Characterization of the LM5 pectic galactan epitope with synthetic analogues of β-1,4-D-galactotetraose

Article abstract of DOI:10.1016/j.carres.2016.10.012

Plant cell wall glycans are important polymers that are crucial to plant development and serve as an important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily on monoclonal antibodies (mAbs) for localizati

Full text of DOI:10.1016/j.carres.2016.10.012

Carbohydrate Research 436 (2016) 36e40
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Characterization of the LM5 pectic galactan epitope with synthetic
analogues of
b-1,4-
D-galactotetraose
a
a
b
c
ꢀ
Mathias C.F. Andersen , Irene Boos , Susan E. Marcus , Stjepan K. Kracun ,
Maja Gro Rydahl c, William G.T. Willats c d, J. Paul Knox b, Mads H. Clausen a
,
, *
a Center for Nanomedicine and Theranostics, Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, Lyngby, Denmark
b Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom
c Department of Plant and Environmental Sciences, Thorvaldsensvej 40, Frederiksberg C, Denmark
d School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Plant cell wall glycans are important polymers that are crucial to plant development and serve as an
important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily
on monoclonal antibodies (mAbs) for localization and visualization of glycans, using e.g. immunofluo-
rescent microscopy. Here, we describe the detailed epitope mapping of the mAb LM5 that is shown to
bind to a minimum of three sugar residues at the non-reducing end of linear beta-1,4-linked galactan.
The study uses de novo synthetic analogues of galactans combined with carbohydrate microarray and
competitive inhibition ELISA for analysis of antibody-carbohydrate interactions.
Received 31 August 2016
Received in revised form
26 October 2016
Accepted 27 October 2016
Available online 30 October 2016
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
Monoclonal antibodies with epitopes found in cell wall poly-
saccharides are important tools for studying plant glycans in situ
Plant carbohydrates are crucial biomass polymers that we either
use directly or process into other products. They are an important
source of food and feed ingredients, and constitute a feedstock for
bio-based materials and fuels [1e3]. Plant glycans show an
extraordinary diversity and complexity leading to a variety of bio-
logical functions as well as physical properties [4,5]. A major class of
highly complex polysaccharides in plant cell walls are pectins,
which can be classified into three types: homogalacturonan (HG)
and rhamnogalacturonan (RG) I and II. RG-I is a heteropolymer with
with immunofluorescence microscopy. For example, this technique
has been applied to determine changes in cell wall polysaccharide
distributions during plant development [6,7]. The frequently used
rat monoclonal antibody LM5 is known to bind b-1,4-linked gal-
actans [8]. LM5 has been applied in immunohistochemical analysis,
immunofluorescence microscopy, live cell labeling and for glycan
microarray screening [8e11]. The antibody was generated by im-
munization of rats with a
been reported that for LM5-binding at least four consecutive
(1 / 4) galactosyl units are required as previous studies indicated
no interaction with -1,4-galacto-biose and etriose [8]. Further-
more, we have previously shown that LM5 does not tolerate -1,6
b-1,4-Gal4-BSA neoglycoprotein. It has
a backbone of alternating
a
-linked
L-rhamnose and
D
-galacturonic
b
acid residues with extensive branching of galactans, arabinoga-
lactans and arabinans [4,5]. Biological studies of individual poly-
saccharides in plant organs and plant-derived materials are
immensely challenging due to their heterogeneity and diversity.
Structurally defined oligosaccharides are useful tools as models for
the more complex glycan polymers and can be used in in-
vestigations of a range of processes, such as cell wall biosynthesis
and degradation as well as protein-carbohydrate interactions in
general.
b
b
branching in order to bind to defined galactans [12]. We were
interested in more detailed insight into the binding mode of LM5.
From previous work, it was not clear whether the non-reducing end
of linear
b
-1,4-galactans is part of the LM5 epitope. In order to
-1,4-tetragalactoside
answer this question, we designed three
b
analogues 1e3 (Fig. 1). In the three synthetic oligosaccharides,
the axial C4eOH in the non-reducing end residue was replaced
with C4eF, C4eOMe and the C4-epimer, respectively. We envi-
sioned that by either replacing the C4 hydroxy group with the
bioisosteric fluorine, blocking it by methylation or changing the
orientation by making the gluco-analogue, we would be able to
* Corresponding author.
E-mail address: mhc@kemi.dtu.dk (M.H. Clausen).
http://dx.doi.org/10.1016/j.carres.2016.10.012
0008-6215/© 2016 Elsevier Ltd. All rights reserved.

