WGA. Sixty five different C-terminal hydrazide-linked neoglycopeptides were printed at various concentra-
tions (2, 1, 0.5, and 0.25 mm) on the epoxide-derivatized glass slide.
Figure 4. The effect of the immobilization density of neoglycopeptides on
lectin binding. Mixtures of a neoglycopeptide (LacNAc-47 (65), GlcNAc-
4
7 (52), Man-47 (39), Manbio-47 (26), and Fuc-47 (13)) and an unligand-
ed molecule, 6-(hydroxyacetamido)hexanoyl hydrazide, were printed at
various ratios (1:0, 1:1, 1:5, 1:10, and 1:20) on the epoxide surface and
the resulting microarrays were incubated with Cy3-labeled AAL (left)
and WGA (right). Values represent the means plus standard deviations
of triplicate experiments.
d
Figure 3. Determination of dissociation constants (K ) for the interaction
between AAL and surface-linked fucose-containing neoglycopeptides
using carbohydrate microarrays (Fuc-27 (5, diamonds), Fuc-37 (9,
squares), and Fuc-47 (13, triangles), see Table 1for their structures).
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Conclusions
Herein, we have developed
strategy to modulate the
a
glycan density on the surfaces
of carbohydrate microarrays.
By using carbohydrate microar-
rays containing neoglycopepti-
des with different numbers of
glycans and defined distances
between the glycans, we have
found that the binding affinities
of lectins for sugars are highly
glycan density-dependent. The
number of glycans on a neogly-
copeptide, the linker length be-
tween the individual sugars, and
the distance between neoglyco-
peptide probes on the surface
are all important factors deter-
mining the binding affinities of
lectins. We believe that our
work provides valuable infor-
mation concerning the design
and preparation of carbohy-
drate microarrays that are used
for functional studies of gly-
cans.
Experimental Section
Synthesis of Boc-Glu(Bn)-tBu
Di-tert-butyl-dicarbonate
.2 equiv) in 10 mL CH Cl
(8.16 mL,
was added
1
2
2
at room temperature to a stirred solu-
tion of Boc-Glu(Bn)-OH (10 g,
2
5
3.50 mmol) and DMAP (0.5 equiv) in
0 mL CH Cl . After 12 h, the mixture
Cl (200 mL),
2
2
was diluted with CH
2
2
washed with water (50 mLꢄ3) and
brine (50 mL), dried over anhydrous
Na SO , and concentrated under re-
2 4
duced pressure. The crude product
was purified by flash column chroma-
tography (n-hexane/EtOAc, 10:1 to
2
9
:1) to give Boc-Glu AHCNUTGTERNNUNG( OBn)-tBu in
1
0% yield.
H NMR (400 MHz,
Figure 5. a) Structure of second- and third-generation dendrons. b,c) Carbohydrate microarrays constructed by
printing Fuc-containing neoglycopeptides (b) and GlcNAc-containing neoglycopeptides (c) on typically modi-
fied (left), second-generation dendron-modified (middle), and third-generation dendron-modified glass slides
CDCl
3
): d=7.34 (m, 5H), 5.12 (d, J=
1.2 Hz, 2H), 4.21 (m, 1H), 2.52–2.36
(m, 2H), 2.22–2.14 (m, 1H), 1.97–1.89
(m, 1H), 1.46 (s, 9H), 1.43 ppm (s,
(
right), were probed with Cy3-labeled AAL and WGA, respectively (see Figure S2 in the Supporting Informa-
1
3
tion for quantitative fluorescence intensity data).
9H); C NMR (100 MHz, CDCl
3
): d=
72.6, 171.3, 155.4, 135.8, 128.5, 128.2,
2.0, 79.6, 66.3, 53.4, 30.3, 28.3,
1
8
+
+
2
4
7.9 ppm; MALDI-TOF-MS calcd for
16.2044, found 416.2164.
C
21
H
31NO
6
Na
[M+Na]
tide probes are of great importance in governing the glycan
binding of lectins.
Synthesis of Boc-Glu(PA)-tBu
Boc-Glu(Bn)-tBu (1.785 g, 4.54 mmol) and 10% Pd/C (178 mg) were
added to a reaction flask and the flask was sealed with a rubber septum.
Subsequently, 30 mL of MeOH/CH
2
Cl
2
(1:1) were added, and the reac-
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Density-Dependent Binding of Glycans by Lectins
tion mixture was stirred at room temperature under 1 atm of hydrogen
gas for 12 h. The mixture was filtrated through Celite to remove Pd/C
and then the filtrate was concentrated under reduced pressure. The resi-
due was then dissolved in 50 mL DMF, followed by sequential addition
of propargylamine (0.269 mL, 4.54 mmol), HOBt (0.695 g, 4.54 mmol),
HBTU (1.635 g, 4.31 mmol), and DIEA (1.87 mL, 11.35 mmol) at 08C.
