Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin

Article abstract of DOI:10.3762/bjoc.10.138

The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors ("synthetic lectins") constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-Dglucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.

Full text of DOI:10.3762/bjoc.10.138

Molecular recognition of surface-immobilized
carbohydrates by a synthetic lectin
Melanie Rauschenberg, Eva-Corrina Fritz, Christian Schulz,
Tobias Kaufmann and Bart Jan Ravoo*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1354–1364.
Organic Chemistry Institute, Westfälische Wilhelms-Universität
Münster, Corrensstrasse 40, 48149 Münster, Germany
doi:10.3762/bjoc.10.138
Received: 19 February 2014
Accepted: 22 May 2014
Published: 16 June 2014
Email:
Bart Jan Ravoo* - b.j.ravoo@uni-muenster.de
* Corresponding author
This article is part of the Thematic Series "Multivalent glycosystems for
nanoscience".
Keywords:
carbohydrates; lectins; molecular recognition; microarrays; multivalent
glycosystems; peptides
Guest Editor: J.-L. Reymond
© 2014 Rauschenberg et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of
synthetic carbohydrate receptors (“synthetic lectins”) constitutes a key advance in biomedical technology. In this article we report a
synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we
prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontan-
eously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-
glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted
in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobi-
lized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluores-
cence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the
carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut
agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins
and that the recognition of synthetic and natural lectins is strictly orthogonal.
Introduction
In comparison to proteins and nucleic acids, carbohydrates have and pathological processes and offer an enormous potential to
traditionally received less attention in the scientific community. encrypt biological information [1-4]. In contrast to the linear
However, it is increasingly apparent that carbohydrates and nucleic acids and proteins, carbohydrates usually form branched
glycoconjugates are involved in a multitude of physiological oligomers or polymers which are joined together by a variety of
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linkages. The vast amount of resulting carbohydrate oligomers nitriloxide-terminated SAMs [36] using μCP. Homogenous
and polymers offers a virtually unlimited number of encodings. spots, high-edge resolution, good reproducibility and short reac-
In nature, carbohydrates are often linked to lipids, peptides or tion times can be easily achieved by using µCP. These advan-
proteins. These conjugates are found inside cells, on cell tages render this method a versatile tool for the fabrication of
membranes as well as in extracellular fluids and matrices. simple carbohydrate microarrays.
Surprisingly, the general understanding of the function of
carbohydrates in cell biology is still lagging far behind our “Synthetic lectins” as artificial carbohydrate receptors would be
knowledge of proteins and nucleic acids. This backlog is mainly highly valuable as drugs in various therapies and as recognition
due to the complexity of biological carbohydrates. Additionally, units in diagnostics and sensing. However, the development of
established analytical and synthetic methods in protein and synthetic lectins poses a phenomenal challenge for supra-
nucleic acid research such as automated sequencing, automated molecular chemistry [37] because a carbohydrate receptor in
synthesis and high-throughput microarray screening are lacking order to be useful must not only compete with the strong hydra-
in glycobiology [5]. However, during the last decade, these tion of carbohydrates in water but also discriminate closely
methods have also been adapted to carbohydrates [6-8].
