On-Chip Screening of a Glycomimetic Library with C-Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin-2 over DC-SIGN/R and Langerin

Article abstract of DOI:10.1002/chem.201802577

A library of mannose- and fucose-based glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.

Full text of DOI:10.1002/chem.201802577

A Journal of
Accepted Article
Title: On-chip screening of a glycomimetic library with C-type lectins
reveals structural features responsible for preferential binding of
dectin-2 over DC-SIGN/R and langerin
Authors: Laura Medve, Silvia Achilli, Sonia Serna, Fabio Zuccotto,
Norbert Varga, Michel Thépaut, Monica Civera, Corinne
Vivès, Franck Fieschi, Niels Reichardt, and Anna Bernardi
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201802577
Link to VoR: http://dx.doi.org/10.1002/chem.201802577
Supported by

10.1002/chem.201802577
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On-chip screening of a glycomimetic library with C-type lectins
reveals structural features responsible for preferential binding
of dectin-2 over DC-SIGN/R and langerin
Laura Medve,[a] Silvia Achilli,[b] Sonia Serna,[c] Fabio Zuccotto,[d] Norbert Varga,[a] Michel Thépaut,[b]
Monica Civera,[a] Corinne Vivès,[b] Franck Fieschi,[b] Niels Reichardt,[c,e] and Anna Bernardi[a]
Abstract: A library of mannose- and fucose-based glycomimetics
was synthesized and screened in a microarray format against a set
of C-type lectin receptors (CLRs) that included DC-SIGN, DC-
SIGNR, langerin and dectin-2. Glycomimetic ligands able to interact
with dectin-2 were identified for the first time. Comparative analysis
of binding profiles allowed to probe their selectivity against other
CLRs.
monosaccharide binding specificity of the CLR. So, a Glu-Pro-
Asn (EPN) motif results in a preference for Mannose (Man), N-
Acetylglucosamine (GlcNAc), Fucose (Fuc) or Glucose (Glc)
residues, whereas a Gln-Pro-Asp (QPD) sequence leads to
recognition of Galactose (Gal) and N-Acetylgalactosamine
(GalNAc).[1] Available data also suggest that the extended
binding sites of CLRs often display higher affinity towards larger
glycan structures, thanks to additional interactions occurring in
the vicinity of the primary Ca2+ site.[2] Thus, complex glycans
exposing the same monosaccharide can bind to CLRs with very
different affinities, depending on accessibility of the recognition
element and on additional features of the lectin binding sites.
Structural studies also demonstrate that secondary binding sites
can alter the affinity and specificity of the CLR towards ligands.
As an example, langerin,[3] a transmembrane CLR expressed on
Langerhans-cells, and the Dendritic-Cell Specific Intercellular
adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), a CLR
expressed by dendritic cells (DCs)[4] share similar primary
binding sites with similar specificity for oligomannosides, but
langerin has an additional calcium-independent sugar-binding
site and binds to large sulfated glycosamino glycans, while DC-
SIGN does not.[5]
Introduction
Lectins are sugar-binding proteins that engage in interactions
with endogenous and exogenous glycans. The interactions
between lectins and carbohydrates are involved in many
fundamental biological events, from cell adhesion to antigen
recognition and internalization, inflammation or quality control in
protein folding. The most abundant class of animal lectins are
the C-type lectin receptors (CLRs) serving a broad range of
functions. They are involved in pathogen recognition and in
prevention of autoimmunity by contributing to the immune
system ability to identify carbohydrate-based pathogen-
associated molecular patterns (PAMP) and damaged-self-
associated molecular patterns (DAMP).They take part in signal
transduction, cell trafficking and in the induction of T-cell
differentiation. Their name, C-type lectins, indicates the
presence of a Ca2+ ion in their carbohydrate recognition domain
(CRD). This ion is the primary site of carbohydrate interaction
and typically coordinates two vicinal hydroxyl groups on a sugar
ring. Many C-type lectin receptors are yet to be explored and
described in detail with regard to their CRD structure,
carbohydrate binding specificities and the molecular factors
governing the interaction with glycans. However, it is known that
the CRDs of CLRs feature evolutionarily conserved groups of
residues that coordinate the Ca2+ ion and determine the
Given the key role played by lectins in biological systems, many
research groups have turned their attention towards the
development of glycomimetic molecules to be used as selective
probes for the study of sugar-protein interactions and/or for
medicinal chemistry purposes.[6] Glycomimetics present several
advantages as drug candidates over natural glycans, since they
can be made metabolically more stable, more bioavailable and
possibly
more
active
and
selective
than
natural
oligosaccharides. Our previous studies have focused on
inhibiting the dendritic cell receptor DC-SIGN, a CLR implicated
in viral and bacterial infections.[7] The primary binding site of DC-
SIGN CRD recognizes mannose oligosaccharides, L-fucose
residues in Lewis-type blood antigens[4] and bi-antennary N-
glycans with terminal GlcNAc moieties[8]. Additional druggable
sites on the lectin have been described recently.[9] DC-SIGN
mediated adhesion to dendritic cells is the first step of several
viral infections, notably by HIV and Ebola viruses.[10]
Glycomimetic antagonists of DC-SIGN have mainly been
designed starting from high mannose glycans, like Man9
(Man)9(GlcNAc)2 (1, Figure 1), or from LeX (Fucα1,3-(Galβ1,4-)-
GlcNAc) (2) type structures. In particular, pseudo-di and tri-
mannoside fragments (3-6) were synthesized[11] as mimics of
the D2 and D3 arms of Man9 (Figure 1). When used in
multivalent constructs, they were found to block DC-SIGN-
mediated infection with activities up to the nanomolar level both
in HIV and Ebola infection models.[12] Notably, the
bisbenzylamide derivatives 6a[11c] and 6b[11d] also exhibited
strong selectivity towards DC-SIGN and did not bind to
[a]
L. Medve, Dr. M. Civera, Prof. A. Bernardi
Dipartimento di Chimica, Università degli Studi di Milano,
Via Golgi 19, 20133 Milano, Italy
E-mail: anna.bernardi@unimi.it
[b]
[c]
S. Achilli, Dr. M. Thépaut, Dr. C. Vivès, Prof. F. Fieschi
Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale,
F-38000 Grenoble, France
Dr. S. Serna, Dr. N. Reichardt
Glycotechnology laboratory, CIC biomaGUNE, Paseo Miramón 182,
20014, Donostia/San Sebastián, Spain
Dr. F. Zuccotto
University of Dundee, Dundee, United Kingdom
Dr. N. Reichardt
[d]
[e]
CIBER-BBN, 20014 Donostia-San Sebastián, Spain
Supporting information for this article is given via a link at the end of
the document.
