Second generation of fucose-based DC-SIGN ligands: Affinity improvement and specificity versus Langerin

Article abstract of DOI:10.1039/c1ob05573a

DC-SIGN and Langerin are two C-type lectins involved in the initial steps of HIV infections: the former acts as a viral attachment factor and facilitates viral invasion of the immune system, the latter has a protective effect. Potential antiviral compounds targeted against DC-SIGN were synthesized using a common fucosylamide anchor. Their DC-SIGN affinity was tested by SPR and found to be similar to that of the natural ligand Lewis-X (Le^X). The compounds were also found to be selective for DC-SIGN and to interact only weakly with Langerin. These molecules are potentially useful therapeutic tools against sexually transmitted HIV infection.

Full text of DOI:10.1039/c1ob05573a

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PAPER
Second generation of fucose-based DC-SIGN ligands : affinity improvement
and specificity versus Langerin†
Manuel Andreini,‡a Daniela Doknic,‡a Ieva Sutkeviciute,‡b,e Jose´ J. Reina,a Janxin Duan,c Eric Chabrol,b,e
Michel Thepaut,b,e, f Elisabetta Moroni,a Fabio Doro,a Laura Belvisi,a Joerg Weiser,c Javier Rojo,d
Franck Fieschi*b,e,g and Anna Bernardi*a
Received 11th April 2011, Accepted 20th May 2011
DOI: 10.1039/c1ob05573a
DC-SIGN and Langerin are two C-type lectins involved in the initial steps of HIV infections: the
former acts as a viral attachment factor and facilitates viral invasion of the immune system, the latter
has a protective effect. Potential antiviral compounds targeted against DC-SIGN were synthesized
using a common fucosylamide anchor. Their DC-SIGN affinity was tested by SPR and found to be
similar to that of the natural ligand Lewis-X (LeX). The compounds were also found to be selective for
DC-SIGN and to interact only weakly with Langerin. These molecules are potentially useful
therapeutic tools against sexually transmitted HIV infection.
(ICAM-3) receptor that plays an important role in establishing the
Introduction
first contact between DC and resting T cells.4 It is a type II trans-
Dendritic Cells (DCs) are instrumental in the development
membrane C-type lectin with a single C-terminal Carbohydrate
of pathogen-specific immune responses.1 DCs are professional
Recognition Domain (CRD) within its sequence. In the cellular
antigen-presenting cells that capture microbes entering skin or
membrane, DC-SIGN is assembled as a tetramer, thanks to an
mucosal tissues and process them to form MHC-peptide com-
extended coiled-coil region that allows simultaneous presentation
of four CRDs.5 This oligomerization influences the lectin avidity in
binding events. DC-SIGN appears to promote dissemination of a
number of viruses (e.g., HIV, hepatitis C virus, Ebola virus)6 and to
participate in suppressing immune responses to some pathogens,
(e.g., Mycobacterium tuberculosis and Helicobacter pylori).7
plexes. After antigen uptake, immature DCs acquire the capacity
to migrate to lymph nodes where they present processed antigens
to T-cells, initiating adaptive immune responses. DCs express a
repertoire of pathogen-recognition receptors (PRRs), including
Toll Like Receptors (TLRs) and C-type lectins that mediate
both signaling by self antigens and, in some cases, pathogen
recognition.2 C-type lectins represent a large family of Ca2+
dependent lectins and recognize pathogen-derived carbohydrate
structures. Many different C-type lectins expressed by DCs have
been described,3 including DC-SIGN.
The various roles attributed to DC-SIGN have generated much
interest towards the identification of ligands that can be used
to explore its different functions and/or to inhibit pathogen
binding.8 However, generation of specific ligands for DC-SIGN
is a challenging task, since many other C-type lectins exist and
share important structural features with DC-SIGN binding site.
Among the list of C-type lectin receptors closely related to DC-
SIGN, Langerin, which is also expressed at the cell surface of
antigen presenting cells, and L-SIGN, expressed on endothelial
liver cell, placenta and lymph nodes, are particularly likely to
interfere with DC-SIGN recognition.9,10 These three lectins are all
calcium-dependent carbohydrate-binding proteins and share the
ability to bind high-mannose oligosaccharides. On the other hand,
the three lectins show different specificity towards fucosylated
oligosaccharides, a fact which may be used to design DC-SIGN
specific ligands. Indeed, contrary to L-SIGN, DC-SIGN is known
to bind the Lewis X (LeX) epitope (Galb4[Fuca3]GlcNAc, 1 in
Fig. 1), as illustrated in the recognition mode of Schistosoma
mansoni egg by these lectins.5d,7,11 Moreover, both DC-SIGN and
Langerin appear to recognize blood group B antigen through its
fucose residue12,13 in the primary Ca2+ binding site, but again the
LeX antigen is specific for DC-SIGN relative to Langerin. As
DC-SIGN (DC-Specific ICAM-3 Grabbing Nonintegrin; CD
209) was originally defined as an intercellular adhesion molecule-3
aUniversita’ degli Studi di Milano, Dipartimento di Chimica Organica e
Industriale and CISI, via Venezian 21, 20133 Milano, Italy. E-mail: anna.
