Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans

Article abstract of DOI:10.1002/chem.201500065

Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays. Automated synthesis meets plant research: Automated synthesis of plant oligosaccharides provides molecular tools for the characterization of important biological probes in plant research. The binding specificities of a collection of xylan-directed monoclonal antibodies have been determined by means of carbohydrate microarrays equipped with synthetic arabinoxylan fragments (see figure).

Full text of DOI:10.1002/chem.201500065

DOI: 10.1002/chem.201500065
Communication
&
Carbohydrates
Automated Synthesis of Arabinoxylan-Oligosaccharides Enables
Characterization of Antibodies that Recognize Plant Cell Wall
Glycans
Deborah Schmidt,[a, b] Frank Schuhmacher,[a, b] Andreas Geissner,[a, b] Peter H. Seeberger,[a, b]
and Fabian Pfrengle*[a, b]
a common backbone consisting of b-1,4-linked d-xylopyrano-
ses. This backbone structure may be partially acetylated and
substituted with l-arabinofuranosyl or d-(4-O-methyl) glucur-
onyl residues.[2b]
Abstract: Monoclonal antibodies that recognize plant cell
wall glycans are used for high-resolution imaging, provid-
ing important information about the structure and func-
tion of cell wall polysaccharides. To characterize the bind-
ing epitopes of these powerful molecular probes a library
of eleven plant arabinoxylan oligosaccharides was pro-
duced by automated solid-phase synthesis. Modular as-
sembly of oligoarabinoxylans from few building blocks
was enabled by adding (2-naphthyl)methyl (Nap) to the
toolbox of orthogonal protecting groups for solid-phase
synthesis. Conjugation-ready oligosaccharides were ob-
tained and the binding specificities of xylan-directed anti-
bodies were determined on microarrays.
High-resolution imaging of cell wall microstructures has pro-
vided insights into the structure, function, dynamics, and bio-
synthesis of plant cell wall polysaccharides.[5] It has been
shown that different glycans are present within subdomains of
a single cell wall and that the occurrence of specific substruc-
tures changes during plant growth.[6] The composition of gly-
cans in the cell wall can be monitored using monoclonal anti-
bodies developed against plant polysaccharides.[7] The specific-
ities of these powerful molecular probes were investigated
using carbohydrate microarrays equipped with poly- and oligo-
saccharides of plant origin.[8] However, the precise epitopes
Plant cells are surrounded by
a
polysaccharide-rich matrix
that constitutes the cell wall of
all higher plants.[1] One of the
main components of plant cell
wall polysaccharides is the hem-
icellulose xylan, the second
most abundant polysaccharide
in Nature (Figure 1).[2] Xylans are
dietary carbohydrates in every-
day food that can provide me-
dicinal benefits including immu-
Figure 1. The hemicellulose xylan cross-links cellulose microfibrils in plant cell walls.
nomodulatory, antitumor, and
antimicrobial effects.[3] In addi-
tion, xylans are potential resources for the production of food
additives, cosmetics, and biofuels.[4] Although the structure of
xylans varies between plant species, they all possess
that are recognized by these antibodies are yet to be deter-
mined.
Despite the potential of xylan fragments as tools for plant
biology and in the development of various practical applica-
tions,[4] access to xylo-oligosaccharides is mostly limited to
their isolation from plants.[9] However, structurally defined and
highly homogeneous samples are obtained by chemical syn-
thesis.[10]
[a] D. Schmidt, F. Schuhmacher, A. Geissner, Prof. Dr. P. H. Seeberger,
Dr. F. Pfrengle
Department of Biomolecular Systems
Max-Planck-Institute of Colloids and Interfaces
Am Mühlenberg 1, 14476 Potsdam (Germany)
Automated oligosaccharide synthesis[11] provides the means
to construct carbohydrate libraries in a facile and timely
manner, as structurally related oligosaccharides can be pro-
duced from common building blocks. Here, we report the au-
tomated synthesis of a collection of arabinoxylan fragments
[b] D. Schmidt, F. Schuhmacher, A. Geissner, Prof. Dr. P. H. Seeberger,
Dr. F. Pfrengle
Freie Universität Berlin, Institute of Chemistry and Biochemistry
Arnimallee 22, 14195 Berlin (Germany)
E-mail: Fabian.Pfrengle@mpikg.mpg.de
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201500065.
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Communication
and their use in determining the binding epitopes of antibod-
ies that are derived from natural xylan polysaccharides.
High coupling efficiencies at every step of the automated
synthesis mandate carefully designed building blocks
(Figure 2). For the construction of the b-1,4-linked xylan back-
bone two different d-xylopyranose building blocks were em-
ployed.