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D1
D2
D3
OH
OH
O
OH
OH
HO
OH
O
OH
O
HO
OH
HO
OH
HO
HO
HO
HO
OH
O
O
O
O
OR
OH
HO
OH
HO
O
O
O
O
OH
HO
NHAc
O
HO
HO
HO
HO
O
O
OH
OH
O
OH
HO
OH
O
HO
O
O
HO
HO
OH
O
OH
O
OH
HO
O
O
O
HO
O
O
OR
NHAc
HO
NHAc
1
2
OH
OH
R
O
HO
HO
OH
OH
O
OH
OH
O
OH
HO
HO
O
HO
HO
HO
HO
O
HO
MeOOC
MeOOC
O
O
N
H
S
O
MeOOC
MeOOC
MeOOC
MeOOC
O
O
N3
OH
NH
O
N3
O
N3
O
HO
HO
O
HO
N3
O
O
3
4
5
6a R=OH
6b R=NH2
Figure 1. Natural DC-SIGN ligands: Man9 (1) – the D1-D3 arms are the template for the design of mimics 3-6; L-Fuc containing LeX blood group antigen (2).
Linear fragment mimics of Man9 arms: pseudo-mannobioside (3) and pseudo-mannotrioside (4); the pseudo-thiomannobioside (5) and two derivatives of pseudo-
mannobioside (6).
langerin, a CLR that share with DC-SIGN a similar set of ligands
but, rather than spreading the infection, facilitates HIV
eradication.[13
Herein, we describe the synthesis of a mannose- and fucose-
based glycomimetic library and its on-chip screening against a
set of human CLRs that led to the discovery of hit ligands able to
interact with dectin-2.[16]
]These results suggested that, with appropriate modifications,
the structure of 6 could represent a general template to generate
a
diverse library of glycomimetics containing one natural
monosaccharide as the lectin-targeting element and a tuning
unit, which could provide additional functional elements for
interaction with the lectin in the proximity of the primary binding
site. As mentioned above, the structure of 6 derives from
mimicry of the Manα1-2Man disaccharide, the terminal unit of
the D1-D3 arms of Man9 and a common disaccharide ligand for
DC-SIGN (PDB 2IT6) and other CLRs of the immune system
with similar specificity, such as DC-SIGNR,[14] langerin (PDB:
3P5F)[15] and dectin-2 (PDB: 5VYB).[16] Screening such a library
against these CLRs in a microarray format may become a potent
tool for glycomimetic drug discovery.[17] Glycan microarrays, as
introduced and developed over the past 15 years, have been an
essential tool in the characterization of lectin specificity,[18] and
have been used to pave the way for the therapeutic exploitation
of vital lectin-sugar interactions.
Results and Discussion
Design and synthesis of the library
The set of bis(benzylamide) compounds previously designed as
DC-SIGN antagonists was directly expanded into a mannose-
based library by adopting the described route[11c] that starts from
diacid 7, with small modifications (Scheme 1). Scale-up to a
multi-gram scale of the protected common scaffold 11, equipped
with a versatile azido-functionality, allowed further derivatization
to a collection of bisamides 12. The glycomimetic library was not
conceived to specifically target one lectin, but rather to broadly
interact with CLRs that contain the EPN motif in their CRD.
Therefore, the required set of amines was selected for diversity,
with the help of chemoinformatic tools and based on commercial
availability. Lead-like physicochemical filters were applied to a
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10.1002/chem.201802577
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large number of amines from available commercial collections
(see Experimental section) and the selection was sifted to
exclude any structural incompatibility. The remaining structures
were then clustered by chemoinformatic descriptors and
representatives of the various subgroups were selected based
on their availability. Overall, a collection of 38 diverse amines
were selected (Figure 2), which allowed us to prepare the Man-
based derivatives 12.1-39 (Scheme 1, Figure 2).
carbohydrate-specificity imparted by the EPN motif. Therefore,
in analogy to the mannobioside mimics, β-fucosylated ligands 15
were synthesized by linking the cyclohexane acceptor (9) with
an appropriately protected fucose-trichloroacetimidate donor
(13) to yield the bis-p-nitrophenylester 14, which was reacted
with a set of primary and secondary amines. This approach
afforded the 11 Fuc-based ligands 15.1-11 (Scheme 1, Figure
2).
As discussed previously, Man-binding CLRs are expected to
recognize L-Fuc residues as well, due to the overlapping
z
O
B
O
z
O
B
H
O
O
z
B
O
z
B
O
H
O
O
H
H
COO
z
B
O
z
B
H
O
O
z
B
O
z
B
H
O
O
T A
O C
COO
,
10
f g
O
'
O
PNP
OC
R RN
OC
7
'
PNP
OC
R RN
OC
d
a
O
O
N3
N3
. -
11
12 1 39
PNP
PNP
CO
,
H
O
c
PNP
OC
PNP
OC
b
CO
O
9
8
z
B
N3
B
H
O
H
O
z
O
O
O
O
3
=
O
PNP
z
B
H
O
O
,
e
f g
O
'
R RN
R RN
OC
O
PNP
PNP
OC
OC
OC
N
O2
'
T A
O C
13
O
z
B
O
O
O
N3
N3
z
B
O
z
. -
15 1 11
B
O
14
Scheme 1. Synthesis and general structure of mannose-(12.1-39) and fucose-derived (15.1-11) members of the glycomimetic library.
a. p-nitrophenyl trifluoroacetate, pyridine, DMF, 50°C, 18 h, 73% b. mCPBA, CH2Cl2, 16h, 94% c. 2-azidoethanol, Cu(OTf)2, CH2Cl2, 70% d. TMSOTf, -30°C, 20
min, 74% e. TMSOTf, -30°C, 3.5 h, 74% f. RR’NH, THF/DMF, (Et3N), 1h-2d g. NaOMe, MeOH, 1.5h.
Ligand immobilization on the microarray surface
linker on lectin binding, compound 19.1 (Figure 2E) was
included as a non-glycosylated “clicked-linker”. Conjugation of 2-
azidoethyl-D-mannoside to 16 provided the control mannose
glycoside 19.2. In addition, amines 20 and 21 (Figure 2) were
prepared by azide reduction of two ligands, 12.1 and 12.2,
respectively. The amines were directly printed on the slides, to
compare their binding with the same ligands immobilized
through the bifunctional linker (17.1 and 17.2, respectively).
The final ligand library, conjugated with the additional linker and
printed onto the microarray surface, consisted of 39 α-
mannosylated (17.1-17.39) and 11 β-fucosylated (18.1-18.11)
Although the azidoethyl functionalized glycomimetics could in
principle be immobilized directly by surface based
cycloaddition,[19] we chose to extend the short linker with an
additional hetero-bifunctional spacer prior to printing the library,
to improve ligand accessibility. This set-up presents the
additional advantage of allowing to print the glycomimetics
alongside existing glycan libraries, that are typically
functionalized with amino-terminated linkers.[20] To this end,
commercially available N-[(1R,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-
ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (16 , Scheme
2) was submitted to selective, rapid and bioorthogonal strain-
promoted azide-alkyne cycloaddition (SPAAC), as described by
Agard et al.[21] yielding conjugates 17 and 18 (Scheme 2, Figure
2). The azido-terminated ligands reacted quantitatively overnight
with 16 and the reaction products were analyzed by MALDI-tof-
MS to assess the identity of the resulting compounds, which
were later robotically printed through the terminal amino
disaccharide mimics (Figure 2) and included
4 control
compounds (19.1, 19.2, 20, 21).