bernardi@unimi.it
bInstitut de Biologie Structurale, Universite´ Grenoble I, 41 rue Jules
Horowitz, 38027 Grenoble, France. E-mail: fieschi@ibs.fr
cAnterio consult&research, Augustaanlage 23, D-68165 Mannheim
dGlycosystems Laboratory, Instituto de Investigaciones Qu´ımicas, CSIC–
Universidad de Sevilla, Ame´rico Vespucio 49, 41092 Sevilla, Spain
eCNRS, UMR 5075, Grenoble, France
f
CEA, Grenoble, France
gInstitut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris,
France
† Electronic supplementary information (ESI) available: . See DOI:
10.1039/c1ob05573a
‡ These authors, listed in alphabetical order, contributed equally to this
work
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improve the binding affinity of the fucosylamides by optimizing
the interactions in the secondary binding site. To achieve these
goals, a library of ca. 40 derivatives of general formula 4 was
designed, synthesized and assayed by SPR to determine the ability
of the compounds to inhibit DC-SIGN binding to immobilized
mannosylated Bovine Serum Albumin (Man-BSA). Moreover,
a preliminary selectivity screening was introduced to test some
library members for inhibition of Langerin, using SPR. Selectivity
for DC-SIGN versus Langerin is specially important to develop
inhibitors of sexually transmitted HIV infections. As discussed
above, interaction with DC-SIGN on mucosal DC is used by
the virus to invade the host immune system. On the contrary,
Langerin is suggested to have protective effects against HIV
infection.16 Indeed, some of the fucosylamides examined displayed
an interesting DC-SIGN selectivity and have the potential of being
developed as antiviral agents.
Fig. 1 The known fucose-based DC-SIGN ligands 1–3a and the general
structure 4 of the library described in this paper. (IC50 from ref. 14).
shown by X-ray structures,12,13 in addition to binding the fucose
residue on the Ca2+ site, DC-SIGN is uniquely able to stabilize LeX
galactose residue in a second binding area due to key residues that
are absent in Langerin.
The existence of a secondary binding site is also suggested by
a glycan array study of over 100 glycan structures.12 This study
demonstrated that the presence of a terminal fucose residue is not
sufficient for DC-SIGN binding, but 14 fucose-bearing glycans
with the structure of Lewis epitopes were found to bind selectively
to DC-SIGN relative to L-SIGN.
Results and discussion
We have recently described the first fucose-based artificial ligand
of DC-SIGN (compound 2, Fig. 1), designed to mimic this
trisaccharide.14 The ligand was built by using an a-fucosylamide
anchor to drive the molecule to the DC-SIGN primary binding
site and connecting it to a galactose mimic using a cyclic cis-b-
amino acid ((1S,2R)-2-amino-cyclohexanecarboxylic acid, Fig. 1).
