Design and synthesis of a "click" high-mannose oligosaccharide mimic emulating Man8 binding affinity towards Con A

Article abstract of DOI:10.1039/c2cc30773a

A dendritic "click" mannooligomer mimicking the high-mannose oligosaccharide Man₈ has been designed by replacing some of the inner mannopyranosyl subunits with triazole moieties; evaluation of its binding affinity towards the mannose-specific lectin concanavalin A revealed striking similarities between the "click" mimic and the natural Man ₈. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc30773a

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COMMUNICATION
Design and synthesis of a ‘‘click’’ high-mannose oligosaccharide mimic
emulating Man8 binding affinity towards Con Aw
a
a
b
a
´
Virginie Cendret,z Marc Francois-Heude,z Alejandro Mendez-Ardoy, Vincent Moreau,*
¸
c
a
´ ´
Jose M. Garcıa Fernandez and Florence Djedaıni-Pilard
´ ¨
Received 3rd February 2012, Accepted 23rd February 2012
DOI: 10.1039/c2cc30773a
A dendritic ‘‘click’’ mannooligomer mimicking the high-mannose
oligosaccharide Man8 has been designed by replacing some of
the inner mannopyranosyl subunits with triazole moieties; evaluation
of its binding affinity towards the mannose-specific lectin concanavalin
A revealed striking similarities between the ‘‘click’’ mimic and the
natural Man8.
adopting the hypothesis that synthetic glycooligomers closely
resembling natural oligosaccharides could be obtained by
replacing internal monosaccharide units by triazole rings.
Reports by Crich and Yang9 and Dondoni and coworkers10
suggest that this structural modification preserves the overall
length of oligomannosyl chains and the orientation of
the external residues. On that grounds, we conceived that
mannosyltriazol segments could actually mimic the critical
Mana(1-2)Man motifs while, at the same time, greatly lowering
the synthetic cost by exploiting the copper catalysed azide–alkyne
cycloaddition (CuAAC), the archetypal ‘‘click’’ reaction.11 As a
proof of concept, the preparation of the ‘‘click’’ Man8 mimic 1
(Fig. 1) has been accomplished and a study of its binding
‘‘High-mannose’’ type N-glycans are known to participate in
quality control and intracellular transportation of glycoproteins.1
Moreover, they cover the surface of many pathogenic micro-
organisms and are the targets of the immune system cells,
including macrophages and dendritic cells, through their
mannose receptors, as well as of soluble circulating proteins such
as collectins.2 They are characterized by the presence of two to
six mannose residues linked to the common pentasaccharidic core
Mana(1–6)[Mana(1–3)]Manb(1–4)GlcNAcb(1–4)GlcNAc (Man5
to Man9, respectively; Fig. 1). For the past three decades, several
groups have reported the synthesis of such structures.3 Their uses
as carbohydrates-based vaccines4 and as recognition moieties in
drug delivery5 or in targeting antigens to dendritic cells6 have also
been investigated. But even if significant progresses have been
made, accessing such complex structures remains very cumbersome
and costly.
Most of the approaches to engineer mimics of the high-
mannose oligosaccharides rely on the synthesis of modified or
truncated fragments and their multivalent presentation.7
Although the incorporation of peripheral unnatural mono-
saccharide moieties with higher affinity than mannose towards
complementary receptors has shown some promise,8 the generation
of simplified core-modified monovalent mimics able to rival
Man8–9 remains elusive. We sought to address this challenge by
a Laboratoire des Glucides FRE-CNRS 3517, Institut de Chimie de
´
Picardie, Universite de Picardie Jules Verne, 33 Rue Saint-Leu,
80039 Amiens Cedex 1, France.
E-mail: vincent.moreau@u-picardie.fr; Fax: +33-3-22827568;
Tel: +33-3-22827661
b
c
´
´
´
Departamento de Quımica Organica, Facultad de Quımica,
Universidad de Sevilla, Apartado 553, E-41071 Sevilla, Spain
´
Instituto de Investigaciones Quımicas, CSIC, Americo Vespucio 49,
Isla de la Cartuja, E-41092 Sevilla, Spain
´
w Electronic supplementary information (ESI) available: Experimental
protocols, NMR and MS spectra. See DOI: 10.1039/c2cc30773a
z These authors equally contributed to the synthesis of the title
compound.