After microarray design and optimization of printing parameters,
the slides were probed with a variety of recombinant and
fluorescently tagged human CLRs, and the binding profile
recorded with
a fluorescence scanner. The fluorescence
intensity of individual spots relates to the amount of bound lectin
and is an estimation of the relative strength of interaction. For a
selected set of ligands, the intensity values from the microarray
functionality
onto
N-hydroxysuccinimidyl
ester
(NHS)-
functionalized glass slides. To detect any interference of the
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10.1002/chem.201802577
Chemistry - A European Journal
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analysis were compared to IC50 values determined in a SPR
competition experiment against DC-SIGN and DC-SIGNR. This
comparison allowed us to critically evaluate the ranking
suggested by the array interaction studies and provided a useful
perspective in the screenings performed with other human
CLRs.
A
B
HO
OH
O
H
H
H
H
H
N
H
H
N
H
N
N
N
N
N
N
N
HO
HO
O
O
O
O
O
R'
O
O
O
O
HO
O
O
O
OH
O
R'
R
12.1
17.1
12.2
17.2
12.3
17.3
12.4
17.4
12.5
17.5
12.6
17.6
12.7
17.7
12.8
17.8
12.9
17.9
12.10
17.10
12, 17
H
H
N
H
N
H
N
H
N
H
N
N
H
H
HN
N
N
F
N
N
N
OH
N
F
S
O
OH
OH
OH
12.11
17.11
12.12
17.12
12.13
17.13
12.14
17.14
12.15
17.15
12.16
17.16
12.17
17.17
12.18
17.18
12.19
17.19
12.20
17.20
H
N
HN
H
N
H
N
HN
N
HN
N
H
N
H
N
H
N
N
N
N
OH
O
N
N
N
OH
CF3
CF3
N
F3C
12.21
17.21
12.22
17.22
12.23
17.23
12.24
17.24
12.25
17.25
12.26
17.26
12.27
17.27
12.28
17.28
12.29
17.29
12.30
17.30
HN
HN
HN
O
N
O
S
N
N
N
H
OH
H
N
S
N
O
N
N
O
NH
N
N
NH2
F
12.31
17.31
12.32
17.32
12.33
17.33
12.34
17.34
monoamide
12.35
17.35
12.36
17.36
12.37
17.37
12.38
17.38
12.39
17.39
methyl ester
D
C
HO
HO
H
H
N
H
N
HN
N
O
H
N
H
O
N
H
N
H
N
O
HO
O
N
O
N
N
N
NH
O
CF3
N
R'
N
O
O
O
OH
HO
O
O
R'
R
15.1
18.1
15.2
18.2
15.3
18.3
15.4
18.4
15.5
18.5
15.6
18.6
15.7
18.7
15.8
18.8
15.9
18.9
15.10
18.10
15.11
18.11
15, 18
HO
OH
O
E
12, 15
R: N3
HO
HO
HO
OH
O
N
-
HO
HO
17 19
R:
O
N
N
O
N
HO
H
O
OH
O
MeOOC
MeOOC
HO
O
HO
O
NH
-
HO
NH2
HO-(CH2 3 R
)
H
H
O
O
O
HO
NH2
R
O
NH2
O
N
O
19.1
19.2
20
21
H
Figure 2. Glycomimetic library of 39 mannosylated, 11 fucosylated and 4 control compounds. A) General structure of mannose glycomimetics. B) Different R’
substituents present in mannose glycomimetics. C) General structure of fucose glycomimetics. D) Different R’ substituents presented in fucose glycomimetics. E)
Control compounds included in the library.
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Experimental design and data analysis
aurantia lectin (AAL, L-Fuc).[23] These experiments allowed the
optimization of the spotting conditions and the validation of the
array. In particular, it was observed that the addition of 10%
DMSO to the printing buffer afforded the most homogeneous
fluorescent images.
Glycan microarrays were printed as previously described[8] on
commercially available NHS-ester activated glass slides. The
immobilization of the ligands was confirmed by incubation with
fluorescently labeled lectins of known glycan specificity: plant
lectin Concanavalin A (ConA, D-Man)[22] and fungal Aleuria
u ar
S g
'
R RN
OC
O
'
R RN
OC
u ar
S g
16
H
H
O
'
N
R RN
OC
O
O
N
N
'
R RN
OC
. -
- -
NH2
O
=
=
α
u ar
S g
an
17 1 39
D
M
N
O
O
H
O
N3
an
. -
- -
u ar
S g
uc
18 1 11
L
F
β
. .
. -
- -
,
,
.
=
=
α
w
r
a e
o
n
u ar
t
RT
12 1 39
D
M
H
H
O
S g
. -
- -
uan
t
q
u ar
S g
uc
15 1 11
L
F
NH2
O
β
N
O
O
H
Scheme 2. Conjugation of azide containing glycomimetics with cyclooctyne 16 via strain-promoted alkyne-azide cycloaddition (SPAAC).
On the array, ConA was found to recognize most of the printed
Man-based glycomimetics 17.n as well as the controls 19.2, 20
and 21, while no binding to the non-mannosylated linker 19.1
and to the fucose based glycomimetics 18.n was observed (see
supporting information Figure SI-1). Interestingly, many of the
Man-based mimics appeared to interact with ConA more
efficiently than mannose itself (19.2), supporting the hypothesis
that secondary interactions can contribute to the overall affinity
of lectin ligands. The intensity of the signals obtained for 17.1
and 17.2 was somewhat higher than that of the short-linker
controls 20 and 21, suggesting a better accessibility of the
former compounds on the array.
DC-SIGN
DC-SIGN is a transmembrane protein expressed primarily on
the surface of dendritic cells in dermal mucosa and on various
other antigen presenting cells of the myeloid lineage. DC-SIGN
has a dual role as a cell surface pattern-recognizing receptor
and as a mediator for T-cell activation. Additionally, a number of
pathogens, most notably the HIV virus, are known to exploit DC-
SIGN in the initial steps of host invasion.[10a,
For these
25]
reasons, DC-SIGN has been actively investigated, both as a
target for discovery of anti-adhesive antiviral therapies and for its
potential role in immunoregulation.[26]
The lectin recognizes highly mannosylated oligosaccharides
often found on viral and bacterial cell surfaces; the four Lewis-
type blood group antigens (Lex Ley, Lea, Leb) and mannan-
As expected, screening the array with fucose-specific AAL
resulted in
a totally different interaction profile, involving
exclusively the Fuc-based ligands 18.n (see supporting
information Figure SI-2). Substitutions to the fucose core
apparently did not affect recognition, as the binding intensity
seems largely the same for all compounds. No binding was
observed to the linker control 19.1 for this or any other of the
tested lectins.