The layout of these residues and more specifically the linker b-
amino acid allowed the molecule to adopt a three-dimensional
shape similar to the LeX trisaccharide (Fig. 1).15 Amide bonds
were chosen to connect the three elements of ligand 2 to achieve
synthetic simplicity as well as chemical and metabolic stability
of the target molecule. DC-SIGN binding studies performed by
SPR showed that ligand 2 and surprisingly its simplified version
3a, which does not contain the galactose-mimic moiety, inhibit
DC-SIGN better than the natural ligand 1 (IC50 = 0.35 mM,
0.5 mM and 0.8 mM, respectively).14 The weak difference of
affinity between 2 and 3a, however, suggested that the galactose-
like fragment in 2 gives a limited contribution to the binding
interaction. Building on this knowledge, the goals of the work
we report in this paper were: 1) to establish a minimal structure
easily accessible in large scale and able to engage the receptor
with an affinity similar to that of the natural ligand LeX; 2) to
In order to select reasonable ligand candidates, the properties of
the protein surface in the vicinity of the Ca2+ site in the LewisX-
DC-SIGN complex (1SL5)12 were examined using GRID.17 Both
the DRY probe and the WATER probe were used to identify
hydrophilic and lipophilic regions of the binding site. The molec-
ular representations shown in Fig. 2 were obtained with the
Maestro graphical interface. Various minima for the WATER
probe were identified in the vicinity of the Ca2+-binding region:
in the crystal structure, they are occupied by crystallographic
water molecules W13, W34 and W36 (Fig. 2a). W13 and W36
are located in two well-defined low interaction energy sites, both
below -11 kcal mol-1. W13 is in the vicinity of the fucose
residue and mediates the interaction of Fuc-O2 with Glu354 and
Lys368, and of Gal-O6 with Asp367 (Fig. 2a). W36 mediates the
interaction of Gal-O4 with Glu358. The W36 site is occupied
by a crystallographic water molecule also in 3 out of 4 known
X-ray structures of DC-SIGN in complex with oligomannosides
(1SL4,12 1K9I,18 2IT519) and it is replaced by one sugar hydroxyl
group in the fourth one (2IT619). The W34 site belongs to a larger
isoenergetic area with a less favorable GRID interaction energy
(ca. -7 kcal mol-1), occupied by water molecules in 2 out of 4
oligomannoside-DC-SIGN complexes (1SL4, 1K9I) and loosely
Fig. 2 Energetic maps of GRID interactions (the Ca2+ ion is shown in yellow) in the structure of the DC-SIGN- LeX complex (pdb code : 1SL5, from
ref. 11). a) WATER probe, -7.0 kcal mol-1 isosurface, showing the binding sites for crystallographic water molecules W13, W34 and W36 in 1SL5. b)
DRY probe, -0.5 kcal mol-1 isosurface, showing hydrophobic areas near the binding region and the groove formed by Phe313 and Leu 371.
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Scheme 1 Retrosynthetic analysis of fucosyl derivative 3.
replaced by mannose hydroxyl groups in the other 2 (2IT5 and
2IT6). In the 1SL5 structure, W34 mediates binding of Gal-O4 to
Ser360 and, together with W36, contributes to the creation of a
secondary binding site involving Leu371, Asp367, Lys373, Glu358
and flanked by Phe313 (Fig. 2a).
GRID analysis with the DRY probe allowed identification of
the hydrophobic areas near the binding region, which are shown in
Fig. 2b. Two of them, formed by Val351 and Asn362/Asn344, are
in the immediate vicinity of the Ca2+-binding site and establish Van
der Waals contact with the ligand. Phe313 and Leu371/Lys368
side chains form a major hydrophobic groove which flank the
W36 crystallographic site.
Docking of mimic 2 in the 1SL5 structure was obtained using
Glide.20 The complex, which included protein, ligand and the two
water molecules W13 and W36, was prepared with the standard
Preparation Wizard routine of Glide, but the final minimization
was performed in implicit (GB/SA21) water with the AMBER*
force field. It was found that this procedure allowed achievement
of a better orientation of the water molecules and optimization
of their hydrogen bonding pattern, which in turn avoided steric
clashes in the following re-docking step. Docking obtained with
this model suggested that optimal interaction is reached with
the ligand in an extended conformation, which would allow the
galactose mimic to place two hydroxyl groups in a hydrophilic
patch of the protein near the side chain of Phe313 while nesting
the cyclohexane ring in the groove formed by Phe313 and Leu371
(Fig. 3).
of lipophilic interactions and to interact specifically with the
secondary hydrophilic regions. Further docking experiments sug-
gested that favorable interactions could also be established by
positively charged groups in the ligands and the negatively charged
regions of the protein created by Asp 367 and Glu 358 side
chains. Following this analysis, candidates for the R group in
4 were selected among commercially available carboxylic acids
featuring aromatic groups and/or hydroxyl groups, amino groups
or acetamides.
The initial set of compounds were synthesized starting from
amine 5,14 which in turn was obtained from tri-O-acetyl-L-
fucosylazide 6 and the protected (1S,2R)-b-amino acid 7, as we
have previously described (Scheme 1).14
The coupling reactions between amine 5 and the acid partners
(RCO2H, Scheme 2) were performed using either HBTU, acid
chloride or EDC/HOBt activation, as described in the Supple-
mentary Information. These conditions afforded the protected
ligands 8 in variable yields (between 43 and 80%) after isolation by
solid phase extraction and chromatographic purification. Removal
of the protecting groups under standard Zemplen’s conditions
gave the required compounds 3. If the acid partner carried a Boc
protection, this was first removed using a mixture of TFA/CH2Cl2
(1/5). In this way ca. 30 different compounds were prepared (see
Supplementary Information for the structure and characterization
of the entire library). Scheme 2 shows the structure of those that
will be used in the following discussion.
