Multivalent glycan arrays

Abstract

Glycan microarrays have become a powerful technology to study biological processes, such as cell-cell interaction, inflammation, and infections. Yet, several challenges, especially in multivalent display, remain. In this introductory lecture we discuss the state-of-the-art glycan microarray technology, with emphasis on novel approaches to access collections of pure glycans and their immobilization on surfaces. Future directions to mimic the natural glycan presentation on an array format, as well as in situ generation of combinatorial glycan collections, are discussed.

Full text

Multivalent glycan arrays
Marco Mende,
a
Vittorio Bordoni,
a
Alexandra Tsouka,
ab
Felix F. Loeffler,
a
Martina Delbianco
a
and Peter H. Seeberger *
ab
Received 11th June 2019, Accepted 26th June 2019
DOI: 10.1039/c9fd00080a
Glycan microarrays have become a powerful technology to study biological processes,
such as cell–cell interaction, inflammation, and infections. Yet, several challenges,
especially in multivalent display, remain. In this introductory lecture we discuss the
state-of-the-art glycan microarray technology, with emphasis on novel approaches to
access collections of pure glycans and their immobilization on surfaces. Future
directions to mimic the natural glycan presentation on an array format, as well as in situ
generation of combinatorial glycan collections, are discussed.
1. Introduction
Glycans decorate the surface of many cells, forming a thick layer (glycocalyx) that
mediates a variety of important cellular processes.
1
This 100 nm–1 mm thick
glycan layer comprises highly diverse structures, including glycoproteins, glyco-
lipids, and glycopolymers. Several complex biological processes, such as protein
folding, cell–cell interaction, cell adhesion, and signaling, are the result of the
interactions of glycans with themselves (carbohydrate–carbohydrate interactions,
CCIs) or with glycan binding proteins (carbohydrate–protein interactions,
CPIs).
2–4
In addition, pathogens use these glycans as receptors for the attachment
to host cells and subsequent invasion.
5,6
At the same time, pathogenic glycans are
recognized by the immune system, which initiate the immune response.
7,8
Pathological events, such as tumor metastasis, inammation, and infections, are
all mediated by glycan–protein interactions.
A better understanding of these CPIs is of fundamental importance. Yet, in
comparison to polynucleotides and proteins, the study of glycans and CPIs has
been slower for multiple reasons: the complexity of carbohydrate synthesis and
their difficult isolation from natural sources has hampered a detailed analysis of
such compounds. The limited access to collections of pure materials precluded
high-throughput screening formats. Even though glycan arrays have become
extremely popular and primary analytical tools for the study of CPIs,
9–11
they are
a
Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am M¨
uhlenberg 1,
14476 Potsdam, Germany. E-mail: peter.seeberger@mpikg.mpg.de
b
Department of Chemistry and Biochemistry, Freie Universit¨
at Berlin, Arnimallee 22, 14195 Berlin, Germany
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limited due to glycan availability. Additionally, CPIs are very weak (typically in the
micromolar range) and glycan binding proteins can oen interact with many
substrates with low specicity. Nature’s strategy to enhance binding strength and
specicity is multivalency, where multiple carbohydrate units bind to one protein
to gain stronger affinity than the sum of the single contributions.
12–14
Chemists
have aimed to reproduce nature, developing several synthetic multivalent systems
that mimic natural supramolecular interactions.
15
Nevertheless, recreating the
binding thermodynamics of natural interfaces in a microarray format, is
extremely challenging.
16
Different approaches aimed to mimic the natural glycan
presentation on an array surface. The most common approach involves the direct
printing of glycans, controlling the density by varying the concentration or by
surface functionalization.
17–19
Alternatively, prearranged multivalent systems,
based on natural or unnatural scaffolds, can be immobilized on surfaces, aiming
at more dened glycan presentation.
9,20
A challenging approach is the direct synthesis of glycans or multivalent glycan
systems on the array. However, in comparison to other biomolecules, chemical
carbohydrate synthesis on surfaces is far more difficult, due to the demanding
reaction parameters. Only the synthesis of disaccharides has been achieved to
date.
21
Enzymatic synthesis on surfaces is more common, e.g., for the synthesis of
N-glycans or the discovery of glycosyltransferases.
22–25
Yet, whether glycans can
also be synthesized in situ in a molecularly dened and multivalent fashion,
remains to be shown.