capped
lipoarabinomannan
and
phosphatidylinositol-
mannosides expressed on mycobacterial surfaces.[27] On the
array (Figure 3A), many of the mannosylated structures, but
essentially none of the β-fucosylated ligands are recognized by
the tetrameric DC-SIGN Extracellular Domain (ECD). Although
DC-SIGN is known to bind fucosylated oligosaccharides, they all
consists of α-fucosides, while, to the best of our knowledge β-
fucosides have not been explored before. The mannose-based
ligands identified by red bars in Figure 4 had originally been
designed as DC-SIGN antagonists and tested by SPR as
inhibitors of DC-SIGN binding to mannosylated BSA.[11c] Both
the SPR data and the microarray results indicate that all these
compounds have similar affinity for the lectin, with the methyl
ester 17.2 being the least effective of the series. None of the
additional modifications of the amide functionality attempted in
this library was found to improve on the previous design. On the
other hand, very low fluorescence intensity was detected for all
tertiary amide derivatives (17.10, 17.20, 17.30, 17.36, 17.37,
17.38, 17.39) on the chip. This observation confirmed early data
Interaction with human CLRs
DC-SIGN and langerin extracellular domains (ECD) were
24]
produced as previously described.[3a,
DC-SIGNR ECD and
dectin-2 ECD were expressed in E.coli and purified as detailed
in the SI section. All CLRs were labeled with Cy3 and the degree
of labeling was estimated as described in the SI.
The analysis was performed in the optimized conditions
described above. Results are reported in Figures 3-5, where
ligands are grouped by chemical features (type of
monosaccharide, degree of amide substitution) rather than by
numbering. A comparative heat-map for the four proteins is
reported in the SI section (Figure SI-3).
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from previous SPR screenings in our groups, which had
indicated that tertiary amides on the pseudo-dimannoside
scaffold were significantly reducing the binding affinity towards
DC-SIGN.[28] The current set of data strongly suggests that this
feature can be reliably used to generate selectivity against DC-
SIGN. Overall, these data allowed the validation of microarray
results and showed that the screening technique implemented is
robust and adequate for fast analysis of binding activity.
Figure 3. A) DC-SIGN glycomimetic ligand binding profile at 50μg/mL DC-SIGN ECD (extracellular domain, tetramer). The red bars indicate previously known
DC-SIGN ligands (ref. 11c) The IC50 values measured for these ligands in ref 11c are collected in Figure SI-4. B) DC-SIGNR glycomimetic ligand binding profile at
150 μg/mL DC-SIGNR ECD. Ligands selected for SPR studies are highlighted in blue. C) Left: ligand Inhibitory potency towards DC-SIGN and DC-SIGNR. IC50
values measured in SPR inhibition assays (Man-BSA immobilized on the surface). Right: the structure of the ligands and the IC50 values for DC-SIGN (grey) and
DC-SIGNR (blue).
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DC-SIGNR
influence on the observed binding selectivity of lectins on the
physical format of the assay was noted early on and repeatedly
confirmed for various systems.[33] Once again, our data suggest
that multiple analytical methods need to be applied to fully
characterize the interaction of lectins with synthetic or natural
ligands. This represents an additional element of complexity for
the discovery of selective lectin antagonists. However it is still
possible, using data from various techniques, to identify ligands
of potential interest in drug discovery programs and to ascertain
the structural features that concur to determine their activity and
selectivity. We have obtained here for the first time microarray
and SPR data on the recognition of glycomimetic ligands by DC-
SIGNR which will be the base for further elaboration of these
structures.
The DC-SIGN related homologue, DC-SIGNR (or L-SIGN) is
expressed primarily on sinusoidal endothelial cells in lymph
nodes, the liver, the lungs, the gastrointestinal tract, and
capillary endothelial cells in the placenta.[29] The two
homologues exhibit 73% identity at the nucleic acid level and
77% identity in the amino acid sequences,[30] but display
differences in the coiled-coil neck domains that affect the spatial
arrangement of the four CRDs. As a result, the two CLRs can
show different avidity towards the same multivalent ligand.[24, 31]
Similarly to DC-SIGN, DC-SIGNR is a pathogen receptor for
HIV-1, HCV, SARS-coronavirus, Mycobacterium tuberculosis,
recognizes influenza A and interacts with lymphocytes.[32]
The microarray screening results are shown in Figure 3B. Many
of the mannose-based ligands interact with the protein more
effectively than mannose itself (19.2) and the binding profile is
rather similar to that observed for DC-SIGN. Four ligands (17.11,
17.15, 17.19 and 17.27, all shown as blue bars in Figure 3B)
were selected for further analysis and the affinity of the
corresponding recognition elements 12.11, 12.15, 12.19 and
12.27 for DC-SIGN and DC-SIGNR was evaluated using an
SPR inhibition assay that measures their ability to inhibit protein
binding to mannosylated bovine serum albumin (Man-BSA)
(Figure 3C). The results show some obvious differences in the
way the ligands are classified by the two assays. In particular,
the IC50 value measured by SPR for 12.11 and DC-SIGN is
higher than expected based on the array profile. This may
simply reflect the intrinsic differences of the physical processes
interrogated in the two assays: in the microarray setup, direct
binding of the tetravalent lectins to a multivalent functionalized
surface is observed; in the SPR inhibition assay the ligands are
scored based on the strength of their monovalent interaction
with the protein. A strong dependence of the affinity values
measured for carbohydrate-protein complexes and even an
Langerin
Langerin is
a
trimeric CLR abundantly expressed on
Langerhans-cells in the epidermis, and at lower levels on CD1c+
myeloid dendritic cells and lamina propria of human colon.[34]
Apart from the protective role against HIV mentioned above, the
lectin binds to other pathogens, such as Candida,
Saccharomyces, Malassezia furfur and Mycobacterium
leprae.[35] Although langerin and DC-SIGN share many of their
natural ligands, differences can be found in their specificity
towards fucosylated glycans. DC-SIGN exhibits
a
good
recognition of many fucose-based Lewis-type ligands (Lex, Lea,
Leb, and Ley), as well as of the A, B and H blood group antigens.
Langerin binds with good affinity only the blood group antigens B
and A, while Lea, Leb, Ley and Lex are poorly recognized.[15, 36]
Moreover, opposite to DC-SIGN, langerin selectively recognizes
sulfated Gal, GalNac and glycosaminoglycans.[5,
Additionally, a divergent structural organization and their distinct
expression locations suggest fundamentally different biological
roles for these two CLRs.
11d, 15]
Figure 4. Langerin glycomimetic ligand binding profile at 25μg/mL lectin concentration.