Scheme 2 Coupling reaction of amine 5 with acid partners.
DC-SIGN affinity for the entire set of 30 compounds of general
formula 3 was estimated using a surface plasmon resonance
(SPR) biosensor in a competition assay which we have previously
described.14 The assay allows an affinity evaluation of all ligands
relative to one another on the basis of their percentage inhibition
Fig. 3 Docking of mimic 2 in 1SL5. (the Ca2+ ion is shown in yellow).
Based on this model, interesting candidates to replace the
galactose moiety should be molecules able to take advantage
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Fig. 4 The dependency of DC-SIGN ECD percent activity on concentrations of corresponding compounds. (see original sensorgrams in SI-Fig.4).
of DC-SIGN binding to immobilized mannosylated bovine serum
albumin (Man-BSA). The commercially available Man-BSA used
in these assays contain an average of 12 glycosylation sites
displaying the Mana1-3[Mana1-6]Man branched trisaccharide.
Man-BSA was covalently attached to a carboxymethyl dextran-
functionalized gold SPR sensor chip CM4. Inhibition studies
were then performed using extracellular domain (ECD) of DC-
SIGN (20 mM) injected alone or in the presence of a constant
concentration (300 mM) of the ligands. At this concentration,
for the particular chip used in the assay, LeX exhibited 25%
of inhibition. All the molecules 3 synthesized showed a similar
efficiency, independent of the nature of the R group, and none
improved significantly over the activity of 3a. To confirm these
data, complete inhibition curves were obtained and IC50 values
were estimated for the selected group of compounds 3a–h shown
in Scheme 2. The results were totally consistent with the previous
observation (Fig. 4).
structural details of the interaction of DC-SIGN with compounds
3.23 To further explore the role of the b-amino acid structure
in defining ligand–protein interaction, the configuration of the
scaffold was varied systematically and a third set of compounds
was synthesized, where the R fragment was kept unchanged and
the b-configuration was systematically permutated.
The new set of compounds 10–12 (Fig. 6) were synthesized
starting from b-amino acids 13–15 (Fig. 6) using the synthetic
sequence employed for 3 and shown in Schemes 1 and 2. The
syntheses of the enantiomerically pure isomeric amino acids
13–15 and of the corresponding fucosylamide derivatives 10–12
are described in the Supplementary Information. The IC50 ob-
tained by SPR analysis of 10–12 are collected in Fig. 7 and
compared to selected data obtained for 3, for LeX and for L-fucose
(see SI-Fig 5 for original sensorgrams).
The IC50 values obtained for 3a and 3b are consistent with
previous measurements (Fig. 4). The data confirm that the activity
of most fucosylamides is close to that of LeX. The series of
compounds 10a–d show a larger increase of the affinity (a factor
of 3) on passing from the acetamide 10a (R = Me) to the aromatic
amides 10b–d, suggesting a possible role of the aromatic group
in the interaction with the protein. This series, which is built on
the (1R,2S)-2-amino-cyclohexanecarboxylic acid scaffold 13, also
contains the strongest ligands so far, the hydroxybenzoic acid
derivatives 10b and 10c (IC50 470 mM) and is therefore the best
current candidate for further optimization.
Interestingly, when an analogous group of compounds 9 (Fig. 5),
obtained by reaction of fucosylazide 6 with b-alanine rather than
with 7, was examined in SPR single point assays at 300 mM
concentration, similar inhibition values (25–30%) were obtained.
Thus, these simple a-fucosyl-b-alanyl amides showed a similar
affinity for DC-SIGN as LeX and all the compounds 3 synthesized.
To analyze the selectivity properties of the compounds screened,
we developed an additional SPR analysis for Langerin binding
properties. As for DC-SIGN, the ability of Langerin to bind to a
surface functionalized with Man-BSA was tested. In the case of
Langerin, binding to Man-BSA as well as to the dextran matrix
(SI-Fig 1) was observed. Therefore, the dextran/Man-BSA surface
was considered as a combined ligand of Langerin ECD. Upon
titration of the surface with Langerin, a similar saturation curve
than for DC-SIGN was obtained (SI-Fig. 2 and 3). Langerin
displayed an apparent Kd for this surface of 10.3 mM. Indeed,
the two lectins exhibited comparable affinity for this surface, thus
the same fixed concentration of Langerin was used in the SPR-
based competition assay, which allowed a direct comparison of
the binding inhibition properties of the compounds for Langerin
versus DC-SIGN.