We review the state of the art of glycan microarrays, from access to glycan
collections, to surface immobilization, and analysis. We will focus on current
approaches to mimic natural interfaces and new directions in surface function-
alization. Moreover, we will describe how simple glycans and more complex
multivalent scaffolds are printed or grown from surfaces to elucidate important
cellular processes.
2. Access to glycan collections
The rst step towards the production of a glycan microarray is the identication
of suitable glycans. Two approaches are available and currently used to access
glycans (see Fig. 1): isolation from natural sources and/or synthesis (enzymatic or
chemical). Natural glycans can be readily obtained from animal tissues, plant
material, or from cultured pathogens.
26
Large collections in terms of size and
diversity could be accessed, when completely uncharacterized binders need to be
identied.
10
Nevertheless, the isolation procedures and characterization of the
nal carbohydrates could be extremely challenging, oen resulting in mixtures of
compounds. Heterogeneous samples, oen containing minor impurities, could
culminate in non-reproducible results. Moreover, extracted glycans generally
require an extra functionalization step for immobilization on surfaces.
Compound collections obtained from chemical synthesis are generally smaller,
more focused, and less diverse. Generating a set of related glycans, with the
possibility of including non-natural glycans, is of great interest for the elucidation
of structure–activity relationships. Chemically obtained compounds are highly
pure, reducing the possibility of false results. A reactive linker can be easily
installed during synthesis, facilitating subsequent immobilization. Using these
two approaches, many glycans were prepared and printed on arrays.
9,10
The
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microarray with the currently largest diversity is represented by the mammalian
array (version 5.3) of the Consortium for Functional Glycomics (CFG), which
includes more than 600 synthetic and isolated compounds.
26
A microbial glycan
microarray is also available, including more than 300 carbohydrates. However,
covering the huge diversity of microbial glycans, oen containing rare sugars,
remains a major challenge.
27
Efforts to access such glycans in a well-dened
manner are still needed.
2.1. Automated glycan assembly
Collections of well-dened glycans are fundamental tools to elucidate glycan
interactions. With the aim to explore the natural and unnatural diversity of
glycans, systematic strategies for the chemical, enzymatic, and chemo-
enzymatic synthesis of glycans were developed. Nevertheless, the challenging
installation of the glycosidic linkage, that requires regio- and stereo-control,
poses a bottleneck. Enzymatic synthesis relies on the specicity of the
enzymes to form the desired glycosidic linkage, but to date, it is limited by the
availability of suitable glycosyltransferases.
28
Such enzymes are extremely
efficient with natural substrates, but oen tolerate only limited substrate
variations, hindering access to chemically modied glycans and unnatural
structures.Inaddition,thein vitro production of functional enzymes is
sometimes troublesome.
29
Despite several challenges, enzymatic synthesis
remains a powerful option, when poorly reactive monosaccharides such as
sialic acid or particularly challenging linkages such as b-mannosides need to
be installed.
30
Efforts to standardize this process resulted in two fully auto-
mated systems.
31,32
Fig. 1 Different approaches to access glycan collections.
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Chemical synthesis offers the unique opportunity to access well-dened
natural and unnatural structures. Collections of complex synthetic glycans,
including heparin sulfate glycans, GPI-anchors, and high-mannose oligosaccha-
rides, were used to create custom arrays to characterize lectin and antibody
specicity and to study the human response to infections and allergies.
33
The
biggest drawback of this approach is the enormous synthetic effort required.
Automated Glycan Assembly (AGA) speeds up the process, allowing for quick and
reliable access to glycans.
34,35
The sequential addition of sugar building blocks
(BBs) on a solid support replaces the purication steps with simple washing
cycles. The coupling cycle, consisting of glycosylation, capping, and deprotection,
has been optimized to achieve nearly quantitative conversion in around 1.5 h.
36,37
Moreover, the glycan is attached to the solid support through a linker that, upon
UV irradiation, liberates the target glycan already equipped with an amino-linker
for subsequent surface functionalization.
38
Collections of natural and unnatural
glycans found applications in vaccine development,
39,40
materials science,
36,41,42
and structural studies
37
(see Fig. 2). Well-dened linear b(1,3) and branched b(1,3)
b(1,6) glucans permitted to conclude that most individuals form antibodies that
bind to both linear (protective) and branched (non-protective) epitope.
43
Synthetic
keratan sulfate (KS) analogues, with different sulfation patterns, helped to iden-
tify the specic interaction between the disulfated KS tetrasaccharide and the
adeno-associated virus AAVrh10 gene-therapy vector (see Fig. 2).