In the microarray assay, the signal for trimeric langerin ECD
raised above noise level only for 4 ligands, three of which (17.7,
17.14, 17.15) are known ligands of DC-SIGN (Figure 4). The
corresponding recognition elements 12.7, 12.14 and 12.15 had
been previously assessed also against langerin, using the SPR
inhibition experiment described above, and found to be poor
competitors of immobilized Man-BSA.[11c] SPR inhibition studies
were repeated for 12.15 and performed for the first time with
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12.21. Neither of them could inhibit langerin binding to
immobilized Man-BSA, up to millimolar concentrations (Figure
SI-5). Some inhibitory activity of 12.15 could be observed in
SPR competition assays, but only when challenging a weaker
interaction using a surface functionalized with Lea-BSA, which is
a poor langerin ligand (Figure SI-5). This experiment allowed
evaluating an IC50 of 1.8 mM. For 12.21, a sharp drop in
langerin activity could be observed above 1 mM concentration of
the ligand, but the data could not be fitted to a binding isotherm.
The fact that ligands displaying little inhibitory activity in the SPR
experiment can still light up on the microarray may depend on
the avidity of the polyvalent presentation generated upon
printing them on the chip. However, we cannot rule out at this
stage that these molecules, through their amide substituents,
may be interacting in a non-competitive fashion, i.e. with a
different site than the carbohydrate binding site on the ECD.
Manα1-2Man recognition.[16] Dectin-2 binds to bacteria such as
Klebsiella pneumoniae and Mycobacterium tuberculosis and
fungi as Candida albicans.[38] Its anti-fungal activity has been
demonstrated in animal-models.[39] Upon ligand binding, dectin-2
is able to promote signaling, cytokine secretion and finally the
initiation of a Th17 immune response.[40]
The binding profile of dectin-2 ECD towards the glycomimetic
library is shown in Figure 5A. It can be observed that some of
the mannosides appear to interact more strongly than mannose
19.2, a weak binder of dectin-2. Remarkably, dectin-2 exhibits
affinity towards some tertiary amide structures (17.10, 17.20,
17.30, 17.36, 17.37, 17.38, 17.39, showcased in Figure 5B) and
β-fucosylated ligands, which are not or barely recognized by DC-
SIGN (Figure 3A). While the two lectins display a similar profile
for mannosides bearing secondary amide structures, they clearly
differ in the fucoside section and more strikingly so in the
mannoside bearing tertiary amide groups section. This suggests
the possibility of an unprecedented selectivity between the two
CLRs towards glycomimetic compounds, which may be related
to the different nature of the two binding sites.[16]
Dectin-2
Dendritic cell-associated C-type lectin 2, dectin-2, is
a
predominantly macrophage and monocyte associated CLR,[37]
with a known specificity for mannose and a preference for
B
N
N
N
N
N
N
O
H
O
H
O
.
.
O
.
.
H
O
17 10
7
1 20
17 35
17 30
H
O
O
O
'
R
N
N
O
N
'
O
R
N
N
R
17
F
.
.
.
17 36
17 38
17 37
Figure 5. Dectin-2 binding profile. Mannosides bearing secondary amide groups are shown as grey bars, mannosides bearing tertiary amide groups are shown as
black bars, fucosides and controls are shown as white bars. B) Structure of mannose ligands of dectin-2 that do not interact with DC-SIGN.
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Indeed, the X-ray structure of dectin-2 in complex with
Man9GlcNAc2 has been recently solved.[16] Figure 6 shows
dectin-2 CRD sumperimposed to the X-ray structure of the DC-
SIGN complex with the pseudo-dimannoside 3.[41] The overlay
shows a highly conserved tertiary structure with a difference in
the loops in close proximity of the Ca2+ binding site which
contain V351 for DC-SIGN and H171 for dectin-2 (to the right of
the ligand in Figure 6A). At the other side of the Ca2+ ion, the X-
ray structure of dectin-2 shows a very shallow surface, lined by a
Trp side chain (W182, Figure 6A). Alignment of the dectin-2 and
DC-SIGN sites shows that the DC-SIGN binding region (blue) is
more confined, by Phe313 on one side and Val351 on the
opposite side of the Ca2+ ion (Figure 6B). The Val351 side chain
is in close contact with the cyclohexane ring of 3 and near the
amide side chains of the amide derivatives (Figure 6B). In
dectin-2 the corresponding loop is more open and the valine
residue is replaced by a histidine in this position (His171). The
different loop orientation, as highlighted in Figure 6, allows more
available space between the protein and the position of the
amide side chain of the mimics (as indicated by the curved
arrow in Figure 6B). As a result, this lectin may be able to
accommodate mannobioside mimics with larger groups, such as
tertiary amides, for the interaction with its CRD. Further
investigation on these ligands is underway.
Figure 6. A) Alignment of DC-SIGN complex with 3 (blue protein, cyan ligand; PDB: 2XR5) and dectin-2 complex of Man9GlcNAc2 (orange protein, ligand not
shown PDB: 5VYB). The Ca2+ ion is shown as a pink sphere. B) Zoom on the Ca2+ binding site viewed from the opposite direction. The curved arrow highlights the
different orientation of the two loops in the two proteins. The small arrow points to the cyclohexene ring of 3 and to the position of the amide group in the
glycomimetic ligands 12.1-n.
potentially of high interest in the search for selective ligands. In
fact, only dectin-2 appeared to interact with the β-fucosides on
Conclusions
the array, although less effectively than with most mannose-
based derivatives.
We have set up and optimized a glycomimetic microarray to use
as a primary screening tool for mannose/fucose selective C-type
lectins. A doubly-functionalized cyclooctyne linker was used for
the fast immobilization of glycomimetic structures carrying an
azide–terminated side chain. The array was validated with plant
or fungal lectins of known specificity and then interrogated with a
set of four human C-type lectins: DC-SIGN, DC-SIGNR, langerin
and dectin-2. Appropriate controls showed that, for all the lectins
examined so far, the linker does not interfere with the binding
process. The glycomimetics used are based on a central
cyclohexane scaffold, carrying either an α-mannose or a β-
fucose residue and further diversified by the presence of
different amide appendages. The mannose based glycomimetics
are structurally derived from the Manα-1-2Man (mannobioside)
natural disaccharide motif. Interestingly, this disaccharide is a
common natural ligand of all the C-type lectins tested in this
study, which on the contrary differ strongly in their ability to
interact with fucose containing oligosaccharides. Thus,
screening of mannose and fucose based glycomimetics is
The screening also revealed that the CLRs studied differentially
respond to the amide substituents of the mimics, generating
different binding profiles. While langerin was found to bind
weakly to most of the structures examined, DC-SIGN and DC-
SIGNR displayed a rather good tolerance to secondary amide
substituents on the pseudo-mannobioside structure and some
similarity in the recognition profile. Most interestingly, a set of
mannosides carrying tertiary amide substituents were found to
selectively recognize dectin-2 over DC-SIGN, which may be
explained by the known structure of the two lectins’ binding site.
Some of the fucose-derived glycomimetics loaded on the chip
also displayed a selectivity for dectin-2 over DC-SIGN and DC-
SIGNR. Thus these screening campaigns simultaneously
provided the first discovery of glycomimetic ligands for dectin-2
and gave important indications for the design and optimization of
dectin-2 selective antagonists.