related isomers. It is obvious that the de-novo design of a “syn-
thetic lectin” is very difficult. The most remarkable synthetic
Carbohydrate microarrays on chips proved to be a particularly lectins to date have been prepared by Davis and co-workers,
useful tool in glycomics [9-11] since their description in 2002 who recently synthesized a cage-like receptor that binds glucose
by several groups [12-17]. Microarrays normally consist of in water with excellent selectivity versus other simple carbohy-
carbohydrates immobilized in an ordered and well defined drates (for example, ~50:1 versus galactose) which also has
format on a flat surface. The arrays can be considered as sufficient affinity for glucose sensing at the concentrations
minimal models of cell surfaces that are compatible with high- found in blood [38]. On the other hand, we have described a
throughput analysis techniques and can be used to study carbo- dynamic combinatorial approach to the identification of
hydrate–protein/antibody interactions, to detect enzymes biomimetic carbohydrate receptors [39]. To this end, we
cleaving glycosidic bonds, to study cell–cell adhesions medi- explored a dynamic combinatorial library of cyclic peptides to
ated by carbohydrates as well as to screen for selective inhibi- select receptors that are assembled from tripeptides under ther-
tors of carbohydrate–protein interactions [18]. The immobiliz- modynamic equilibrium. Amongst others, we identified a syn-
ation of carbohydrates can be divided into noncovalent and thetic lectin (HisHis) that binds N-acetylneuraminic acid
covalent attachment routes. While carbohydrates simply adhere (NANA) [18]. HisHis is a cyclic hexapeptide which is easily
to the surface when using the noncovalent strategy, the cova- obtained from the air oxidation of the tripeptide Cys-His-Cys
lent attachment leads to the fabrication of highly stable arrays (see Figure 1). It should be noted that in this case HisHis is
because a chemical bond is formed between the substrate and obtained as a mixture of two isomers, in which the tripeptides
the carbohydrates. Fabrication of carbohydrate microarrays can are either oriented in parallel or antiparallel direction. The
be achieved either by microspotting of carbohydrates on acti- parallel isomer is shown in Figure 1. We have recently synthe-
vated surfaces or by using printing techniques on activated sized and isolated the parallel and antiparallel isomers of HisHis
substrates. The first approach can be realized by robotic printers and found that both isomers can bind NANA in a cooperative
[19,20] generating high density chips with a large number of 1:2 complex (1 molecule of HisHis and 2 molecules of NANA)
different spots. Read out is performed with an array scanner [40]. The binding constants are K1 = 143 M−1 and
using fluorescence microscopy or surface plasmon resonance. K2 = 5.08 × 103 M−1 for the parallel isomer and K1 = 94 M−1
and K2 = 990 M−1 for the antiparallel isomer at neutral pH in
In recent years, microcontact printing (μCP) [21-24] has gained water [40]. We also found that the parallel isomer of HisHis
importance as a replication method for biological microarrays (but not the antiparallel isomer) binds β-D-galactose in a 1:1
such as protein [25,26] and DNA microarrays [27-30]. Our complex with K = 7.96 × 103 M−1 [40]. NMR spectroscopy and
group has shown that also simple carbohydrate microarrays can DFT calculations indicate that the interaction of the peptide
be conveniently prepared by μCP if reactive glycosides are with the carbohydrates is based primarily on hydrogen bonding
printed on a suitable target self-assembled monolayer (SAM) [40]. It should be emphasized that NANA is a particularly
[31,32]. Amongst others, we reported carbohydrate microarrays interesting carbohydrate since it belongs to the class of sialic
fabricated by cycloaddition of alkynes on azide-terminated acids which are often the terminal carbohydrates in glyco-
SAMs [33], by Diels–Alder reaction of cyclopentadienes and proteins and glycolipids on the cell surface. Sialic acids are
furans on maleimide-terminated SAMs [34], by thiol–ene click involved in the communication of cells with their environment
reaction of functionalized thiols on alkene-terminated SAMs [41] and selective detection, binding and blocking of sialic acids
[35] as well as by strain promoted cycloadditions on azide- and on the cell surface is of significant biomedical interest.
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Figure 1: Molecular structures of carbohydrates (NANA, Glc, Gal, Man) immobilized on epoxide SAMs, NANA-binding synthetic lectin (HisHis), and
the FITC-labeled synthetic lectin (FITC HisHis). For simplicity, only the parallel isomer of HisHis is shown.
It is the aim of this report to investigate whether the synthetic thetic lectin FITC-HisHis as well as two natural lectins on the
lectin HisHis is able to selectively bind to NANA immobilized immobilized carbohydrates provides insight into the affinity and
on a substrate which serves as a model for a cell surface. To this selectivity of lectin–carbohydrate interaction.