Fig. 1 Structures of the high-mannose glycans Man5–9 and of the
‘‘click’’ pseudo-Man8 mimic prototype 1. The generally used notation
for the mannopyranoside rings and arms in Man8 is indicated.
c
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Chem. Commun., 2012, 48, 3733–3735 3733

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capabilities towards the mannose-specific lectin concanavalin
A (Con A), a commonly used model receptor showing strong
affinity for high-mannose oligosaccharides,12 in comparison
with the series of natural oligosaccharides Man5–9 is reported.
The pseudo-Man8 prototype 1 keeps the three peripheral units
H, F, D and the disaccharide AB in relative positions that are
analogous to those encountered in the natural Man8 (Fig. 1). We
reasoned that the latter could act as a scaffold which should
preserve the overall orientation of the D1, D2 and D3 arms. A
simultaneous multi ‘‘click-coupling’’ approach involving the
known a-D-mannopyranosyl azide 213 and disaccharide 3 should
then generate triazole connectors at the position of the inner
carbohydrate units G, E and C (Scheme 1).
a-directing participating groups in mannopyranosylation
reactions.16 Deacetylation of 10 gave triol 11, which was
reacted with propargyl bromide in the presence of NaH to
afford 3. The triple CuAAC reaction of 3 and azide 2 was
carried out using sodium ascorbate (NaASc) and copper
sulfate as catalyst in a 4 : 1 mixture of DMF–water under
microwave heating,17 leading to the protected pseudo-Man8 12
in 72% yield. Deprotection of the anomeric STol group in 12
(-13) was easily performed after treatment with NBS. The
final de-O-benzylation step under catalytic hydrogenation
conditions in the presence of the triazole moieties proved
problematic, however, as already observed by others.18 The target
fully unprotected ‘‘click’’ Man8 mimic 1 could be obtained in 40%
yield after transfer hydrogenolysis in the presence of ammonium
formate18a,19 (Scheme 2).
The requested tri-O-alkynyl disaccharide 3 was prepared via
a glycosylation step implying the trichloroacetimidate donor 4
and the orthogonally protected acceptor 5. Both 4 and 5 were
obtained from the known p-tolyl 2-O-benzyl-4,6-O-benzylidene-
1-thio-a-D-mannopyranoside14 6 (Scheme 2). Reaction of 6 with
benzoyl chloride (-7) followed by reductive ring opening of
the 4,6-O-benzylidene group15 gave 5. Acetylation (-8) followed
by cleavage of the thiotoluene (STol) group in the presence of
N-bromosuccinimide (NBS) (-9) and subsequent reaction with
trichloroacetonitrile under DBU catalysis afforded donor 4.
The next glycosylation reaction between 4 and 5 led exclusively
to the a(1-6)-disaccharide 10 in high yield (87%), in agreement
with the proposed role of acyl substituents at O-3 and O-6 as
Con A has been shown to be very well-suited for glycan
profiling.20 The most important structural feature for high
affinity is that the glycan must possess the trimannose group
Mana(1-6)[Mana(1-3)]Man at the reducing end, which is
present in all the high-mannose oligosaccharides.21 We have
recently demonstrated that altering this key branched motif
has dramatic consequences in the ability to bind Con A.22 The
mannose substitution pattern of the trimannose group also
affects the binding affinity.23 A comparative evaluation of the
affinity of compound 1 and Man5-9 towards Con A will thus
provide an indication of the potential of ‘‘click’’ high-mannose
mimics to emulate the recognition properties of their natural
partners. We have conducted this study by enzyme-linked
lectin assay (ELLA).24 The IC50 value for the inhibition of
horse radish peroxidase-labelled Con A (Con A-HRP) binding
to immobilized yeast mannan is assumed to be inversely
proportional to the ligand–Con A affinity. Most interestingly,
it has been recently shown that affinity data for multimannosides
towards Con A-HRP obtained by ELLA closely resembled data
towards the human macrophage mannose receptor (hMMR).25
Results indicated that Con A strongly binds Man5–9 (Fig. 2)
with affinities 33 to more than 290-fold higher as compared with
methyl a-D-mannopyranoside (MeaMan). In agreement with
literature data,20,23 the higher glycoforms Man8 and Man9 were
the best ligands, exhibiting 3- to 7-fold higher affinities as
compared to Man5–6. We were delighted to see that the ‘‘click’’
Scheme 1 Retrosynthetic pathway to ‘‘click’’ pseudo-Man8 mimic 1.
