Selection of a synthetic glycan oligomer from a library of DNA-templated fragments against DC-SIGN and inhibition of HIV gp120 binding to dendritic cells

Article abstract of DOI:10.1039/c1cc13213j

We report the synthesis of a nucleic acid-encoded carbohydrate library, its combinatorial self-assembly into 37485 pairs and a screen against DC-SIGN leading to the identification of consensus ligand motifs. A prototypical example from the selected pairs

Full text of DOI:10.1039/c1cc13213j

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COMMUNICATION
Selection of a synthetic glycan oligomer from a library of
DNA-templated fragments against DC-SIGN and inhibition
of HIV gp120 binding to dendritic cellswz
Mihai Ciobanu,a Kuo-Ting Huang,a Jean-Pierre Daguer,a Sofia Barluenga,a Olivier Chaloin,b
Evelyne Schaeffer,b Christopher G. Mueller,b Daniel A. Mitchellc and Nicolas Winssinger*a
Received 31st May 2011, Accepted 30th June 2011
DOI: 10.1039/c1cc13213j
We report the synthesis of a nucleic acid-encoded carbohydrate
library, its combinatorial self-assembly into 37 485 pairs and
a screen against DC-SIGN leading to the identification of
consensus ligand motifs. A prototypical example from the
selected pairs was shown to have enhanced binding. A dendrimer
incorporating the selected motifs inhibited gp120’s binding to
dendritic cells with higher efficiency than mannan.
glycans as shown in Fig. 1. A resin containing three orthogonally
protected branch points was prepared (1): a Fmoc-protected amine
for PNA oligomerization, and the a- and e-nitrogen of lysine
protected as an azide and with Mtt, respectively, for sequential
coupling of different glycan fragments. The library synthesis
commenced with the introduction of the first PNA codon followed
by reduction of the azide and coupling of the first glycan fragment.
Five glycan fragments (Fig. 1, panel II) derivatized with 2-hydroxy
acetic acid at the reducing end were coupled by standard amide-
bond formation. Four of the glycan fragments contain one or two
azides for further diversification. After the second cycle of split and
mix, the azides were modified by either, a simple reduction and
temporary protection (Boc), copper catalyzed cycloaddition14 with
different alkynes or reduction and acetylation or benzoylation
(Fig. 1, panel III) followed by PNA-encoding. Deprotection of
the Mtt and two more cycles of split and mix reiterating the glycan
coupling and azide diversifications afforded the final library.
Cleavage from the resin (TFA) followed by global deprotection
of the glycans (NH3 in MeOH) afforded the final library of 441
different PNA-encoded glycan dimers [while the library is made up
of 5 ꢀ 5 ꢀ 5 ꢀ 5 building blocks affording 625 permutations, the fact
that fragment E (Man-a1,2-Man) does not contain an azide reduces
the library size while providing an oversampling of this fragment].
We have recently shown that PNA-encoded small molecule
fragments could be combinatorially assembled onto a library of
DNA templates and screened against an immobilized target to
identify the fittest fragment pairs.15 We reasoned that the same
approach could be used to screen a large collection of assemblies
of the PNA-encoded glycan (Fig. 2). Thus, a library of single
strand DNA was used to combinatorially pair a subset of the
glycan library (105 members containing arbitrarily J2 as the last
element of diversity) with a different subset (357 members omitting
J2 as the last element of diversity) to afford a solution theoretically
containing 37 485 glycan pairs. While this library does not cover
the entire solution set of permutations, all library members
are minimally paired with Man-a1,2-Man disaccharide, which
represents the starting point of the library. Next, DC-SIGN
was immobilized on magnetic beads and presented with the
DNA-templated library in an HBS–CaCl2 buffer for 30 min.
Following the removal of the supernatant and accompanying
washes, the resin was heat-denatured to recover the DNA
Despite early success with the synthesis and screening of carbo-
hydrate libraries,1 most attention has been devoted to glycan arrays
containing collections of natural glycan motifs.2 In this study, we
aimed at preparing a library of modified synthetic glycans in a
combinatorial fashion using peptide nucleic acid3 (PNA) encoding,4
assembling the library in different dimeric combinations and
screening for improved binding to DC-SIGN (dendritic cell-specific
intercellular adhesion molecule-3-grabbing nonintegrin). DC-SIGN
is a tetrameric C-type lectin mainly present at the surface of
immature dendritic cells, which modulates the immune response
against different pathogens.5,6 Its affinity for high mannose glycans
has been exploited by several opportunistic pathogens,7 including
HIV8 which displays a high density of the Man-a1,2-Man motif on
its gp120 envelope protein.9 This has engendered a significant
interest in developing strategies to antagonize this interaction in
order to inhibit the pathogen entry.10 Good success has been
achieved with scaffolds which display multiple copies of mannose-
based glycans,11 notably with dendrimers which are particularly
effective to recapitulate the avidity of multimeric interactions.12
The library design was based on the previous observation that
mannose disaccharides appended at the a- and e-nitrogen of lysine
could be dimerized onto a DNA-template to recapitulate the
binding of gp120’s high mannose glycan.13 To further probe
binding to DC-SIGN, we designed a library of PNA-encoded
a Institut de Science et Ingenierie Supramoleculaires (ISIS),
´
Universite de Strasbourg—CNRS, 8 allee Gaspard Monge,
67000 Strasbourg, France. E-mail: winssinger@isis.u-strasbg.fr
b Institut de Biologie Moleculaire et Cellulaire Universite´ de
Strasbourg—CNRS (UPR9021), Strasbourg, France
c Warwick Medical School, University of Warwick, UK
w This article is part of the ChemComm ‘Glycochemistry and glyco-
biology’ web themed issue.
z Electronic supplementary information (ESI) available: Detailed
experimental procedures. See DOI: 10.1039/c1cc13213j
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9321–9323 9321