end, we prepared a set of simple carbohydrate microarrays as
well as fluorescein-isothiocyanate labeled HisHis (FITC- Results and Discussion
HisHis) and studied the selectivity and affinity of HisHis The synthetic lectin HisHis was prepared by air oxidation of the
towards immobilized NANA in comparison with the glycosides tripeptide Cys-Hys-Cys (synthesized by solid phase peptide
of glucose (Glc), galactose (Gal) and mannose (Man) synthesis) as described previously [39]. Fluorescein-labeled
(Figure 1). In this study, we exploit the epoxide ring opening FITC-HisHis was obtained by labeling of Cys-His-Cys with
reaction of amine-tethered carbohydrates on epoxide-termi- fluorescein isothiocyanate, which was achieved by using an
nated SAMs [42] to print carbohydrate microarrays on silicon Fmoc-protected oligo(ethylene glycol) spacer synthesized in
and glass substrates. Epoxide-terminated SAMs are particularly four steps from commercially available ethylenediamine (see
versatile for the fabrication of biological arrays [43,44] and we Supporting Information File 1). The introduction of this water
have recently demonstrated that epoxide-terminated substrates soluble spacer should ensure the unhindered formation of the
are easily modified using µCP [45]. The incubation of the syn- cyclic synthetic lectin FITC-HisHis from the FITC-labeled Cys-
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His-Cys by air oxidation. The spectroscopic and analytical data step from an epoxy-terminated trimethoxysilane. The most
obtained for FITC-HisHis are fully consistent with the molec- common approaches to fabricate epoxide SAMs is by dip-
ular structure (see Supporting Information File 1). Successful coating or vapor condensation. However, this leads to the for-
incorporation of the fluorophore was also evident in the UV–vis mation of thick films of aggregated molecular layers with
spectrum of the precursor FITC-labeled Cys-His-Cys (see undefined surface morphology [47-50]. Therefore, the
Figure S1 in the Supporting Information File 1). We note that if epoxide SAMs were prepared by the method of Julthongpiput
prepared directly by air oxidation from Cys-His-Cys, HisHis as et al. because well-defined monolayers of epoxides on glass
well as FITC-HisHis consist of a mixture of two isomers, in and silicon substrates can be obtained by using (3-glycidoxy-
which the two tripeptides are arranged in parallel or antiparallel propyl)trimethoxysilane in toluene [42]. The successful surface
direction, respectively. We have recently synthesized and modification was verified by contact angle and XPS measure-
isolated the parallel and antiparallel HisHis isomers and found ments (see Supporting Information File 1) which are in accor-
that both isomers can bind NANA in a 1:2 complex with dance with literature data [42].
slightly stronger binding by the parallel HisHis compared to the
antiparallel HisHis [40]. Isothermal titration calorimetry (ITC) The µCP protocol to fabricate carbohydrate microarrays was
confirmed that the interaction of FITC-HisHis (mixture of optimized to provide a highly effective combination of stamp,
isomers) and NANA is characterized by the same stoichio- ink and substrate. In view of the fact that the epoxide ring
metry (1:2) and nearly the same binding constants (K1 = 163 opening reaction with amines is much faster at elevated
M−1 and K2 = 5.36 × 103 M−1) were obtained. ITC data are temperatures, the surface of oxidized, patterned PDMS stamps
provided in Supporting Information File 1. These findings indi- was coated with 2-[methoxy(polyethyleneoxy)propyl]trimethyl-
cate that the introduction of the FITC label does not affect the silane (PEG silane) [51]. The PEG coated stamps provided
formation of the synthetic lectin HisHis and its interaction with optimal wetting of the stamp by the carbohydrate ink solution,
NANA. Since the mixture of isomers is much more easily while preventing contamination of the substrate by PDMS
synthesized than the individual isomers and since both isomers residues. Moreover, it was observed that oxidized stamps
are potent binders of NANA, all experiments described in this without such a PEG coating adhered irreversibly to the epoxide
report where performed with the mixture of parallel and antipar- substrates, possible due to reaction of the epoxide substrate with
allel isomers of FITC-HisHis.