Scheme 1. Synthesis of xylose building blocks 6a and 6b. Reagents and
conditions: a) TrCl, DMAP, NEt3, DMF; b) RBr, NaH, TBAI; c) TsOH, MeOH/Et2O/
H2O (100:10:1), 2a: 72%, 2b: 62% (3 steps); d) H2O/AcOH, reflux; e) Ac2O,
DMAP; f) HSTol, BF3·OEt2, 08C, 4a: 62%, 4b: 48% (3 steps); g) NaOMe,
MeOH, CH2Cl2; h) Bz2O, 5–10 mol% Yb(OTf)3, dioxane, 5a: 71%, 5b: 61% (2
steps); j) FmocCl, pyridine, CH2Cl2, a: 79%, b: 76%; k) HOP(O)(OBu)2, N-iodo-
succinimide, triflic acid, 6a: 87%, 6b: 95%.
and 6b in good overall yields. The arabinose building blocks
were synthesized following literature procedures.[15]
Figure 2. Monosaccharide building blocks for the automated solid-phase
synthesis of arabinoxylan fragments.
With the required building blocks in hand a set of eleven
plant arabinoxylan fragments was chosen to be produced
using an automated oligosaccharide synthesizer.[16] We focused
on specific substructures of naturally occurring arabinoxylans
comprised of a linear b-1,4-linked xylan backbone substituted
with a-1,3-linked single l-arabinofuranosyl and b-1,2-d-xylopyr-
anosyl-a-1,3-l-arabinofuranosyl disaccharide residues. Linear
oligoxylans (9–12) and oligoarabinoxylans of different com-
plexity (13–19) were produced via the iterative addition of
building blocks 6a,b and 7a,b to linker-functionalized resin 8
(Scheme 2). The photocleavable linker ensures smooth cleav-
age of the assembled oligosaccharides from the solid support
using UV light in a microfluidic photoreactor.[17] The linker is
designed to withstand strong acidic and basic conditions and
delivers conjugation-ready glycans after cleavage from the
solid support. The glycosylation steps were performed in dupli-
cate using five equivalents of glycosyl donor and either equi-
molar TMSOTf or N-iodosuccinimide together with catalytic
amounts of TfOH for activation. Each glycosylation using
a xylose building block was followed by the removal of either
the temporary Fmoc protecting group at C-4, allowing for
elongation of the xylan backbone, or of the Nap protecting
group at C-3, for installation of arabinose substituents. After
optimization of the reaction conditions on a model disacchar-
ide, we established a reliable protocol for the selective cleav-
age of Nap groups on solid support.[18] Thus, a new temporary
protecting group was added to the toolbox of orthogonal pro-
tecting groups for automated oligosaccharide synthesis. The
Nap ether differs from the routinely used Fmoc and levulinoyl
protecting groups by exerting no disarming effect on the re-
spective building block, facilitating the fine-tuning of glycosyl
donor reactivity in solid-phase syntheses.[19] For the synthesis
of pentasaccharide 15, which contains a b-1,2-d-xylopyranosyl-
a-1,3-l-arabinofuranosyl substituent, the linear xylan backbone
was capped by reaction with acetic anhydride after assembly.
After removal of the Nap-group at the central xylose unit and
installation of the arabinose residue using building block 7b,
the side chain was selectively elongated by deprotection of
Each of the C-4 hydroxyls was protected with a fluorenylme-
thoxycarbonyl (Fmoc) group because it can be selectively re-
moved during the automated synthesis using an amine base.
The required b-selectivity in the glycosylation steps was en-
sured through the installation of benzoate esters on the C-2
hydroxyls. The C-3 hydroxyl was either equipped with a benzyl
ether as permanent protecting group or with a (2-naphthyl)-
methyl (Nap) group as temporary protecting group for substi-
tution of the backbone with arabinose residues. Selective
cleavage of Nap ethers in the presence of benzyl ethers has
been reported under oxidative conditions using dichlorodicya-
nobenzochinone (DDQ),[12] and we envisioned that similar reac-
tion conditions might be applicable to solid-phase synthesis.
We chose the Nap ether because of its similar electronic prop-
erties to benzyl ethers and the relatively straightforward syn-
thesis of the respective xylose building block. Finally, dibutyl-
phosphate was chosen as the anomeric leaving group because
glycosyl phosphates gave the best results in the assembly of
large oligomeric glycans previously.[11e,f] Installation of the ara-
binose substituents on the xylan backbone relied either on
a perbenzoylated or a 2-Fmoc protected l-arabinofuranosyl
building block depending on further side-chain elongation.
The synthesis of the desired xylose building blocks 6a and
6b was accomplished starting from the commercially available
d-xylofuranose derivative 1 (Scheme 1). By following a strategy
reported by Paquette et al. we were able to install either
a benzyl or a Nap ether in 3-position of thioglycosides 4a and
4b, respectively.[13] After removal of the acetyl groups using
sodium methoxide selective protection of the 2-position was
performed following an ytterbium triflate-catalyzed benzoyla-
tion protocol.[14] Protection of the 4-hydroxyl group of thiogly-
cosides 5a and 5b with fluorenylmethoxycarbonyl chloride
(FmocCl) in the presence of pyridine was followed by the con-
version of the thioglycoside leaving group into the corre-
sponding phosphate affording the desired building blocks 6a
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Scheme 2. Automated solid-phase synthesis of arabinoxylan fragments 9–19. The letter code below the structures represents the reaction sequence applied
in the respective synthesis. Reagents and conditions: a) or b) 2”5 equiv of BB 6a or 6b, TMSOTf, DCM, À358C (5 min)!À158C (30 min); c) or d) 2”5 equiv
of BB 7a or 7b, NIS, TfOH, DCM/dioxane, À408C (5 min)!À208C (40 min); e) 3 cycles of 20% NEt3 in DMF, 258C (5 min); f) 7 cycles of 0.1m DDQ in DCE/
MeOH/H2O (64:16:1), 20 min; g) 3 cycles of Ac2O, pyridine, 258C (30 min); h) hn (305 nm); i) NaOMe, THF/MeOH, 12 h; j) H2, Pd/C, EtOAc/MeOH/H2O/HOAc,
12 h. 9: 15%, 10: 27%, 11: 20%, 12: 43%, 13: 23%, 14: 19%, 15: 42%, 16: 8%, 17: 15%, 18: 7%, 19: 21% (yields are based on resin loading).