44
Frameshisof
the S. pneumoniae serotype 8 (ST8) capsular polysaccharides were used to identify
the glycotopes recognized by antibodies against ST8. The insights were essential
for the preparation of a semisynthetic Streptococcus pneumoniae serotype 8 gly-
coconjugate vaccine candidate.
40
AGA was exploited to determine the binding
epitopes of many plant cell-wall glycan-directed mAbs.
45,46
A total of 88 synthetic
Fig. 2 Applications of glycan collections synthesized with AGA.
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oligosaccharides, including arabinogalactan-, rhamnogalacturonan-, xylan-, and
xyloglucan were printed on a microarray aiming to comprehensively map the
epitopes of plant cell-wall glycan-directed antibodies (see Fig. 2).
47
3. Printing on surfaces
Once the glycan collection has been produced, the glycans are printed onto the
array surface. High accuracy and reproducibility are essential for a reliable
microarray. Two technologies are mainly used to deposit bioactive molecules,
such as carbohydrates, on a reactive surface. These rely on contact and non-
contact printing (see Fig. 3).
48
Contact technologies (see Fig. 3A) are naturally more precise, mainly relying on
pin printing and microstamping of arrays. A pin printing setup consists of
a robotically controlled print head, equipped with one to dozens of differently
shaped solid pins. The pins soak a certain volume of a spotting solution (dis-
solved biomolecule or building blocks) from wells of a microtiter plate upon
dipping. Nanoliters of the solution can then be deposited as a droplet on the
reactive surface by bringing the pins in contact with the surface. The transfer
process relies on favorable surface energies between the spotting solution, the
surface, and the pin. An alternative to pin printing is microcontact printing,
where crosslinked polydimethylsiloxane (PDMS) microstamps with micro-
features are used.
49
Spray-on or robotic feature–feature ink transfer is applied
to coat the stamp with the spotting solution. The substance is then transferred to
the surface upon contact between the stamp and the surface. This technique is
mainly used to array one compound on a surface, while pin printing allows for the
deposition of different molecules at the same time. In both cases, extensive
washing steps and relling aer iterative cycles are necessary.
Non-contact printing technologies (see Fig. 3B) rely on the ejection of spotting
solution from a reservoir through an orice as a droplet or stream onto the
microarray surface. Common inkjet printing technology uses a solution of a dis-
solved biomolecule or building block, serving as the “ink”. The solutions are
ejected from a cartridge by a print head nozzle at a distance of 1–5 mm from the
surface. The ejection process can be triggered by mainly three different methods:
piezo actuation, valve-jet, or thermal inkjet. All three methods are based on
a reversible and rapid change of pressure within the cartridge to release small
droplets of the spotting solution. Non-contact printing approaches are highly
exible, since they allow for a fast switching between various cartridges and
frequent relling is avoided. Furthermore, because the method is contact-free,
Fig. 3 Schematic illustration of contact (A) and non-contact printing (B).
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there is no risk of surface disruption. Possible clogging of the nozzle and process-
related contaminations are the main drawback of this technique.
The combinatorial laser-induced transfer method (cLIFT) helps to circumvent
contamination and clogging issues.
50
Currently the method is restricted to the
chemical synthesis of peptide and peptoid
51
arrays, but may be expanded to
glycan array synthesis. Novel micro- and nanoprinting technologies exploit
cantilevers from atomic force microscopy to pattern surfaces, such as the well-
known dip-pen nanolithography.
52
Meanwhile, the technique evolved to sophis-
ticated microuidic and lithographic setups, enabling photochemical patterning
of surfaces with different monosaccharides in high resolution.
53
Another recent
scanning probe approach shows the layer-by-layer printing and synthesis of
peptides with a resolution of 50 mm.
54
Aer deposition of the compounds onto the
surface, immobilization can be achieved in many different approaches.
3.1. Non-covalent immobilization
The rst immobilization of glycans onto a surface was reported in 2002,
following a non-covalent adsorption approach.
55
Non-covalent attachment of
either modied or unmodied glycans to a surface is mediated by electrostatic
interactions, hydrogen bonds or van der Waals forces. Today the selective
covalent attachment of sugar molecules to a microarray is preferred, because it
results in more stable and well-dened binding sites, enabling more precise
biomolecular interactions.
Site-nonspecic immobilization. The easiest way to immobilize a glycan on
a surface is the non-covalent, site-nonspecic approach (see Fig. 4A). Since no
extra-functionalization of the sugar is required, this method is only suitable for
longer glycans that maintain a large contact area with the surface. The binding
site of the molecule to the surface is random, which makes screening of biomo-
lecular interaction less precise. Moreover, there is a constant risk of losing the
compounds during the washing steps.