M.C.F. Andersen et al. / Carbohydrate Research 436 (2016) 36e40
37
Fig. 1. Retrosynthetic analysis of the desired tetragalactan analogues 1e3.
learn more about the LM5 epitope.
The chemical synthesis of -1,4-D-galactans can be problematic
due to low reactivity of the axially disposed C4eOH of the corre-
sponding galactosyl acceptors. Thus, few approaches have been
reported so far [13e15]. Our group recently published a convergent
strategy for the synthesis of linear and branched b(1 / 4)-D-gal-
actans [12]. This approach inspired our pursuit of the three
different analogues required for studying the LM5 epitope.
84% yield without side product formation [20]. Unfortunately,
neither pentenyl donor 14 nor 17 could be used as donors, as they
proved to have low reactivity and afford unsatisfactory coupling
yields in glycosylation reactions. Instead, the pentenyl glycosides
were converted to the more reactive imidate donors 7 and 8 in two
steps. The perbenzoylated glucosyl imidate 9 was synthesized
following a literature procedure [16].
Next step was the synthesis of the trisaccharide acceptor 22.
Starting from pentenyl glycoside 15, regioselective benzylation of
C3eOH was performed via the stannylene acetal in a yield of 79%
(Scheme 3). Pivaloyl-protection of C2eOH afforded donor 10, which
was transformed to galactosyl imidate 18 in two steps.
b
2. Results and discussion
It was intended to synthesize the analogues with the building
blocks 7e11 shown in Fig. 1. Thereby, N-phenyltrifluoroacetimidate
donors could be reacted with pentenyl glycosides as acceptors.
Pentenyl glycosides where chosen as intermediates, due to their
stability under methylation and fluorination conditions.
Fluorinated galactoside 14 was prepared in four steps from the
known pentenyl glucoside 12 (Scheme 1) [16,17]. The common way
of synthesizing fluorinated sugars is the use of diethylaminosulfur
trifluoride (DAST) [18]. Unfortunately, this method was not appli-
cable for C4eF galactose analogues. Instead, the C4eOH of 13 was
converted to the corresponding triflate and fluorine was introduced
by an SN2 substitution with tetrabutylammonium fluoride (TBAF)
to give 14 in 59% (over two steps).
The O-4 methylated pentenyl galactoside donor 17 was obtained
in three steps from the known diol 15 (Scheme 2) [19]. Alkylation of
the C4eOH of 16 turned out to be challenging due to the low
reactivity and the tendency of the neighboring benzoyl group to
either migrate or undergo hydrolysis. A combination of Meer-
weins's salt and Proton-sponge® gave smooth conversion to 17 in
Acceptor 19 was obtained from 10 by regioselective opening of
the benzylidene acetal with triethylsilane/trifluoroacetic acid
(Et3SiH/TFA) in 80% [21]. The regioselectivity was validated by
acetylating the liberated C4eOH, which resulted in a characteristic
downfield shift from 3.98 ppm to 5.08 ppm of the H-4 proton in the
NMR spectrum, whereas the H-6 protons resonated at the same
frequencies as for the starting material [22,23].
Trimethylsilyl trifluoromethanesulfonate (TMSOTf) catalyzed
coupling of donor 18 and acceptor 19 afforded disaccharide 20 in an
excellent yield of 81% (Scheme 4). Disaccharide 20 was used as
donor in the following glycosylation of monosaccharide acceptor
11, synthesized according to Andersen et al. [12] After the glyco-
sylation reaction, the pivaloyl groups were exchanged by benzyl
ethers in a two-step procedure in 82% to give trisaccharide 21. This
maneuver was selected, because we were concerned that the
relatively harsh basic conditions needed for deesterification could
lead to elimination of hydrogen fluoride for the fluorinated
analogue. Regioselective opening of the benzylidene acetal of 21
Scheme 1. a) i. BzCl, Et3N, DMAP, CH2Cl2, 89%, ii. Et3SiH/TFA, CH2Cl2, 74%; b) i. Tf2O, pyridine, CH2Cl2, ii. TBAF, CH2Cl2, 85% over two steps; c) i. NBS, MeCN/H2O (20:1), ii. 2,2,2-
trifluoro-N-phenylacetimidoyl chloride, Cs2CO3, CH2Cl2, 5 ꢀC, 59% over two steps.