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Bis(4-nitrophenyl) (1S,2S)-cyclohex-4-ene-1,2-dicarboxylate (8)
The affinity of selected compounds for DC-SIGN, DC-SIGNR
and langerin was also measured by SPR inhibition experiments.
The ligand ranking obtained in these solution assays differed
quantitatively from that inferred from array binding profiles. As
often observed in the study of sugar-protein interactions, the
affinity and binding selectivity measured for lectins can strongly
depend on the format of the assay, in ways that complicate the
discovery process. In the case at hand, the clustered ligand
presentation on the array can strongly influence the avidity of the
system in ways that cannot be reproduced by binding inhibition
experiments, where the monovalent ligand in solution is
competing against an immobilized glycoprotein. Thus, the SPR
and array binding data should be regarded as complementary
information, describing different features of the ligand-lectin
interaction, The microarray format of the test we propose here
allows to analyze the binding profiles of lectins even if they are
available only in minute quantities, as it is the case for dectin-2
in this study, and may provide structural information useful in the
design of multivalent inhibitors that mimic the dense ligand
presentation of the array surface. Further characterization of the
binding properties of dectin-2 binders, as well as their structural
optimization will be the object of active investigation in our
laboratories.
Diacid 7 (4.23 g, 24.86 mmol, 1 mol equiv) was dissolved in dry DMF
under N2 and pyridine (5.23 mL, 64.63 mmol, 2.6 mol equiv) was added
dropwise to the solution. 4-nitrophenyl trifluoroacetate (14.03 g, 59.66
mmol, 2.4 mol equiv) was added to the mixture and the reaction was
stirred overnight at 50°C. After completion (Rf (product) = 0.58 in
toluene : EtOAc = 8 : 2 + 0.1% acetic acid), the reaction was diluted with
200 mL dichloromethane and washed twice with 100 mL 0.5M HCl, twice
with 50 mL cold, saturated NaHCO3 and twice with 50 mL water. The
organic phase was dried over Na2SO4, filtered and concentrated in
vacuo. The obtained crystals were washed with cooled diethyl ether and
filtered to yield the pure product 8 as a white powder. Yield: 73%. [αD]:
+130 (c = 1 in CHCl3); 1H NMR (400 MHz, CDCl3): δ = 8.28 – 8.22 (m,
4H; H10), 7.27 – 7.22 (m, 4H; H9), 5.83 (app d, J=2.8 Hz, 2H; H4, H5),
3.27 – 3.19 (m, 2H; H1, H2), 2.78 – 2.68 (m, 2H; H3ps–eq, H6ps–eq), 2.48 –
2.37 (m, 2H; H3ps–ax, H6ps–ax); 13C NMR (100 MHz, CDCl3): δ = 172.8 (C7);
155.4 (C11); 145.7 (C8); 125.5 (C10); 124.9 (C5, C4); 122.5 (C9); 41.5 (C1,
C2); 28.0 (C3, C6); MS (ESI): m/z calculated for [C20H16N2O8Na]+: 435.08
[M+Na]+; found: 435.33; m. p. 171°C
2,3,4-tri-O-benzoyl-α-L-fucopyranosyl-1-trichloroacetoimidate 13
L-(-)-fucose (200 mg, 1.22 mmol, 1 mol equiv) was dissolved at -40°C in
1 mL pyridine under N2 atmosphere and benzoyl chloride (636 μL, 5.48
mmol, 4.5 mol equiv) was added dropwise to the solution. After 2 h the
starting unprotected sugar was not detected by TLC anymore (Rf = 0.1 in
toluene : EtOAc = 9 : 1), so the reaction was left to warm to room
temperature and stirred until there was only one major spot visible on the
TLC plate (Rf = 0.24 in toluene : EtOAc = 97 : 3). Upon completion, the
reaction mixture was diluted with water and extracted with DCM. The
joint organic phases were dried over Na2SO4, filtered and concentrated in
vacuo, to yield the product 1,2,3,4-tetra-O-benzoyl-L-fucopyranose as a
white foam. Yield: quant (α : β = 9 : 1). Under these conditions only a
small amount of fuco-furanose form is obtained, typically around 1% by
1H NMR , so that no additional purification is needed before the next
step. 1H NMR (400 MHz, CDCl3): α-anomer: δ = 8.17 – 7.21 (m, 20H,
Experimental Section
Chemicals were purchased from Sigma-Aldrich or Acros Organics, and
specific amines from Key Organics, Crea-Chim, Vitas M Labs, Life
Chemicals, Alinda Chemicals, Chem Bridge or Enamine BB (suppliers
indicated in the Supporting Information, section Characterization of the
ligands) and were used without further purification. All reaction solvents
were dried over activated 4Å or 3Å molecular sieves. Thin layer
chromatography was carried out using 60 F254 TLC plates and visualized
by UV irradiation (254 nm) or by staining with cerium molibdate,
potassium permanganate or ninhydrin solution. Canavalia ensiformis
lectin (ConA) and Aleuria aurantia lectin (AAL) were purchased from
VectorLabs and labeled with Alexa Fluor® 555 NHS Ester (Succinimidyl
Ester) (Thermo Fisher Scientific). Human CLRs were prepared and
labeled as described below.
OBz), 6.87 (d, 1H, H1, J1-2 = 3.7 Hz), 6.08 (dd, 1H, H3, J3-4 = 3.2 Hz, J3-2
=
10.8 Hz), 5.99 (dd, 1H, H2, J2-3 = 10.8 Hz, J2-1 = 3.7 Hz), 5.90 (dd, 1H, H4,
J4-5 = 1.1 Hz, J4-3 = 3.2 Hz), 4.64 (dd, 1H, H5, J5-6 = 6.6 Hz, J = 12.9),
1.32 (d, 3H, H6, J = 6.4 Hz); β-anomer: δ = 8.17 – 7.21 (m, 20H, OBz),
6.21 (d, 1H, H1, J1-2 = 8.3 Hz), 6.06 (m, 1H, H2), 5.81 (dd, 1H, H4, J4-5
=
1.0 Hz, J4-3 = 3.4 Hz), 5.72 (m, 1H, H3), 4.34 (dd, 1H, H5, J5-6 = 5.7 Hz, J
= 12.5 Hz), 1.39 (d, 3H, H6, J = 6.4 Hz). MS (ESI): m/z calculated for
[C34H28O9Na]+(M + Na+): 603.16; found: 603.35.
Library design
1,2,3,4-tetra-O-benzoyl-L-fucopyranose (707.3 mg, 1.218 mmol, 1 mol
equiv) was dissolved in dry THF and cooled to 0°C under N2 atmosphere.