With this approach, unmodied polysaccharides were spotted onto a nitro-
cellulose-coated glass slide.
55,56
Charged polysaccharides like heparin are partic-
ularly suitable for this approach, since the negatively charged sulfate groups can
be efficiently attached to positively charged poly-L-lysine coated glass slides via
electrostatic interactions.
57,58
Site-specic immobilization. Reproducibility can be enhanced by specic
binding to a surface at a distinct position of the glycan (see Fig. 4B). The chemical
modication of the glycan is generally carried out at the reducing end. Glyco-
conjugates, such as glycolipids, can be easily immobilized on a surface, resem-
bling the natural presentation of glycans.
Nitrocellulose or PVDF (polyvinylidene diuoride) membranes were used to
immobilize lipid-conjugated glycans (neoglycolipids) via hydrophobic interac-
tions (van der Waals forces). The neoglycolipids were prepared by reductive
amination of the sugar compound and an amino-conjugated lipid.
59–68
A similar
attachment strategy used uorous tagged glycans for immobilization on a Teon/
epoxy coated glass slide.
69–71
The peruorinated alkyl chain allows for easy puri-
cation and permits strong binding to the surface that survives extensive washing
steps. Another uorous approach was carried out on aluminum oxide coated glass
slides, which were covalently functionalized with a phosphonate, tagged with
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a peruoroalkyl chain.
72,73
On-spot analysis via mass spectrometry was also
possible. Importantly, when hydrophobic surfaces are used, a blocking step
before biomolecular screenings is required.
The strong biotin–streptavidin interaction (K
d
10
15
M) was exploited to
manufacture glycan arrays. Streptavidin-coated surfaces in combination with
biotinylated glycans were utilized.
74–77
Similarly, DNA hybridization was employed
to prepare glycan microarrays. The glycans were functionalized with an oligo-
nucleotide that was hybridized with the complementary oligonucleotide attached
to a surface.
78
3.2. Covalent immobilization
The covalent attachment of a glycan to a surface is usually preferred, because it
minimizes the risk of compound leaching during the washing steps. Glass slides
coated with a silane or thin polymer lm are employed, which are functionalized
with various functional groups for the coupling reaction.
Site-nonspecic immobilization. The simplest and fastest way to couple
unmodied glycans to a surface is the covalent site-nonspecic approach (see
Fig. 4C). However, the random binding of the sugar can be problematic for the
validity of the biomolecular binding screenings. Photochemical reactions, where
the functionalized glass slide bears a photo-activatable group, are commonly
used.
Photo-labile groups such as aryl(triuoromethyl)diazirine
79
or 4-azido-2,3,5,6-
tetrauorophenyl
80
are common functionalities that, upon UV irradiation, turn
Fig. 4 Different immobilization strategies for glycan microarray production. (A) Non-
covalent, site-nonspecific glycan binding; (B) non-covalent site-specific glycan binding;
(C) covalent site-nonspecific glycan binding; (D) covalent site-specific glycan binding.
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into reactive carbene or nitrene species. These reactive compounds are able to
react easily with the spotted unprotected sugar compounds via simple insertion
reactions to form stable covalent bonds. Another approach makes use of
phthalimide-modied surfaces.
81,82
Upon UV irradiation, the carbonyl groups of
phthalimide can readily undergo a photochemical hydrogen abstraction reaction
with the desired sugar which ends in stable covalent bonds between the
compound and the surface. The reaction between boronic acid functionalized
surfaces and diols of the sugars was also exploited to produce carbohydrate
microarrays.
83
Site-specic immobilization. The covalent site-specic attachment of chemi-
cally modied glycans is now the method of choice for carbohydrate microarray
production (see Fig. 4D), with many different available reactions. These coupling
reactions have to be highly selective, easy to manipulate and mild. Selective
surface attachment renders the binding studies with biomolecules more reliable
when compared to site-nonspecic approaches. The nature of the linker between
the sugar and the surface plays a crucial role, inuencing protein binding.
Hydrophilic oligo or poly(ethylene glycol)-based linkers oen show better results
compared to the hydrophobic analogues. Additionally, the linker affects the
nonspecic adsorptions of the proteins and its length is important for the
accessibility of the attached glycan.
84,85
The most challenging part of the covalent
site-specic method is the functionalization of the sugar, which oen requires
multiple steps and well-wrought synthetic strategies.