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Fig. 1 Split and mix synthesis of a PNA-encoded library of glycans.
templates of the best binders. The same experiment was performed
with underivatized beads as a negative control. The sequence of
the DNA, hence the structure of the selected fragment, was first
determined by microarray hybridization. The microarray contains
625 sequences complementary to each PNA but does not provide
information as to the pairing of the PNAs. As can be seen in
Fig. 2, while there was no obvious selection against the beads
alone (red, false coloring), a clear pattern emerged for the selection
against DC-SIGN (green, false coloring). Notably Man(6N3)-
a1,2-Man was highly selected as the first (B1) and second (B2)
glycan while azides derivatized as a phenyl triazole (G1, G2) or
benzoyl amide (I1, I2) were preponderant. To obtain further
information about the pairing of selected assemblies, the selected
DNA was cloned and 10 random clones were sequenced. Analysis
of the sequences showed a consensus motif in good agreement
with the microarray data with a very strong selection for B1 as the
first glycan in both PNAs but a weaker selection for the second
glycan in either position. Nevertheless, Man(6N3)-a1,2-Man is
still the most or second most selected fragment. Notably, under-
ivatized Man-a1,2-Man is clearly underrepresented in the results
of the selection despite its oversampling in the library.
In order to validate the results of the screen, three assemblies
were prepared as single entities to measure their binding kinetics
by surface plasmon resonance and calculate a dissociation
constant (KD). The first assembly (4, see Fig. 3 for the structure)
was present in the clone sequencing and is representative of the
selected motifs identified on the microarray; the second (5) is made
up of two units of a Man-a1,2-Man dimer and as such constitutes
a positive control and a starting point of the library; the third (6) is
a homodimer of the first PNA from assembly 4. As the DC-SIGN
interaction with a carbohydrate is known to be calcium-dependent,
the affinities were evaluated in a buffer with and without
calcium in order to validate that the measured affinity is indeed
a product of the foreseen interaction. All three assemblies showed
a measurable binding affinity only in the presence of calcium
Fig. 2 DNA-templated combinatorial assembly of the glycan library and
screening against DC-SIGN. (I) The library of 441 PNA-encoded glycans
is assembled onto a library of DNA templates and the best binders
are obtained by affinity purification against immobilized DC-SIGN.
(II) Microarray analysis of the selected population: the selection against
DC-SIGN is shown in green whereas a control selection against BSA is
shown in red (false color scheme). The microarray contains all the
complementary sequences to the library of glycans. Each PNA encodes
for a dimer of glycans denoted as A1-E1 for the first one and A2-E2 for
the second one. Each glycan derivatization is denoted as F1-J1 for the
functional group corresponding to the first glycan and F2-G2 for the
functional group corresponding to the second glycan (see Fig. 1 for
structures). (III) Sequencing of 10 clones from the selection against
DC-SIGN was analyzed providing the shown consensus motif.
c
9322 Chem. Commun., 2011, 47, 9321–9323
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To the best of our knowledge, this is the first example of a split
and mix combinatorial synthesis of a nucleic acid encoded glycan
library. The ability to pair such encoded glycan library in a
combinatorial fashion onto DNA provides a rapid means to
generate a large diversity of assemblies to probe and decode
binding leveraged on multimeric interactions. While the present
pilot library focused specifically on mannose, and only explored
limited modifications, the results do suggest that a substitution
with an aryl group at the 6-position of the terminal mannose is
beneficial for DC-SIGN binding. In the larger context, the results
also suggest that this approach should be broadly applicable to
different glycoconjugates including mammalian oligosaccharides
and microbial peptidoglycans.
This work was supported by a grant from the European
Research Council (ERC 201749). The Institut Univervsitaire
de France (IUF) is gratefully acknowledged for its support.
Notes and references
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Fig. 5 Inhibition of gp120 binding to dendritic cells (DC cells).
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9321–9323 9323