2M MeNH2 in THF (731 μL, 1.462 mmol, 1.2 mol equiv) was added
dropwise to the solution. After one hour, the major spot on the TLC had
slightly lower Rf than the starting material and a new spot started to
appear further below. The solution was concentrated in vacuo, and the
product was purified by flash chromatography (Rf = 0.18 and 0.15 for α
and β anomers in toluene : EtOAc = 9 : 1) to yield pure 2,3,4-tri-O-
benzoyl-L-fucopyranose as a colourless oil. Yield: 74% (α : β = 2 : 1). 1H
NMR (400 MHz, CDCl3): α-anomer: δ = 8.18 – 7.23 (m, 15H, OBz), 6.04
(dd, 1H, H3, J3-2 = 10.7 Hz, J3-4 = 3.2 Hz), 5.81 – 5.77 (m, 2H, H1, H4),
5.68 (m, 1H, H2), 4.69 (dd, 1H, H5, J5-6 = 6.5 Hz, J5-4 = 13.1 Hz), 1.30 (d,
3H, H6, J6-5 = 6.5 Hz); β-anomer: δ = 8.18 – 7.23 (m, 15H, OBz), 5.73
(dd, 1H, H4, J4-5 = 1.0 Hz, J3-4 = 3.5 Hz), 5.71 – 5.66 (m, 1H, H3), 5.58
(dd, 1H, H2, J2-1 = 7.9 Hz, J2-3 = 10.4 Hz), 4.99 (d, 1H, H1, J1-2 = 7.9 Hz),
4.13 (dd, 1H, H5, J5-6 = 6.1 Hz, J5-4 = 12.8 Hz), 1.39 (d, 3H, H6, J6-5 = 6.5
Hz); MS (ESI): m/z calculated for [C27H24O8Na]+(M + Na+): 499.14; found:
499.84
Markush structures were used to search the compound collections from
eMolecules (6,585,694 compounds, August 2013) and Molport
(10,010,542, December 2013) for primary and secondary amines. In
particular, only amines with 150 <= MW <= 275, number of 9 <= heavy
atoms <= 20, number of rotatable bonds < 6, number of rings > 0 and no
undefined stereocentres were considered. Amines containing potentially
reactive species[42] and PAINS[43] were also removed. The resulting
compounds were clustered and the cluster center selected (ECFP4
fingerprints as molecular descriptors, maximum distance between cluster
members Tanimoto = 0.6). To reduce the compounds to a number
amenable to visual inspection 20% of the cluster centers was selected
maximizing molecular diversity (based on FCFP4 fingerprints). This
resulted in a set of 1116 primary amines (630 aliphatic and 486 aromatic)
and 796 secondary amines (472 aliphatic and 324 aromatic). After visual
inspection and confirmed commercial availability a set of 38 compounds
was selected for acquisition.
Synthesis of the ligands
2,3,4-tri-O-benzoyl-L-fucopyranose (460 mg, 0.969 mmol, 1 mol equiv)
was dissolved in dry dichloromethane (5 mL) and cooled to 0°C. DBU (58
μL, 0.388 mmol, 0.4 mol equiv) and trichloroacetonitrile (972 μL, 9.694
Mannosylated scaffold 11 was prepared according to ref[11c] from
acceptor 9 and the known donor 10. Compound 9, in turn, was prepared
from 8 in two steps, according to ref.[11c]
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mmol, 10 mol equiv) were added to the solution which was left to warm to
room temperature and stirred until the starting material disappeared. The
solution was concentrated in vacuo, and the product was purified by flash
chromatography (Rf = 0.34 in hexane : EtOAc = 9 : 1) to yield the pure
product as a colourless oil (only α-anomer). Yield: 93%. 1H NMR (400
MHz, CDCl3): δ = 8.60 (s, 1H, NH), 8.18 – 7.25 (m, 15H, OBz), 6.83 (d,
1H, H1, J1-2 = 3.5 Hz), 6.04 (dd, 1H, H3, J3-2 = 10.9 Hz, J3-4 = 3.5 Hz),
5.91 (dd, 1H, H2, J2-3 = 10.9 Hz, J2-1 = 3.5 Hz), 5.89-5.86 (m, 1H, H4),
4.65 (dd, 1H, H5, J5-6 = 6.5 Hz, J5-4 = 6.3 Hz), 1.30 (d, 3H, H6, J6-5 = 6.5
Hz); MS (ESI): m/z calculated for [C29H24O8ClNa]+(M + Na+): 642.05;
found: 642.37
Conjugation of the ligands with the bifunctional linker 16
A 10 mM solution of the ligands was prepared in water and when
necessary for complete solubilization, 5 % DMSO (Thermo Scientific
Molecular Probes™) was added. The compounds were stirred overnight
at room temperature with equimolar amounts of 16 (N-[(1R,8S,9S)-
Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-
dioxaoctane, BCN-amine, Sigma Aldrich) in water and the conversion of
the SPAAC-reactions was monitored by MALDI-Tof mass spectrometry
using 2,5-dihydroxybenzoic acid (DHB) as matrix (5 mg/mL in
CH3CN:0.1% aqueous TFA, 3:7 containing 0.005% NaCl). The MALDI
data are reported in the SI section.
1,2-Cyclohexanedicarboxylic acid, (1S,2S,4S,5S)-4-(2-azidoethoxy)-
5-[(2,3,4-tri-O-benzoyl-β-D-fucopyranosyl)oxy]-1,2-bis(p-
nitrophenylester) (14):
Ligand printing and screening
Stock solutions of the conjugates 1 mM in water were diluted with sodium
phosphate buffer (300 mM, pH 8.5, 0.005 % Tween® 20, 10 % DMSO) to
a final concentration of 50 µM. 40 µL of each solution was placed into a
384 well source plate (Scienion, Berlin, Germany) which was stored at -
20°C and reused if necessary. These solutions (750 pL, 3 drops of 250
pL) were spotted onto NHS functionalized glass slides (Nexterion® Slide
H - Schott AG, Mainz, Germany).Ligands were spotted in 4 replicates (9
different ligands per row), establishing the complete microarray which
was printed in 7 copies onto each slide After printing, the slides were
placed in a 75% humidity chamber (saturated NaCl solution) at room
temperature overnight. The unreacted NHS groups were quenched by
placing the slides in a 50 mM solution of ethanolamine in sodium borate
buffer 50 mM, pH 9.0, for 1h.