A very powerful strategy exploits the thiol–maleimide chemistry. The reaction
is very fast under mild conditions and highly selective. Glycans are either func-
tionalized with maleimide groups and coupled to thiol-coated surfaces,
84–86
or
thio-sugars are attached to maleimide-coated surfaces (see Fig. 5A).
87–94
With this
approach, even challenging glycans like glycosylphosphatidylinositols (GPIs)
could be easily printed onto microarrays.
95,96
The formation of disulde bonds,
either between thiosulfonate-conjugated glycans and thiol-functionalized
surfaces or between thiol-conjugated glycans and pyridyl disulde-modied
surfaces, was also successful.
97,98
Nevertheless, the possible oxidation of the
thiol due to exposure to air can create problems.
Fig. 5 Important coupling reactions for site-specific covalent bond formation. (A) Thiol-
functionalized sugar and maleimide surface; (B) amine-functionalized sugar and N-
hydroxysuccinimide ester surface; (C) free reducing end glycan and hydrazide surface.
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The most widely used immobilization strategy employs amino-functionalized
glycans and N-hydroxysuccinimide (NHS) ester-coated surfaces (see Fig. 5B). The
reaction between these two compounds under slightly basic conditions (pH 8.5)
leads to the formation of a very stable amide bond with very good selec-
tivity.
11,99–102
The NHS ester-coated glass slides are commercially available and the
synthesis of amine-functionalized glycans follows standard protocols.
103
Amine-
modied glycans can be readily accessed also from automated strategies.
35
Alternatively, the amino-modied glycans can be attached to cyanuric chloride-
functionalized surfaces via nucleophilic aromatic substitution.
104,105
Unprotected linker-free glycans can be immobilized in a site-specic way,
using hydrazide- (see Fig. 5C) or oxyamine-modied surfaces.
106,107
These func-
tional groups are highly nucleophilic and able to react easily with the reducing
ends of the glycans to form stable adducts. Similarly, an aldehyde-functionalized
surface and oxyamine-modied sugars were used to prepare glycosaminoglycan
microarrays.
108
Another glycosaminoglycan microarray was produced on an
amine-coated surface, using a deaminated heparin, bearing an aldehyde
functionality.
109
Epoxide-coated surfaces in combination with hydrazide-functionalized sugars
offer a valuable alternative to form stable covalent bonds (see Fig. 6C).
110–115
Moreover, epoxide-coated surfaces are very versatile and can be used in combi-
nation with many different nucleophiles such as amine- or thiol-conjugated
carbohydrates (see Fig. 6A and B).
116,117
Cycloadditions with dienophile-
conjugated carbohydrates also show good selectivity and can be carried out
under mild conditions. Diels–Alder reaction between a cyclopentadiene-linked
Fig. 6 Versatility of epoxide-coated surfaces. (A) Thiol-functionalized sugar; (B) amine-
functionalized sugar; (C) hydrazide-functionalized sugar.
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sugar and a benzoquinone-coated surface,
118
as well as the very fast tetrazine–
norbornene inverse electron demand Diels–Alder reaction were applied.
119
Nevertheless, the lack of long-term stability of some of these compounds limits
their applications.
The copper-catalyzed azide–alkyne click reaction (CuAAC) was applied in
glycan microarray production, because of its high selectivity and compatibility
with a broad range of functional groups. Glycans functionalized with azide groups
are coupled to alkyne-functionalized surfaces (or inverted functionalization).
120–124
In addition, azide-modied glycans were used to prepare microarrays through
chemoselective Staudinger ligation.
125
Photochemical attachment of a 4-azido-
2,3,5,6-tetrauorophenyl-conjugated sugar to a polymer monolayer offered
a very mild alternative.
80,126
Aer spotting the compound onto the surface, irra-
diation with UV light converts the azide functionality to a reactive nitrene species,
which is able to react with the polymer monolayer to from a stable covalent bond.
4. Multivalent presentation
Carbohydrate–protein interactions are very weak. However, usually multiple
simultaneous interactions between several carbohydrate ligands and one receptor
occur, which increases the binding strength. This concept is called multivalency.
For a multivalent interaction to take place, the spatial distribution and orienta-
tion of the sugar groups are crucial.
127–129
To translate this to carbohydrate
microarrays, the glycan presentation and, especially the density and orientation,
need to be considered in detail.
In conventional array platforms, single monovalent glycans are randomly
attached to the array surface via a linker, yielding a certain –but uncontrolled –
multivalent display, which may be sufficient to elicit a high-avidity binding event.