the oxidized PDMS surface. In the optimized µCP protocol, the
PEG coated stamps were wetted with a 20 mM ethanolic solu-
In order to study the affinity of the synthetic lectin HisHis on tion of carbohydrate inks (NANA, Glc, Gal or Man) and triethy-
the surface, four carbohydrates (NANA, Glc, Gal, Man, see lamine, and dried after 1 min incubation time. After placing the
Figure 1) were selected for the fabrication of carbohydrate stamps on the epoxide-terminated SAMs on cleaned and acti-
arrays. To provide carbohydrate inks suitable for microcontact vated glass or silicon substrates, the substrate and stamp were
printing (µCP), NANA was conjugated via its C1 carboxylic placed in an oven at 60 °C for 4 h. Finally, the stamp was
acid moiety, whereas the other carbohydrates were conjugates removed from the substrate at room temperature. In order to
as β-glycosides (Glc, Gal) and α-glycosides (Man), respective- prepare microarrays with two carbohydrates, the printing step
ly. In order to flexibly attach the carbohydrates on the substrate was repeated with a second carbohydrate ink on a flat stamp to
and to ensure unhindered carbohydrate–lectin interactions, functionalize the remaining epoxide surface (Figure 2). After
oligo(ethylene glycol) spacers were introduced. Oligo(ethylene printing, the surfaces were rinsed extensively to remove any
glycol) chains are flexible, water-soluble and do not interact residual physisorbed material.
with lectins [46]. A nucleophilic primary amine function was
introduced on the terminus of the spacer in order to ensure reac- Immobilization of carbohydrates on the epoxide-terminated
tion with the epoxide-functionalized substrate upon µCP. The SAMs was investigated by several methods. Printing of a polar
NANA ink was prepared by solution phase peptide coupling carbohydrate on the epoxide surface should lead to increased
using the DIPCDI/Oxyma pure® coupling protocol. The glucose hydrophilicity exclusively in the printed areas which can be
(Glc), galactose (Gal) and mannose (Man) inks were synthe- detected by water condensation experiments. Figure 3 shows
sized as described in Supporting Information File 1.
optical microscopy pictures of epoxide SAMs onto which
NANA ink or Gal ink were printed with a structured stamp
The effective immobilization of the amine-terminated carbohy- (10 μm dots spaced by a 5 μm gap). It is obvious that the water
drate inks requires a surface functionalized with epoxides. condensed on the surface is found exclusively in the areas
Epoxides are stable under ambient conditions yet highly reac- where the hydrophilic NANA or Gal were printed. Furthermore,
tive towards amines at elevated temperatures and epoxide quantification of the surface hydrophilicity was performed by
SAMs on silicon oxide surfaces can be obtained in only one water contact angle measurements. To this end, a flat PDMS
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Figure 2: Schematic representation of the preparation of a simple carbohydrate microarray by μCP of amine-functionalized carbohydrate inks on
epoxide-terminated SAM (“microcontact chemistry”). The first carbohydrate (red) is printed with a patterned PDMS stamp, the second carbohydrate
(green) is printed in the interspaces with a flat PDMS stamp.
Figure 3: Optical microscopy images of water droplets selectively condensed in the areas where (A) the NANA ink and (B) the Gal ink were patterned
by μCP on an epoxide-terminated SAM.
stamp was used to obtain a homogenous carbohydrate surface are spaced by a 5 μm gap) as well as a cross printed pattern
after μCP for 4 h at 60 °C. After printing, the contact angles obtained by a successive print at 90° angle with two identically
decreased from 61°/33° (advancing/receding) for the epoxide- patterned stamps (5 μm stripes that are spaced by a 10 μm gap)
terminated SAMs to ~30°/~12° for the carbohydrate-functional- using the Glc ink and the Gal ink are shown in Figure 4. A clear
ized SAMs (with minor differences for the different carbohy- pattern in accordance with the shape and dimensions of the used
drate inks). Data for all contact angle measurements can be stamps can be seen. If printing of the carbohydrate ink results in
found in Table S1 in Supporting Information File 1.
a very dense layer of carbohydrates aligned mostly perpendic-
ular to the surface, the height difference of printed and
Further information on the printing process was obtained from nonprinted areas should be around 1 nm. The observed height
X-ray photoelectron spectroscopy (XPS) of Si-wafers function- differences are significantly less which indicates that the carbo-
alized with an epoxide-terminated SAM and printed with hydrates are tilted relative to the surface normal and that the
NANA ink using a PEG coated flat stamp (see Figure S3 and printed layers exhibit a lower than maximal surface coverage.