38
M.C.F. Andersen et al. / Carbohydrate Research 436 (2016) 36e40
Scheme 2. a) i. BzCl, Et3N, DMAP, CH2Cl2, 89%, ii. Et3SiH/TFA, CH2Cl2, 0 ꢀC, 72%; b) Me3O$BF4, Proton sponge, CH2Cl2, 84%; c) i. NBS, MeCN/H2O (20:1), ii. 2,2,2-trifluoro-N-phe-
nylacetimidoyl chloride, Cs2CO3, CH2Cl2, 0 ꢀC, 74% over two steps.
Scheme 3. a) i. Bu2SnO, then BnBr, CsF, toluene, 79%, ii. PivCl, Et3N, DMAP, CH2Cl2, 92%; b) i. NBS, acetone/H2O (9:1), 2,6-lutidine, 50 ꢀC, ii. 2,2,2-trifluoro-N-phenylacetimidoyl
chloride, Cs2CO3, CH2Cl2, 75% over two steps; c) Et3SiH/TFA, CH2Cl2, 80%.
Scheme 4. a) TMSOTf, CH2Cl2, -40 ꢀC, 81%; b) i. 11, NIS/TESOTf, CH2Cl2/MeCN (1:1), À40 ꢀC, 86%, ii. KOH, DMSO, iii. BnBr, NaH, TBAI, DMF, 82% over two steps; c) i. ethanethiol, p-TSA,
CH2Cl2, 94%, ii. Ac2O, Et3N, CH2Cl2, 88%.
proved challenging. No conversion was observed upon treatment
with NaCNBH3/HCl [24] or NaCNBH3/I2 [25], whereas Et3SiH/TFA
[21] and Et3SiH/Cu(OTf)2 [26] only resulted in slow hydrolysis.
Instead, the acetal was hydrolyzed with ethanethiol and p-tolue-
nesulfonic acid (p-TSA) followed by a selective acetylation of
C6eOH to yield 22 (Scheme 4).
TMSOTf-catalyzed glycosylation of acceptor 22 with the three
donors 7e9 afforded the corresponding tetrasaccharides 4, 5 and 6
in good yields of 74e78% (Scheme 5). The experiments were per-
formed under identical reactions conditions in dichloromethane
with 5% TMSOTf as promoter. The three reactions were allowed to
reach 0 ꢀC, since no conversion was observed at lower tempera-
tures. The methylated donor 8 reacted faster (2.5 h to full conver-
sion) than the electron-poor, fluorinated galactoside 7 and the
glucoside 9 (4 h to full conversion for donors 7 and 9).
Transesterification with sodium methoxide in methanol fol-
lowed by hydrogenolysis with Pearlman's catalyst gave the un-
protected target molecules 1, 2 and 3 (Scheme 5). In order to
remove traces of Pd(OH)2/C, purification by C18 reverse-phase
column chromatography on C18-modified silica was necessary.
The compounds 1e3 were printed and analyzed using microarrays
as described previously [12]. The result of the microarray analysis is
shown in Table 1. The galactans
previously synthesized in our lab, were used as controls [27].
-Gal4 was used to normalize the signals (highest fluorescence in-
tensity set at 100). The controls showed interaction with LM5, but
not with LM6. LM6 is an antibody with high affinity against -1,5-L-
b
-1,4-D-Gal3 and
b
-1,4-
D-Gal4,
b
-1,4-
D
a
Ara and was used as control. We have previously shown that LM5
does not tolerate branching when binding to galactans and that
introduction of arabinose as the terminal, non-reducing end