These systems usually rely on two-dimensional arrangements of monovalent
glycans, with very little control over spatial organization. However, carbohydrate–
protein interactions vary quite signicantly and high glycan density may either
enhance it via multivalency or suppress it via steric hindrance. Therefore, several
studies were conducted to identify the optimal presentation of carbohydrates by
varying the glycan concentration during printing and the exibility of the
attachment point.
130,131
To date, full control on spatial organization is still a big
challenge and clustering effects can cause unreproducible results.
To improve control over glycan presentation, multivalent glycoconjugates with
various valencies and spatial arrangements have been designed and immobilized
on arrays. Scaffolds, based on natural glycoproteins, neoglycoproteins/
neoglycopeptides, glycodendrimers, multivalent display on DNA, glycoclusters,
and glycopolymers, have been used (Fig. 7).
13,132–144
Natural glycoproteins, such as the heavily glycosylated mucins, were used as
multivalent glycan systems for the production of arrays. This microarray retained
the three-dimensional presentation of mucin oligosaccharides, without modi-
cations of the protein backbone and permitted the discovery of biologically
important motifs for bacterial–host interactions.
145
A similar approach uses
natural proteins, such as bovine serum albumin (BSA) or human serum albumin
(HSA), for the production of neoglycoproteins/neoglycopeptides (proteins or
peptides with glycans covalently attached via non-native linkage), which display
multiple copies of each glycan. Presynthesized glycoconjugates can be
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immobilized on epoxide slides. This strategy permits immobilization of both
synthetic carbohydrates as well as natural carbohydrates, presented on glyco-
proteins. Important factors that affect the binding are the number of glycans on
a neoglycopeptide, the linker length between the individual sugars, the distance
between neoglycopeptide probes on the surface, and the type of protein.
113,146–148
These parameters can be tuned to affect the recognition process. Variations in the
neoglycoprotein density revealed differences in specicity for antibodies that
were not apparent at low density.
149
Oligonucleotide hybridization permits to tailor spatial geometry. The rigidity of
the double strand nucleic acid with well-dened nucleotide spacing permits to
adjust the ligand presentation on this supramolecular scaffold.
150–152
Similarly,
peptide nucleic acids (PNAs) have been used to tag glycans and evaluate their
multivalent interactions with lectins. From an assembly stand-point, stable PNA–
DNA duplexes can be achieved with shortersequences than the corresponding DNA
homoduplexes (10–14-mer PNA typically provides sufficient duplex stability).
153
Fig. 7 Schematic glycan presentation on microarrays. (A) High density arrangement of
glycans. (B) Low density arrangement of glycans. (C) Multivalent glycoconjugates to
modulate glycan presentation on microarray surfaces.
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Chemical ligation of sugars at different positions within a PNA oligomer has
been achieved
154
by using thiol moieties embedded in the backbone of the PNA,
chemoselectively conjugated to a maleimide-glycan. DNA microarrays permitted
the combinatorial pairing of diverse PNA-tagged glycan conjugates. The use of
adjacent hybridization sites produced assemblies, emulating the diversity of di-,
tri- and tetra-antennary glycans, mimicking the geometry of the HIV gp120 glycan
epitope. The combinatorial synthesis of an extended library of PNA-encoded
glycoconjugates represents the largest array of heteroglycan conjugates reported
to date.
155–157
Unnatural scaffolds, like dendrimers, were used for microarray analyses of
CPIs.
158–161
Well-dened 3D saccharide arrangements on microarrays were con-
structed upon covalent binding of the dendrimers to the chip surface. Carbohy-
drates were attached to the dendrimer arm via “click”chemistry, prior or
following the attachment of the whole construct to the chip. The multivalency can
be precisely controlled with the structure of the glycodendrimer, with valencies
ranging from one to eight sugars. Other unnatural alternatives used for multi-
valent presentation are glycoclusters. Calix[4]arenes are a suitable platform that
can be easily derivatized at the upper and lower rims, resulting in well-organized
three-dimensional architectures.
162,163
With such systems, the primary impor-
tance of the spatial arrangement, compared to the number of carbohydrate
residues, was highlighted.
164
The importance of spatial orientation was observed by 16 different fucosylated
glycomimetics, bearing one to eight fucose moieties, synthesized with antenna-
like, linear (or comb-like), or crown-like arrangements.