Figure S4 in Supporting Information File 1). While nearly no This is most likely due to a combination of factors, i.e., quality
N(1s) signal was detected in the epoxide-terminated SAM, a of the base epoxide SAM, reaction time with the amine inks,
clear N(1s) signal can be observed when the NANA ink is and steric bulk of the carbohydrates compared to the triethylene
printed. The C(1s) peak shows a splitting into the C–C glycol linker. We note that these observations are consistent
(285 eV), C–O (287 eV), C=C, C=O and residual epoxide C–O with our earlier reports on the immobilization of glycosides on
signals (289 eV) as expected for the NANA-terminated SAM SAMs by µCP [33-36].
[52].
Having established that one or more carbohydrate inks can be
In addition, atomic force microscopy (AFM) of patterned patterned on epoxide-terminated SAMs by µCP, we turned our
epoxide SAMs was performed to verify the success of the attention to the investigation of the interaction of synthetic and
printing process. The AFM height profiles of a pattern of natural lectins with the immobilized carbohydrates. To this end,
NANA ink printed with a patterned stamp (10 μm stripes that bifunctional surfaces were fabricated by µCP of a the first ink in
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Figure 4: (A) AFM height image (zoom) of NANA ink in 10 μm stripes on an epoxide-terminated SAM; (B) Height profile of the black line shown in (A);
(C) AFM height image of Glc ink and Gal ink cross printed in 5 μm stripes on an epoxide-terminated SAM; (D) Height profile of the black line shown in
(C).
a dot pattern (10 μm dots spaced by a 5 μm gap) and filling up observed that the parallel isomer of HisHis binds to β-galacto-
the interspaces with the second ink by using µCP with a flat sides [40].
stamp. In a first set of experiments, NANA ink was printed in a
dot pattern and the remaining area was functionalized by
printing Man ink with a flat stamp. In a reverse experiment,
Man ink was printed in dots and the remaining area was func-
tionalized with NANA ink. After incubation of these carbohy-
drate arrays with the synthetic lectin FITC-HisHis, fluores-
cence was observed exclusively in the areas where the NANA
ink had been printed. No significant fluorescence was observed
in the area where the Man ink had been printed (Figure 5). This
observation confirms – for the first time – that the synthetic
lectin HisHis binds selectively to NANA, and not to Man, also
when the carbohydrate is immobilized on a surface. We note
that no fluorescence was detected when the inks were printed
onto bare glass slides (instead of epoxide-functionalized glass
slides) or when printing was performed without inking the PEG
Figure 5: Fluorescence images of bifunctional carbohydrate microar-
rays incubated with FITC-HisHis. (A) NANA (dots 10 × 5 μm) and Man
coated PDMS stamp. In a second set of experiments, NANA ink
was printed in dots and the remaining area was functionalized
with Glc ink, and vice versa. Again it was observed that HisHis
binds exclusively to NANA, and not to Glc (see Figure 8A). In
a third set of experiments, Gal ink was printed in dots and the
remaining area was functionalized with Man ink, and vice
versa. As expected, it was observed that FITC-HisHis binds to
Gal, and not to Man (Figure 5). In our preceding work, we had
(background); (B) Man (dots 10 × 5 μm) and NANA (background);
(C) Gal (dots 10 × 5 μm) and Man (background); (D) Man (dots
10 × 5 μm) and Gal (background).
In order to confirm the interaction between HisHis and NANA
with surface plasmon resonance (SPR) spectroscopy a commer-
cially available polycarboxylate hydrogel sensor surface was
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employed, which is known to be particularly advantageous for with Man background (Figure 7). If the recognition process
low molecular weight compounds due to the signal amplifica- between FITC-HisHis and NANA or Gal is selective, incuba-
tion caused by multiple binding events in the hydrogel on the tion with a large excess of the two carbohydrates should lead to
sensor. The functionalization of the polycarboxylate hydrogel release of the surface-bound synthetic lectin from a microarray
with amine terminated NANA was performed by N-hydroxy- displaying NANA or Gal. In this case, the detected fluores-
succinimide (NHS) activation and subsequent peptide coupling. cence pattern should vanish after washing the microarrays with
Indeed, using the hydrogel sensor a small but significant SPR concentrated solutions of NANA and Gal. As is shown in
signal increase was observed upon the addition of HisHis (see Figure 7B,C, FITC-HisHis is indeed displaced from the
Figure S5 in Supporting Information File 1). The initial rate and microarray when a solution of 250 mM NANA or a solution of
extent of surface binding correlated with the concentration of 500 mM methyl β-D-galactoside solution is used to rinse the
HisHis (0.5–2.0 mM) applied to the sensor. However, it was not microarray. The original fluorescence pattern can be restored by
possible to obtain sufficiently reproducible data to perform a incubation with FITC-HisHis (Figure 7D) and this process is re-
quantitative analysis of the peptide–carbohydrate interaction. versible over several cycles. Upon simultaneous incubation of
The poor quality of the SPR signal is certainly due to the low the microarray with NANA or methyl β-D-galactoside and
molecular weight of HisHis which limits any further SPR FITC-HisHis, no fluorescent pattern was detected
investigations.