The reaction mixture was warmed to room temperature and stirred for
another 6 h. Subsequently, it was diluted with EtOAc (200 mL), washed
Preparation of Epoxy-derivatized Glass Slides
Amine-coated normal glass slides (Nuricell, GS Nanotech, Korea) and
amine-coated second- and third-generation dendron-modified glass slides
(
NSB POSTECH, Korea) were immersed into 3% poly(ethylene glycol)
diglycidyl ether in 10 mm NaHCO (pH 8.3) for 30 min under gentle
3
shaking. The resulting epoxide-derivatized slides were washed with dis-
tilled water and then dried by purging with argon gas.
with water (50 mLꢄ3) and brine
and concentrated under reduced pressure. The crude product was puri-
fied by flash column chromatography (n-hexane/EtOAc 5:1 to CH Cl
EtOAc 4:1) to give Boc-Glu(PA)-tBu in 80% yield. H NMR (400 MHz,
CDCl ): d=4.15 (m, 1H), 4.04 (dd, J=5.2, J=2.4 Hz, 2H), 2.32 (t, J=
.6 Hz, 2H), 2.24 (t, J=4.8 Hz, 1H), 2.16–2.12 (m, 1H), 1.94–1.87 (m,
2 4
ACHTUNGTRNENUG( 50 mL), dried over anhydrous Na SO ,
Fabrication of Carbohydrate Microarrays
Neoglycopeptide probes were dissolved in 100 mm sodium phosphate
buffer (pH 5.4) containing 40% glycerol. Aliquots of the resulting solu-
tions (10 mL) were placed into the wells of a 384-well plate. Next, each
probe (1 nL, 0.25–2 mm) from the 384-well plate was microspotted at pre-
determined places onto the epoxide-derivatized glass slide with a distance
of 240 mm between the centers of adjacent spots by using a pin-type mi-
croarrayer (MicroSys 5100 PA, Cartesian Technologies). After comple-
tion of printing, the slide was placed into a humid chamber (55–60% rel-
ative humidity) at room temperature for 5 h. The slide was then divided
into several blocks by using a compartmentalized plastic film that was
coated with adhesive on one side (thickness: 0.1–0.2 mm) to avoid cross-
2
2
/
1
3
7
1
3
1
H), 1.46 (s, 9H), 1.44 ppm (s, 9H); C NMR (100 MHz, CDCl
3
): d=
1
72.0, 171.4, 156.0, 82.2, 79.9, 79.7, 71.4, 53.6, 32.4, 29.2, 28.3, 28.0 ppm;
+
+
MALDI-TOF-MS calcd for C17
28 2 5
H N O Na [M+Na] 363.1890, found
3
63.1767.
Synthesis of Fmoc-Glu(PA)-OH
Boc-Glu(PA)-tBu (1.319 g, 3.85 mmol) was dissolved in 9.5 mL TFA and
contamination. A solution of 3% 2-aminoethanol in 10 mm NaHCO
pH 8.3) was then dropped onto the blocks and then left at room temper-
3
0
.5 mL H O at room temperature. After stirring for 2 h, the volatile ma-
terial was removed under reduced pressure. The resulting NH -Glu(PA)-
OH was dissolved in 20 mL water containing sodium bicarbonate
646 mg, 7.7 mmol), and then Fmoc-OSu (3.4 g, 4.04 mmol) in 20 mL di-
2
(
2
ature for 30 min to remove unreacted epoxide groups. Next, the slide was
washed with PBS buffer (pH 7.4) containing 0.1% Tween 20 (wash
buffer, 3ꢄ3 min). After drying by purging with argon gas, a solution of
(
oxane was added at 08C. The reaction mixture was then warmed to room
temperature and stirred for further 12 h. The organic solvent was re-
moved under reduced pressure and the remaining aqueous layer was
washed with EtOAc (20 mLꢄ2) to remove compounds that were dis-
solved in the organic solvent. The aqueous layer was acidified with 1n
0
.1% Tween 20 containing 1% BSA was dropped onto the slide and then
left at room temperature for 30 min. Subsequently, the slide was washed
with wash buffer (3ꢄ3 min) and dried by purging with argon gas.
Detection of Carbohydrate–Protein Interactions on Carbohydrate
Microarrays
HCl to pH 2. The white solid was filtered off and dried to give Fmoc-
1
Glu(PA)-OH in 90% yield. H NMR (400 MHz, DMSO-d
6
): d=12.78 (s,
Solutions of Cy3-labeled lectins in 1x PBS buffer (pH 7.4) containing
1
2
2
5
H), 8.31 (t, J=5.2 Hz, 1 H), 7.89 (d, J=7.6 Hz, 2H), 7.74 (d, J=7.2 Hz,
0
.1% Tween 20 were dropped onto each block compartmented by a plas-
H), 7.66 (d, J=8.0 Hz, 1H), 7.42 (t, J=7.2 Hz, 2H), 7.34 (t, J=7.6 Hz,
H), 4.28 (m, 2H), 4.23 (m, 1H), 3.94 (m, 1H), 3.85 (dd, J=2.4 Hz, J=
.2 Hz, 2H), 3.08 (s, 1H), 2.22 (t, J=7.6 Hz, 2H), 2.02 (m, 1H), 1.82 ppm
tic film and then incubated for 1 h at room temperature. The unbound
proteins were removed by washing the slide with PBS buffer containing
1
3
0
.1% Tween 20 (30 mL, 3ꢄ3 min). The slide was scanned using an Ar-
(
m, 1H); C NMR (100 MHz, DMSO-d
6
): d=173.7, 171.0, 156.2, 143.8,
rayWoRx biochip reader (Applied Precision, Northwest Issaquah, WA).