165
Binding properties
using DNA directed immobilization (DDI)-based glycan microarrays showed that
no chelate effect was present, with a one to one interaction between fucose and
the lectin. Synthetic glycopolymers have been used to generate mucin-like
structures which, as do natural ones, possess rigid extended structures.
124,166
Polymers of low polydispersity, displaying a-GalNAc residues, were produced by
reversible addition-fragmentation chain transfer (RAFT) polymerization. This
new class of orthogonally end-functionalized mucin-mimetics was printed on
a microarray, where GalNAc valency and interligand spacing could be controlled.
This system again proved that glycan valency and organization are critical
parameters that determine the modes through which these interactions occur.
5. Characterization and binding measurements
The readout of a glycan microarray is an important step to obtain precise and
convincing data. To detect binding events of glycan binding proteins (GBPs) or
successful enzymatic glycosylations on the array, different methods are available.
The most frequently applied method is the detection of uorescently-labeled
binders, which directly or indirectly bind to the glycans on the microarray (see
Fig. 8). The binding event can be visualized with a uorescence scanner in several
ways: either the GBP is uorescently labelled or a uorescently tagged secondary
reagent (e.g. antibody) is used to bind to the GBP or to a tag (e.g. biotin, His tag) on
the GBP. As discussed, multivalency plays a crucial role for CPIs and the glycan
density on the microarray is essential to achieve differential binding. If the
density is too low, the GBPs are sometimes unable to properly bind to the glycans,
which results in a loss of signals and thus to misleading results.
167
Additional
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problems can be caused by the label, which can reduce the activity or inuence
the selectivity of the GBPs.
168
Unfortunately, indirect labeling of GBPs is oen not
possible, because uorescently-labeled secondary reagents are not available.
9
Mass spectrometry is a label-free method to monitor chemical or enzymatic
glycosylations directly on an array. Thiol-linked sugars were deposited on a gold
surface, whereby self-assembled monolayers are formed. Elongation reactions
were then monitored by an on-slide mass spectrometry technique named SAMDI-
TOF-MS.
21,24,169
With a similar non covalent approach, glycosylation of carbohy-
drates immobilized on modied gold surfaces using van der Waals forces
between aliphatic
170,171
or peruorinated
172
carbon chains was monitored.
Multiple detection techniques could be used as proven by a multifunctional
microarray platform consisting of a glass surface coated with an indium-tin oxide
layer. Matrix-assisted laser desorption/ionization time-of-ight (MALDI-TOF)
mass spectrometry, uorescence spectroscopy, and optical microscopy can be
employed on the same surface.
25
Surface plasmon resonance (SPR) imaging is an alternative label-free method
for the analysis of glycan microarrays that allows for determination of the
thickness of layers on a metal surface in the nanometer range. SPR has the
advantage of real-time monitoring of GBP binding events, which allows for
measuring of kinetic and thermodynamic parameters. Metal surfaces (e.g. gold)
are mandatory for this approach to excite surface plasmons within the metal by
irradiation with polarized light. SPR was used to screen interactions between
GBPs and glycans of the pathogen Schistosoma mansoni.
146
BSA–mannose-
conjugates with different mannose substituents were attached to a gold surface
and incubated with ConA to measure K
D
values and relate it to multivalency.
173
Additionally, SPR permitted to identify ligand specicity of plant lectins
174
and to
better understand siglec-8 (ref. 76) or ConA
175
binding specicities.
The above mentioned analysis technologies are the most common, but many
others, such as evanescent-eld uorescence,
176–178
ellipsometry,
179
electro-
chemoluminescence,
180
detection of radioactivity,
181,182
oblique-incidence reectivity
(OI-RD) microscopy,
183
frontal affinity chromatography,
184,185
isothermal calorim-
etry,
186
and cantilever-based detection
187
exist. Nevertheless, multivalency
cannot be detected directly with one of the analytical techniques. Experiments
using multivalent scaffolds have to be compared to those with the monovalent
analogue. New technologies to systematically vary the glycan density directly on
the microarray are required, to understand multivalent events.
Fig. 8 Detection of directly or indirectly fluorescently labeled glycan binding proteins
(GBPs) binding to specific glycans on a microarray by fluorescence scanning.
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6. Conclusions and outlook
The direct in situ synthesis on surfaces is already well-established and commer-
cialized for oligonucleotides (e.g.,Affymetrix, Agilent, Illumina) and peptides (e.g.,
Intavis, JPT, PEPperPRINT). A big part of this success is based on the enabling
technologies, that had a major impact on high-throughput analysis and
screening. To translate these technologies to in situ glycan synthesis will be far
more challenging: to date, only the chemical synthesis of disaccharides on
a“macroarray”surface has been shown,
21
whereas enzymatic synthesis is more
promising.