(Figure 7E,F). This indicates that the synthetic lectin HisHis is
saturated by the excess of carbohydrate which is present in solu-
Additionally, bifunctionalized carbohydrate surfaces were incu- tion and can therefore no longer bind to the carbohydrates on
bated simultaneously with FITC-HisHis, TRITC-ConA, and the microarray. Conversely, it would be expected that carbohy-
FITC-PNA to elucidate whether selective binding of the syn- drates which do not show any interaction with HisHis should
thetic lectin to glycosides is also observed in the presence of not lead to a decrease in the fluorescence of FITC-HisHis on the
natural lectins. In Figure 6, the overlays of fluorescence images microarray and this is indeed observed for methyl α-D-manno-
are shown. In each experiment, FITC-HisHis is detected exclu- side, methyl β-D-glucoside, methyl β-D-fucoside, trehalose,
sively on the NANA and the Gal areas, whereas TRITC-ConA sucrose as well as N-acetylglucosamine (see Figure 7G–L).
binds to Man and FITC-PNA binds to Gal only. These observa- These experiments demonstrate two important points: firstly,
tions demonstrate that selective recognition between HisHis and they attest the selectivity as well as the affinity of HisHis for
NANA or Gal is not in any way disturbed by the presence of a immobilized NANA and Gal, and secondly, these results show
second lectin and that the synthetic lectin HisHis and the natural that even a very simple microarray provides a versatile
lectins ConA and PNA operate orthogonally.
screening tool.
Finally, in addition to the mono- and disaccharides described
above, the selectivity of the synthetic lectin HisHis was tested
by using two heparins. These polymers display a particularly
high carbohydrate epitope density. While one polymer (wild
type, WT, 6.61 µg µL−1) has a negatively charged sulfonate
group at the terminus, the second one (KO, 6.12 µg µL−1) does
not possess this group and is therefore charge neutral. After 5 h
incubation of a microarray of NANA (10 μm dots spaced by a
5 μm gap) and Glc (background) with three different dilutions
of WT and KO, the fluorescence images were recorded
(Figure 8). No significant effect is observed when the polysac-
charides are diluted by a factor of 1000 or a factor of 100, but a
10 times dilution of KO and WT (0.661 µg µL−1 and
0.612 µg µL−1) leads to a strong decrease of the FITC-HisHis
fluorescence on the microarray (Figure 8C,F). No loss in fluo-
rescence was detected when the wafer was incubated for 5 h
with the same amount of Milli-Q water. These data demon-
strate that heparin-type polysaccharides can compete with the
interaction of the synthetic lectin HisHis and the carbohydrate
Figure 6: Overlay of fluorescence images of bifunctional carbohydrate
microarrays; (A) NANA (dots 10 × 5 μm) and Man (background) incu-
bated with FITC-HisHis and TRITC-ConA; (B) Man (dots 10 × 5 μm)
and NANA (background) incubated with FITC-HisHis and TRITC-
ConA; (C) Gal (dots 10 × 5 μm) and Man (background) incubated with
FITC-HisHis and TRITC-ConA; (D) Man (dots 10 × 5 μm) and Gal
(background) incubated with FITC-PNA and TRITC-ConA.