1
2
4
40.7, 127.6, 127.1, 125.3, 120.1, 81.2, 72.9, 65.7, 53.5, 46.7, 31.5, 27.8,
+
+
6.6 ppm; MALDI-TOF-MS calcd for
29.1421, found 429.1994.
C
23
H
22
N
2
O
5
Na
[M+Na]
Analysis of Density-dependent Binding of Glycans by Lectins
Neoglycopeptide probes (LacNAc-47 (65), GlcNAc-47 (52), Man-47 (39),
Manbio-47 (26), and Fuc-47 (13)) were dissolved in 100 mm sodium phos-
phate buffer (pH 5.4) containing 40% glycerol. An unliganded molecule,
6-(hydroxyacetamido)hexanoyl hydrazide, was added to the solution of
neoglycopeptide to make mixtures at various molar ratios (1:0, 1:1, 1:5,
Solid-phase Synthesis of C-Terminal Hydrazide-Linked Neoglycopeptides
Hydrazide-linked neoglycopeptides were synthesized by using the Fmoc/
À1
tBu strategy on Wang resin (0.9 mmolg ). A solution of 4-nitrophenyl
chloroformate (4 equiv) in CH
in 9 mL CH Cl and 2,6-lutidine (8 equiv) at 08C. After shaking for 12 h,
the resin was washed with 10% DMF in CH Cl . Subsequently, a solution
2 2
Cl was added to Wang resin (0.9 mmol)
1
:10, and 1:20, with a final concentration of neoglycopeptide of 2 mm).
2
2
These solutions (10 mL) were placed into the wells of a 384-well plate
and were microspotted at predetermined places onto an epoxy-derivat-
ized glass slide and then the resulting microarrays were incubated with
Cy3-labeled AAL and WGA.
2
2
of hydrazine (5 equiv) and DIEA (10 equiv) in DMF was added to the
resin. After shaking for 4 h, the resin was washed with DMF. 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).
After each coupling reaction, the unreacted amino group was capped
with an acetyl group. The Fmoc group was removed by treatment with
Determination of Dissociation Constants for Lectin–Surface-linked
Neoglycopeptide Interactions Using Carbohydrate Microarrays
2
0% piperidine in DMF and the resin was washed with DMF and CH
several times. For click chemistry, azide-linked mono- and disaccharides
2.0 equiv per one triple bond) dissolved in DMF (300 mL) were added to
the peptide resin and then sodium ascorbate (1.0 equiv per one triple
bond) and CuSO (1.0 equiv per one triple bond) in water (50 mL) were
2
Cl
2
To determine dissociation constants, fucose-containing neoglycopeptides
5, 9, and 13 (1 mm) were printed onto the epoxide-derivatized surfaces
and the resulting microarrays were probed with various concentrations
(0.61 nm–5 mm) of Cy3-AAL. After incubation for 1 h at room tempera-
ture, the microarrays were washed with 1x PBS buffer containing 0.1%
Tween 20 to remove unbound lectin (30 mL, 3ꢄ3 min). Fluorescence in-
tensities of microspots were quantitated by using an ArrayWoRx biochip
reader. Kd values were calculated by using the following equation: Fl=
(
4
added to the reaction mixture. After 24 h, the hydrazide-linked neoglyco-
peptides were cleaved from the solid support by treatment with TFA/trie-
thylsilane (98:2, v/v) for 2 h. The hydrazide-linked neoglycopeptides were
analyzed by analytical RP-HPLC using a gradient of 5–100% CH
3
CN
d 0
Flmax·[P]0/ ACHUTNTGNERNUG( K +[P] ), in which Fl denotes the fluorescent intensity of the
(
0.1% TFA) in water (0.1% TFA) over 45 min, and the purified products
carbohydrate immobilized on the surface, Flmax is the maximum fluores-
were characterized by MALDI-TOF MS (Table S1 in the Supporting In-
formation).
cent intensity of the carbohydrate immobilized on the surface, and [P] is
the initial concentration of the lectin.
0
Chem. Asian J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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Injae Shin et al.
FULL PAPERS
Acknowledgements
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Published online: && &&, 0000
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

FULL PAPERS
Carbohydrates
Xizhe Tian, Jaeyoung Pai,
Injae Shin*
&&&& — &&&&
Analysis of Density-Dependent
Binding of Glycans by Lectins Using
Carbohydrate Microarrays
Finding the sweet spot: Carbohydrate
microarrays were designed, con-
structed, and applied to analyze the
density-dependent binding of glycans
by lectins. It was found that the
tide, the linker length between the
individual sugars, and the distance
between neoglycopeptide probes on
the surface are all important factors
that determine the binding affinity of
lectins.
number of glycans on a neoglycopep-
Chem. Asian J. 2012, 00, 0 – 0
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
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These are not the final page numbers! ÞÞ