A mixture of acceptor 9 (250 mg, 0.4013 mmol, 1 mol equiv) and donor
13 (207 mg, 0.4013 mmol, 1 mol equiv) was co-evaporated from toluene
three times. Powdered and activated 4Å molecular sieves (acid washed)
were added; and the mixture was kept under vacuum for a few hours and
then dissolved with dry DCM (4 mL). The solution was cooled to -30°C
and TMSOTf (9 μL, 0.0401 mmol, 0.2 mol equiv) was added to the
mixture. The reaction was stirred at -30°C for 2 h and at RT for an
additional 1.5 h and was then quenched with Et3N. The mixture was
filtered over a celite pad. The solution was concentrated in vacuo, and
the product was purified by flash chromatography (Rf = 0.22 in hexane :
EtOAc = 6 : 4) to yield the pure product 14 as a colourless foam. Yield:
72%. 1H NMR (400 MHz, CDCl3): δ = 8.23 – 6.83 (m, 23H, HAr), 5.75 –
5.63 (m, 2H, H2, H4), 5.57 (dd, 1H, H3, J3-2 = 10.5 Hz, J3-4 = 3.4 Hz), 4.86
(d, 1H, H1, J1-2 = 8.0 Hz), 4.14 (m, 1H, C2), 4.06 (q, 1H, H5, J5-4 = 12.9
Hz, J5-6 = 6.3 Hz), 3.87 (m, 1H, C1), 3.84 – 3.77 (m, 1H, H7a), 3.69 – 3.62
(m, 1H, H7b), 3.41 – 3.31 (m, 2H, H8a,b), 3.22 – 3.13 (m, 1H, C4), 3.02 –
2.92 (m, 1H, H5), 2.36 – 2.27 (m, 1H, C3 or 6eq), 2.21 – 2.12 (m, 1H, H3 or
6eq), 2.08 – 1.90 (m, 2H, H3ax, 6ax) ; 13C NMR (100 MHz, CDCl3): δ =
172.5, 172.3 (C9); 166.3, 165.9, 165.7 (COBz); 155.5, 155.3 (C10); 145.8,
145.7 (C13); 133.9, 133.7, 133.5 (CHBz); 130.3, 130.0, 130.0 (CHBz);
129.5, 129.1 (CquatBz); 128.9, 128.7, 128.6 (CHBz); 125.5, 125.4 (C12);
122.7, 122.6 (C11); 100.2 (C1); 75.0 (CC1); 72.7 (CC2); 72.0 (C3); 71.3 (C2);
70.4 (C5); 70.1 (C4); 69.6, 68.7 (C4); 51.2 (C8); 39.0, 39.0 (CC4,CC5); 27.5,
The immobilized ligands were probed with solutions of fluorescently
labeled (Alexafluor555) plant and fungal lectins. Solutions of
Concanavalin A (ConA-555, 1 µg/mL) and Aleuria aurantia lectin (AAL-
555, 15 µg/mL) were prepared in PBS containing 2 mM CaCl2, 2 mM
MgCl2 and 0.005% Tween-20. For incubations, 200 µL of the lectin
solution was applied to each microarray using 8 Well ProPlate™ Slide
Modules incubation chambers for 1h in the dark at room temperature.
The slides were washed under standard conditions (PBS and water),
dried with argon and the introduced fluorescence was analyzed with a
microarray scanner.
27.2 (CC3
,
CC6); 16.9, 16.7 (C6);
MS (ESI): m/z calculated for
[C49H43N5O17]+: 996.26; found: 996.86
The immobilized ligands were probed with solutions of fluorescently
labeled C-type lectins. Solutions of Cy3-labelled DC-SIGN ECD-Cy3 (50
µg/mL, DOL: 0.3) and DC-SIGNR ECD (150 µg/mL, DOL: 0.95), langerin-
Cy3 (25 µg/mL, DOL: 0.7) and dectin-2-Cy3 (50 µg/mL, DOL: 0.4) were
diluted in TBS (50 mM Tris·HCl, 150 mM NaCl, pH=8.0) containing 4 mM
CaCl2, 0.5% BSA and 0.005% Tween® 20. For incubations, 200 µL of
each lectin solution was applied to each subarray using 8 Well ProPlate
Module incubation chambers. The microarray was incubated under
gentle shaking overnight in the dark at 4°C. The slides were washed
using TBS containing 4 mM CaCl2 and water, dried with argon and the
fluorescence was analyzed with a microarray scanner.
General procedure for the synthesis of bisamides 12 and 15
Amine coupling. Scaffold 11 or 14 (1 mol equiv) was dissolved in dry
THF or DMF see SI section) under N2 and the amine (3 mol equiv) was
added to the solution. For amines sold as ammonium salts and amines
with low reactivity (see SI section) 3 mol equiv. of Et3N were also added.
The mixture was stirred at RT from 1 h to 2 days, and monitored by TLC
or NMR. Upon completion, the solution was washed with 1M HCl, 1M
NaOH and water on supported liquid extraction cartridges (Biotage
ISOLUTE® HM-N). The crude was purified by flash chromatography
(DCM with gradient of methanol from 0 to 20%) or used without
purification in the following Zemplén-deprotection if the purity was
satisfying.
Expression and Purification of CLRs
DC-SIGN extracellular domain (DC-SIGN ECD) and langerin extracellular
domain (langerin ECD) constructs were produced and purified as
previously described.[3a, 24]
Zemplén debenzoylation. The benzoyl-protected bisamide (1 mol
equiv) was dissolved in distilled MeOH and 1M freshly prepared NaOMe
in MeOH was added to the solution (1.5 mol equiv NaOMe) to 0.1M final
concentration of the substrate. After completion, the reaction was
neutralized with Amberlite® IR120 hydrogen form ion-exchange resin,
filtered and concentrated in vacuo. The crude was purified by direct or
reverse phase flash cromatography, yielding the pure product 12 or 15.
DC-SIGNR ECD and dectin-2 ECD were expressed in E. coli BL21(DE3)
in 1 liter of LB medium supplemented with 50 μg/mL kanamycin at 37 °C.
Expression was induced by addition of
1 mM isopropyl 1-thio-D-
galactopyranoside (IPTG) when the culture had reached an A600 nm of 0.8
and maintained for 3h. The protein was expressed in the cytoplasm as
inclusion bodies. Cells were harvested by a 20-min centrifugation at 5000
g at 4 °C. The pellet was resuspended in 30 mL of a solution containing
150 mM NaCl, 25 mM Tris-HCl, pH 8 and one anti-protease mixture
tablet (Complete EDTA free, Roche). Cells were disrupted by sonication
Library characterization. Ligands 12.1, 12.3, 12.4, 12.7, 12.12, 12.14,
12.15, 12.18, 12.19 were previously described by Varga et al.;[11c] ligand
12.2 was described in Reina et al.[44] while the characterization of the
other ligands is detailed in the Supporting Information.
This article is protected by copyright. All rights reserved.

10.1002/chem.201802577
Chemistry - A European Journal
FULL PAPER
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4 °C in a Beckman 45Ti rotor. The pellet was solubilized in 30 mL of 6 M
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Acknowledgements
This project has received funding from the European Union’s
Horizon 2020 research and innovation program under the Marie
Sklowdowska-Curie
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grant
agreement
No.
642870
(Immunoshape). Support by the Italian Ministry of Research
through a PRIN grant (prot. 2015RNWJAM_002) and the
Spanish Ministry of Economy, Industry and Competitiveness
(grant CTQ2017-90039-R to N.-C. Reichardt) is acknowledged.
HMRS analyses were obtained from the UNITECH “COSPECT”
platform at the University of Milan. For human CLRs ECD
production, this work used the Multistep Protein Purification
Platform (MP3) and the SPR platform for the competition test of
the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-
EMBL) with support from FRISBI (ANR-10-INSB-05-02) and
GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership
for Structural Biology. FF also acknowledges the support of the
French ANR for Glyco@Alps (ANR- 15-IDEX-02).
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