Additionally, the selectivity of the FITC-HisHis was tested with NANA. The concentration dependence of displacement assay
a set of competition experiments on a microarray of NANA dots indicates the high affinity of HisHis for NANA in the
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Figure 7: Fluorescence images of a microarray consisting of NANA (dots 5 × 3 μm) and Man (background). (A) Incubation with FITC-HisHis;
(B) washing of (A) with 250 mM NANA; (C) washing of (A) with 500 mM methyl β-D-galactoside; (D) incubation of (B) with FITC-HisHis; (E) simulta-
neous incubation with FITC-HisHis and NANA; (F) simultaneous incubation with FITC-HisHis and methyl β-D-galactoside; (G) washing of (A) with 500
mM methyl α-D-mannoside; (H) washing of (A) with 500 mM methyl β-D-glucoside; (I) washing of (A) with 500 mM methyl β-D-fucoside; (J) washing
of (A) with 500 mM trehalose; (K) washing of (A) with 500 mM sucrose; (L) washing of (A) with 500 mM N-acetylglucosamine.
Figure 8: Fluorescence images of a microarray of NANA (dots 5 × 3 μm) and Glc (background), first incubated with FITC-HisHis and subsequently in-
cubated for 5 h with (A) a 1000-fold dilution of KO (0.00612 µg µL−1); (B) a 100-fold dilution of KO (0.0612 µg µL−1); (C) a 10-fold dilution of KO
(0.612 µg µL−1); (D) a 1000-fold dilution of WT (0.00661 µg µL−1); (E) a 100-fold dilution of WT (0.0661 µg µL−1); (F) a 10-fold dilution of WT
(0.661 µg µL−1).
microarray. Thus, the results obtained from the microarray Conclusion
support our earlier finding that the interaction of HisHis and In summary, we have investigated the molecular recognition of
NANA is 1:2 [39], so that HisHis is likely to bind divalently to surface immobilized carbohydrates by a synthetic lectin. To this
the microarray surface.
end, amine-tethered carbohydrates were printed on epoxide
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SAMs by μCP. Using tailor-made PDMS stamps and an opti- Inc.) for 55 min after which they were stored under water.
mized printing protocol, simple carbohydrate microarrays could Coating of the stamps with 2-[methoxy(poly(ethylene-
readily be obtained by μCP. Consistent with measurements in oxy))propyl]trimethylsilane was done by incubating the
solution, the microarrays showed high selectivity of the syn- oxidized and dried stamps in a 1 vol % solution of the silane in
thetic lectin HisHis for NANA and Gal versus Glc and Man. abs. ethanol for 2 h, after which they were washed with abs.
Both the selectivity and the high affinity of the synthetic lectin ethanol and dried [51]. Flat PDMS stamps were fabricated as
could be demonstrated in competition experiments with mono- described above by using a flat silicon wafer as master.
saccharides, disaccharides and heparin-type polysaccharides.
The results obtained here demonstrate – for the first time – the Microcontact printing: The surface of the freshly oxidized or
high selectivity and affinity of a synthetic lectin for two coated PDMS stamp was covered with 2 to 3 drops of ethanolic
different carbohydrates immobilized in a simple microarray. solutions of the ink (20 mM) and triethylamine (20 mM) and in-
We contend that this is a significant development in the cubated for 1 min. After blow-drying, they were placed on the
search for synthetic lectins that operate under physiological according SAM. Printing was performed by placing the
conditions.
substrates together with the stamp in an oven which was
tempered to 60 °C for 4 h. After removing the stamp from the
substrate, with dichloromethane (DCM), abs. ethanol and Milli-
Experimental
General: Chemicals were purchased from Sigma Aldrich, Q water and dried. Subsequent printing steps were
Acros Organics, Iris Biotech and ABCR and used without carried out as described above. After the last print, the
further purification. Silicon wafers (B-doped, 100-orientation, substrates were sonicated in DCM, abs. ethanol and Milli-Q
resistivity 20–30 Ω) were kindly donated by Siltronic AG. The water, and dried.