the Fmoc-substituted arabinose residue and a final glycosyla-
tion with xylose donor 6a.
analytical HPLC, NMR spectroscopy, and high-resolution mass
spectrometry.[18]
Following oligosaccharide assembly, the resin was removed
from the synthesizer and the crude oligoarabinoxylosides were
cleaved from the solid support.[17] Subsequent removal of the
benzoates with sodium methoxide and hydrogenolytic cleav-
age of the benzyl ethers and the Cbz protecting group afford-
ed the fully deprotected oligoarabinoxylans 9–19 in 7–43%
overall yield, based on the calculated loading of linker-func-
tionalized resin 8. The products were purified by preparative
reversed-phase HPLC, either at the stage of the semi-protected
oligosaccharides before hydrogenation, or at the final stage
yielding the pure products. All products were characterized by
With arabinoxylan fragments 9–19 in hand, microarray
slides[20] were printed and used as tools to probe the binding
preferences of 31 anti-xylan monoclonal antibodies.[21] Selected
examples are shown in Figure 3. As expected all linear oligoxy-
lans were detected by monoclonal antibody LM10 that was
originally raised against a synthetic pentaxyloside–BSA conju-
gate.[22] LM10 also bound to all arabinose substituted struc-
tures containing a terminal xylose residue. Similarly CCRC-
M150, raised against a natural corn stover xylan, recognized
almost all oligoxylans independent of backbone substitution.
In some cases the binding epitopes were precisely character-
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ized. CCRC-M147 recognized selectively all structures contain-
ing an unsubstituted xylan disaccharide and CCRC-M154
bound exclusively to oligoxylans substituted with a-1,3-linked
arabinose residues. The antibodies used in this study were pre-
viously grouped into four clades based on their binding prop-
erties towards natural xylan polysaccharides.[21] Interestingly,
we observed different binding patterns towards the synthetic
oligoarabinoxylans not only for antibodies from the same
clade but also for antibodies raised against the same polysac-
charide antigen. For example, while CCRC-M140 detected only
the linear hexa- and octaxylosides, CCRC-M153 also bound to
arabinose substituted xylosides. Both antibodies were original-
ly raised against the same natural oat xylan and bind the same
group of natural xylans. None of the antibodies showed either
strong or selective binding to pentasaccharide 15 containing
the b-1,2-d-xylopyranosyl-a-1,3-l-arabinofuranosyl substituent
making this antigen a promising candidate for immunizations
and production of monoclonal antibodies towards this other-
wise undetectable epitope. Our results demonstrate that libra-
ries of synthetic plant oligosaccharides can be used to deter-
mine the binding specificities of cell wall glycan-directed anti-
bodies as exemplified by the identification of antibody CCRC-
M154 as a selective marker for xylans that are densely substi-
tuted with a-1,3-linked arabinose residues.
Employing automated solid-phase synthesis enabled us to
rapidly procure a collection of oligosaccharide fragments of
plant arabinoxylans. The library of synthetic arabinoxylan frag-
ments was printed as microarray and probed with anti-xylan
antibodies, which are valuable probes for immunolabeling
studies of plant cell walls. The binding specificities of several
antibodies were determined, and currently other oligoxylans
such as glucuronoxylans are being prepared for characteriza-
tion of antibodies that did not bind the structures presented
in this report. With the synthetic ability to rapidly create libra-
ries of precision plant carbohydrates, future glycan array stud-
ies will provide the essential information required for a detailed
interpretation of immunolabeling studies of plant cell walls.
Acknowledgements
We gratefully acknowledge financial support from the Max
Planck Society, the Fonds der Chemischen Industrie (Liebig Fel-
lowship to F.P.) and an ERC Advanced Grant (AUTOHEPARIN to
P.H.S.). We thank Dr. M. Schlegel for helpful discussions and Dr.
K. Gilmore for critically editing this manuscript. Generation of
the CCRC series of monoclonal antibodies used in this work
was supported by a grant from the NSF Plant Genome Pro-
gram (DBI-0421683).
Keywords: arabinoxylan · carbohydrates · microarray · plant
cell wall · solid-phase synthesis
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Received: January 7, 2015
Published online on February 26, 2015
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