Fig. 2 ELLA plots (logarithm scale) for the inhibition of Con
A-HRP binding to yeast mannan by increasing concentrations of the
‘‘click’’ Man8 mimic 1 in comparison with the natural high-mannose
glycans Man5–9. The corresponding IC50 values (mean of three
independent determinations ꢀ SD) and the potencies relative to
methyl a-D-mannopyranoside are indicated.
Scheme 2 Synthesis of compound 1.
c
3734 Chem. Commun., 2012, 48, 3733–3735
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Fig. 3 Structures of the trivalent mannopyranosyl conjugates 14 and 15
and of the C7-symmetric 21-valent conjugate 16. The corresponding IC50
values for inhibition of Con A-HRP binding to yeast mannan obtained in
the same set of ELLA experiments collected in Fig. 2 are indicated.
Man8 mimic 1 was also a very strong ligand for Con A, stronger
than Man7 and only 2.5-fold weaker as compared with Man8.
Compound 1 might be seen as a trivalent a-D-mannopyr-
anosyl conjugate, the observed affinity enhancement relative to
MeaMan being then ascribable to the multivalent or cluster
effect26 rather than to its structural resemblance to Man8. To
test this possibility, a second comparative ELLA study
was performed including the previously reported trivalent
conjugates 1427 and 15.28 In addition, conjugate 16,28 in which
a trivalent mannopyranosyl dendron is presented in seven
copies, was also assayed. The data proved the much higher
efficiency of the pseudo-Man8 mimic as a Con A ligand
as compared to the other trivalent derivatives, its binding
potency being similar to that of the 21-valent conjugate 16
(Fig. 3). It also compares favourably with other Con A-binding
‘‘click’’ clusters already in the literature.29 Altogether, the
current body of results strongly suggests that some mannopyr-
anosidic units of the high mannose part of N-glycans could
be replaced by triazole rings while preserving their binding
capabilities towards receptor partners. The biological significance
of this family of oligosaccharides warrants further research on
related ‘‘click’’ mimics.
This work was supported by the ‘‘Conseil Regional de Picardie’’,
the ‘‘Ministere de l’Enseignement et de la Recherche’’, the ‘‘Spanish
Ministerio de Innovacion y Ciencia’’ (MICINN, contract numbers
SAF2010-15670, and CTQ2010-15848), the ‘‘Junta de Andalucıa’’
and the ‘‘European Union’’ (FEDER and FSE). We also thank the
CITIUS for technical support. The authors acknowledge Dr David
Lesur (FRE 3517) and Dr Serge Pilard (Plateforme Analytique,
UPJV, France) for MS and HPLC.
25 A. Dıaz-Moscoso, N. Guilloteau, C. Bienvenu, A. Mendez-Ardoy,
J. L. Jimenez Blanco, J. M. Benito, L. Le Gourrierec, C. Di
Giorgio, P. Vierling, J. Defaye, C. Ortiz Mellet and J. M. Garcıa
Fernandez, Biomaterials, 2011, 32, 7263.
26 J. J. Lundquist and E. J. Toone, Chem. Rev., 2002, 102, 555.
27 J. M. Benito, M. Gomez-Garcıa, C. M. Ortiz Mellet, I. Baussanne,
J. Defaye and J. M. Garcıa Fernandez, J. Am. Chem. Soc., 2004,
126, 10355.
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c
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Chem. Commun., 2012, 48, 3733–3735 3735