heparins KO and WT were kindly donated by Prof. Dr. Rupert
Hallmann (Westfälische Wilhelms-Universität Münster). Milli- Lectin carbohydrate interactions: As described in [34], in
Q water was prepared from distilled water using a PureLab order to reduce non-specific protein adsorption, the arrays were
UHQ deionization system (Elga). Tetramethylrhodamine isoth- incubated with a 3% bovine serum albumin (BSA) solution in
iocyanate-labeled concanavalin A (TRITC-ConA) was PBS buffer (pH 7.5, 0.1% Tween 20) for 30 min and washed
purchased from Sigma Aldrich. Fluorescein isothiocyanate- two times with PBS buffer prior to lectin screening. The
labeled peanut agglutinin (FITC-PNA) was obtained from surfaces of the carbohydrate arrays were covered by a solution
Vector Laboratories. Detailed information concerning the syn- of 1 mM FITC HisHis and 1 μg of labeled lectin (TRITC ConA
thesis and analysis of carbohydrates and peptides is provided in or FITC PNA) in 100 μL of HEPES buffer (20 mM HEPES, pH
Supporting Information File 1.
7.5, 0.15 M NaCl, 1.0 mM CaCl2). In the case of ConA, MnCl2
was added to a concentration of 1 mM. After 90 min, the arrays
SAM preparation: Epoxide-terminated SAMs were prepared were washed with the same buffer, rinsed with Milli-Q water,
as reported in literature [42]. Glass slides as well as Si-wafers dried, and analyzed.
were cut into small pieces of around 1.6 × 2.6 cm2, cleaned with
detergent and dried. After sonification in pentane, acetone and Selectivity studies: The carbohydrate chips were incubated
Milli-Q water, the slides were put into a freshly prepared with a 1 mM solution of FITC HisHis for 1 min, washed with
piranha solution (H2O2/H2SO4 = 1:3) for 30 min. The wafers phosphate buffer (100 mM, pH 7.4) to remove unbound
were then thoroughly washed with water, dried and immersed in receptor, dried and analyzed. Afterwards, the wafer was washed
a 0.3 vol % solution of (3-glycidoxypropyl)trimethoxysilane in three times with 500 μL mono- or disaccharide solution, dried
toluene for 24 h. Excess silane was removed by sonification in and analyzed again. 100 μL of the heparin polymer (KO and
abs. ethanol for 20 min. After washing the slides with abs. WT) solutions were put on glass slides which were afterwards
ethanol, and drying, they were kept at least 24 h under ambient covered with a beaker. After 5 h at room temperature, the
conditions before printing.
glass slides were washed with Milli-Q water, dried and
analyzed.
Stamp preparation: Poly(dimethylsiloxane) (PDMS) stamps
were prepared by mixing PDMS with a curing agent (Sylgard Contact angle measurements: Water contact angles were
184, Dow Corning) in a 10:1 ratio and pouring the viscous mix- measured using the sessile drop method on a DSA 100
ture on a patterned silicon master. After removing the air in goniometer (Krüss GmbH Wissenschaftliche Laborgeräte,
vacuum, the PDMS was cured at 80 °C overnight and peeled off Hamburg/Germany). The advancing and receding contact
after cooling. The patterned areas were cut out with a knife and angles were measured on glass and silicon substrates, and at
oxidized in a UV-ozonizer (PSD-UV, Novascan Technologies least three measurements were performed for every sample.
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Beilstein J. Org. Chem. 2014, 10, 1354–1364.
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Shape Analysis.
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X-ray photoelectron spectroscopy (XPS): X-ray photoelec-
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(Kratos Analytical, Manchester/UK). Monochromatized Al Ka
radiation (1486.6 eV) as the excitation source and a pass energy
of 20 meV for narrow scans were used. The obtained spectra
were analyzed using the Casa XPS (version 2.3.15, Casa soft-
ware Ltd, Teignmouth/UK) software and were referenced to the
C(1s)-peak of the saturated hydrocarbons by setting it to
285 eV. All measurements were carried out on silicon
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Experimental procedures, characterization data and
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Acknowledgements
Silicon wafers were kindly donated by Siltronic AG
(Burghausen, Germany) and the heparin polymers WT and KO
were kindly donated by Prof. Dr. Rupert Hallmann
(Westfälische Wilhelms-Universität Münster). The Deutsche
Forschungsgemeinschaft is acknowledged for financial support
(SFB 858). This work was supported by COST CM1005
“Supramolecular Chemistry in Water”.
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