Fig. 5 b-Alanine derivatives 9.
These observations, that are in striking contrast with the
expectations derived from docking studies, strongly suggest that
the R substituent in 3 is not reaching the secondary binding site
identified by the docking algorithm and may not be interacting
at all with the protein. The unexpected results obtained with a-
fucosyl-b-alanyl amides 9, whilst providing us with very simple
ligands of high efficiency,22 confirm that the (1S,2R)-2-amino-
cyclohexanecarboxylic acid scaffold selected for the synthesis of 3
does not enforce optimal interaction of the secondary residue with
the protein. NMR studies are currently in progress to assess the
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Fig. 6 Library of compounds with different stereochemistry in the cyclohexane scaffold.
Fig. 7 The IC50 values obtained for compounds 10–12 by SPR inhibition assay. IC50 of L-fucose, LeX, 3a and 3b measured in the same conditions are
shown for comparison.
Compounds initially tested for their DC-SIGN inhibitory
potency (Fig. 7) were evaluated with Langerin ECD. The results
are shown in Fig. 8A (see also SI-Fig.6). The inhibitory potency
of the fucosylated mimics is so low against Langerin that it
was not possible to determine an IC50. A crude comparison of
the inhibitors’ properties towards DC-SIGN and Langerin was
obtained by comparing the residual activity of both lectins at the
highest concentration tested for each compound (Fig. 8B).
The data show that many of the fucosylamides tested display a
larger DC-SIGN selectivity than LeX and confirm 10b as one of
the most interesting elements of this group, both for its DC-SIGN
affinity and for its specificity.
molecules to the DC-SIGN CRD binding site. We were able to
identify many compounds that, compared to the natural ligand
LeX and its previously reported mimic 2, display a similar DC-
SIGN inhibition efficiency at a fraction of the synthetic cost.
In particular, a-fucosylamides 9, derived from b-alanine, are
interesting candidates for polyvalent presentations8d–k due to their
high synthetic accessibility and good ligand efficiency.22 A second
group of compounds, a-fucosylamides 10 derived from (1R,2S)-
2-amino-cyclohexanecarboxylic acid 13, were also of interest
because they yielded the most active and selective ligand of this
group (10b). Indeed, it may be important that molecules directed
to block the action of DC-SIGN do not interfere with the action
of other lectins. We have recently shown that a-N-fucosylamides
of general formula 3 and 9 interact strongly with the L-fucose
binding lectin PA-IIL of Pseudomonas aeruginosa.24 In particular,
to develop inhibitors of sexually transmitted HIV infections it may
be necessary to select DC-SIGN antagonists that do not interfere
with the action of Langerin. Like DC-SIGN, Langerin is a
Conclusions
In this paper, we have presented a new library of fucose-based
ligands of DC-SIGN, all characterized by the presence of a b-
amino acid tether and of a fucosylamide anchor, able to direct the
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Fig. 8 A) Inhibition of Langerin ECD binding to Man-BSA immobilized on the dextran surface. B) Residual lectin activity at the highest tested
concentration of the ligands (4.6 mM). Langerin, white bars; DC-SIGN, black bars.
membrane C-type lectin known to bind to HIV-1. However, whilst
interaction with DC-SIGN is used by the virus to invade the host
immune system, Langerin is suggested to have protective effects
against HIV infection.16,25 Indeed, Langerhans cells, which are the
first dendritic cells to encounter HIV via genital mucosa, have been
described as a natural barrier for HIV-1 transmission, which is
dependent on Langerin expression.15 The importance of Langerin
in HIV protection has been again emphasized in the context of
HIV-1/Herpes Simplex Virus type II (HSV-2) co-infection.26 In
this last case, it has been demonstrated that HIV susceptibility
of Langerhans cells, and the subsequent virus transmission,
could be promoted by HSV-2-dependent abrogation of Langerin’s
functions. Conversely, DC-SIGN has a well-established role in
dendritic cells-mediated HIV-1 transmission.6 Thus, the selectivity
of the ligands for DC-SIGN relative to Langerin was also tested.