M.C.F. Andersen et al. / Carbohydrate Research 436 (2016) 36e40
39
Scheme 5. a) TMSOTf, CH2Cl2, À20 ꢀC / 0 ꢀC; b) i. NaOMe, MeOH, ii. H2, Pd(OH)2, MeOH, THF.
smaller oligosaccharides more effectively inhibited the binding of
LM5 to an immobilized antigen. Firstly, lupin galactan was titrated
in the soluble phase to determine its capacity to inhibit binding of
Table 1
Binding of LM5 monoclonal antibody to galactan analogues 1e3 and linear galactans
(
b-1,4-D-Gal3/4) on microarray; LM6 antibody (affinity to a-1,5-L-Ara) was used as a
control.
LM5 to immobilized antigen (lupin galactan) and 20
mg/ml was
Galactan
Antibody
LM5
chosen as the amount to act as soluble galactan inhibitor. In order to
increase the ratio of non-reducing ends in the solution, in a set of
separate tubes the soluble galactan was hydrolyzed with a range of
LM6
b
b
-1,4-D-Gal3
-1,4-D-Gal4
55
100
34
31
0
0
0
0
0
0
36
concentrations of endo-b
-1,4-galactanase for 15 min at 37 ꢀC. Re-
actions were stopped by heating at 95 ꢀC for 5 min and samples
were then decanted to act as the soluble phase in LM5 ciELISAs.
Fig. 2 shows that at low enzyme concentrations enzyme-treated
galactan was a more effective inhibitor than the untreated sam-
ple, demonstrating a higher capacity for binding to LM5. Increasing
the enzyme concentration leads to less inhibition, this is due to
1 e C4eF
2 e C4eOMe
3 e C4-epimer
a
-1,5-L-Ara4
0
residue precludes binding [12]. According to the original report,
LM5 requires at least four consecutive galactose units to bind [8].
degradation to b-1,4-D-Gal2, which is not recognized by LM5. These
Surprisingly, we could see binding with the trisaccharide b-1,4-D-
Gal3. This result shows that three galactose units are enough for
antibody recognition. Binding studies with the three synthesized
analogues 1e3 showed noteworthy differences. Moderate intensity
was detected with C4eF galactan 1. As an isostere of the
Gal4 similar binding behavior was expected and could be seen even
though lesser in intensity than -1,4- -Gal4. Similar results were
b-1,4-D-
b
D
seen with the methylated galactan 2. It was proposed that
methylation will block the C4 position and thus hinder the antibody
binding. That could not be confirmed. Interestingly, no binding was
detected with the epimeric analogue 3. This result suggest that the
non-reducing end residue of the galactans is a pivotal part of the
binding epitope. The configuration at the terminal C4 appears to be
important for the LM5-binding, which explains the similar
behavior of the fluorinated and methylated analogues 1 and 2
compared to epimeric analogue 3.
In order to confirm that the non-reducing end residue of the
galactans is recognized by LM5, we designed a competitive inhi-
bition enzyme-linked immunosorbent assay (ciELISA) experiment
to demonstrate that the presence of a higher concentration of
Fig. 2. LM5 ELISA showing capacity of the pre-treatment of 20
mg/ml lupin galactan
(soluble galactan) with a range of endo- -1,4-galactanase concentrations to modulate
b
LM5 binding to immobilized antigen. At low concentrations of enzyme the inhibitory
capacity of soluble galactan increases. Means and SD of three independent experi-
ments shown.

40
M.C.F. Andersen et al. / Carbohydrate Research 436 (2016) 36e40
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We acknowledge financial support from the Danish Council for
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standing enzymatic degradation of complex polysaccharide sys-
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Council (GlycAct and SET4Future projects), the Villum Foundation
(PLANET project), the Novo Nordisk Foundation (Biotechnology-
based Synthesis and Production Research) and the Bio-Value Stra-
tegic Platform founded by the Danish Council for Strategic Research
and the Danish Council for Technology and Innovation (Grant Case
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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