22–25
Furthermore, multivalency is usually neglected in oligonucleotide
and peptide synthesis.
In contrast, multivalency is essential for GBPs, because of the naturally weak
(K
D
mm) protein–glycan affinity, compensated by multiple binding sites.
188
Since the advent of glycan microarrays, the main focus has been on the analysis of
glycans on surfaces, with less interest in the control of molecular density and
spacing. Yet, a dened way of presenting glycans on microarrays is the key step to
strong GBP binding. Therefore, strategies are required to display glycans in
a molecularly dened spatial order.
Different scaffolds or density variations have been proposed and quite
successfully applied for multivalent glycan display.
166,167
Especially, the density
variation presentation on surfaces leads to random and non-homogeneous
systems, which lack reproducibility. Most scaffolds offer dened spacing, but
Fig. 9 Peptide array with peptide tetramers, synthesized via laser transfer, derivatized with
up to four a-D-mannose azides clicked to the peptide backbone. Sixteen different
peptides (quadruplicate spots) show differential lectin (ConA) binding, due to different
multivalent display.
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lack exibility, because the spacing cannot be changed easily. An elegant solution
is DNA technology to display glycans. Only recently, this was shown for the display
different molecules.
189,190
Using DNA-origami structures as scaffolds, multiple
glycan structures can be placed in a wide variety of 2D and 3D congurations at
exact positions in a controlled and reproducible way. Moreover, it may be used to
exactly space the glycans to generate a perfectly matching template for the
binding sites of multivalent GBPs. Thereby, control in the screening processes for
pathogen interaction with a large variety of structures is possible.
The dened generation of many diverse scaffolds with dened glycan spacing
will be one of the future research goals in glycan array technology. Progress in the
eld of in situ synthesis of scaffolds has been made. By growing brush-like gly-
copolymers directly on the surface via in situ photo-polymerization, glycan
microarrays with multivalent display were generated.
20
Different polymer lengths
were produced with different amounts of sugar units on the polymer scaffold, by
changing the irradiation time.
We recently employed a novel laser-based transfer setup
50
to generate peptide
scaffolds for multivalent display. We synthesized arrays of peptide tetramers,
containing all 16 possible sequences of L-glycine and L-propargylglycine. The
propargylglycine offers an alkyne group for copper catalyzed click chemistry to
attach up to four glycan azides to the peptide backbone. Depending on the
amount and position of the a-D-mannose, we obtained differential binding of the
lectin concanavalin A (see Fig. 9). This approach may serve as a basis to generate
large and complex compound collections for the multivalent display of many
different glycans in an orthogonal synthesis strategy. With our laser-based
approach, molecules can be synthesized directly on surfaces step-by-step, by
“printing”and stacking solid polymer nanolayers,
51,191
which embed all kinds of
different chemicals and building blocks. Especially for peptide synthesis and
applications in disease research, this offers a rapid strategy to generate diverse
microarrays.
192–197
In the future, this technology may be exploited for the in situ
synthesis of glycopeptides, glycans, and DNA in a microarray format.
A large gap remains in the multivalent display and analysis of complex glycans
that needs to be lled. Advances accessing glycans and their synthesis and
immobilization on surfaces show promising directions for future glycan micro-
array research. Precisely dened multivalent arrangements on DNA or other
structural scaffolds will enable the identication of cooperative effects between
identical or diverse collections of glycans. Simultaneously, novel tools based on
the presentation of single or multiple glycan molecules in specic arrangements
and stoichiometry on the surfaces will be developed.
In the future, newly developed platforms will enable highly parallelized
screenings, testing tens of thousands of combinations simultaneously in
a microarray-based assay format. The eld of protein–glycan interactions will
benet as researchers will be able to uncover the conformation of glycans in
a biological environment and open new roads to develop efficient vaccines.
Conflicts of interest
P. H. S. declares a signicant nancial interest in GlycoUniverse GmbH & Co
KGaA, the company that commercializes the synthesis instrument, building
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blocks and other reagents. F. F. L. is named on a pending patent application
related to laser-based microarray synthesis.
Acknowledgements
We thank the Max-Planck Society, the Minerva Fast Track Program, and the MPG-FhG
Cooperation Project Glyco3Dysplay, and the German Federal Ministry of Education
and Research (BMBF, grant no. 13XP5050A) for generous nancial support.
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