Indeed, we described here for the first time, simultaneous screening
of artificial compounds towards both lectins. In agreement with
literature data on the natural ligands of these lectins, a low
inhibitory potency of LeX towards Langerin has been observed
in contrast to DC-SIGN. The capacity of LeX and of the other
fucosylated derivatives to inhibit Langerin binding is so low that
we could not perform a full inhibition curve in a reasonable range
of concentration. Therefore, selectivity of the fucose-based ligands
tested was assessed by comparing residual lectin activity at the
highest ligand concentration tested. Most of the a-fucosylamides
assayed were found to be more DC-SIGN selective than the
natural ligand LeX and therefore they are more likely to be turned
into therapeutically useful tools against sexually transmitted HIV
infection.8
Surface plasmon resonance analysis
All experiments were performed on a Biacore 3000 using func-
tionalized CM4 sensor chips and the corresponding reagents
from Biacore. Two flow cells were activated as previously
described.28 Flow cell one was then blocked with 30 mL of
1 M ethanolamine and used as a control surface. The sec-
ond one was treated with BSA-Mana1-3[Mana1-6]Man (Man-
BSA, Dextra) (60 mg mL-1) in 10 mM acetate buffer, pH 4.
Remaining activated groups were blocked with 30 mL of 1 M
ethanolamine. The final density immobilized on the surface of
the second flow cell was 2000 RU. The Man-BSA used to func-
tionalize CM4 chip harbours 12 glycosylation sites according to
manufacturer.
Two types of SPR tests were set up for the evaluation of
glycomimic compounds. The single point inhibition assay was
used for fast screening of compound selectivity. Here, either DC-
SIGN or Langerin at concentration of 20 mM were incubated with
corresponding compounds (300 mM final concentration) and 20 ml
of the samples were co-injected over Man-BSA surface. The lectin
steady state binding responses were extracted from the sensor-
grams and compared with the responses of compound-free lectin
injections and converted to inhibition percent values. In the case of
Langerin inhibition assay, even with parallel functionalization of
the reference surface with non-glycosylated BSA, some interaction
of Langerin with the dextran matrix still remains. Indeed, the
dextran/Man-BSA surface has been considered as a combined
ligand of Langerin ECD (see Supplementary Information for more
details).
The second type of SPR test was used to estimate the
relative compound affinity to the lectins on the basis of their
IC50 values. This was accomplished in the same manner as in
the single point inhibition assay, except that both lectins were
incubated with increasing concentrations (from 0 to 5000 mM)
of the corresponding compounds, the injected sample volumes
were 13 ml, and the resulting binding responses were con-
verted to residual lectin activity values, which were plotted
against concentration values of the compounds. The relative
IC50 values for each compound were determined by fitting
four parameter logistic model (eqn (1)) to the experimental
data.
Experimental
Langerin and DC-SIGN ECD expression and purification
Langerin ECD constructs (comprising residue 68–328) have been
overexpressed using a pET30b derived vector in BL21(DE3) as
described previously.27 The protein was expressed as inclusion
body, refolded and purified to homogeneity in a functional form
as already described.22 DC-SIGN ECD protein (residue 66–404)
has been overexpressed and purified as described previously.5a
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Docking calculations. Docking calculations were performed
using the program GLIDE 4.5.20 The initial setup for the
receptor preparation before docking runs was performed using
Schro¨dinger’s ‘Protein Preparation Wizard’, starting from the
X-ray crystal structure of the DC-SIGN-LeX complex. All crys-
tallographic water molecules, except for W13 and W36, were
deleted, bond orders assigned and hydrogen atoms added. The
assignments of protonation states for basic and acidic residues
were based on the optimization of hydrogen bonding patterns.
The final minimization of the complex was not performed with
the Preparation Wizard default, but the complex was minimized
(500 steps, Truncated Newton Conjugate Gradient method) in
implicit water (GB/SA21 model) using MacroModel29 with the
AMBER* force field (dielectric constant 1, cut off extended,
convergence on gradient with threshold of 0.05). The oxygen
atoms of W13 and W36 as well as the Ca2+ ion were an-
chored to the original position through a harmonic potential
during minimization. At the end of the minimization, the root-
mean-square deviation (RMSD) of all heavy atoms was within
top− bot
y = bot +
slope
⎛
⎜
⎜
⎞
(1)
x
⎟
⎟
⎟
⎟
⎠
1+
⎜
⎜
⎝
IC
50
where y is the percent activity, x is the corresponding concentra-
tion, bot and top are the lowest and the highest values of percent
activity, respectively.
In both type of experiments 5 ml flow rate was used and the
running buffer was 25 mM Tris-HCl, pH 8, 150 mM NaCl,
4 mM CaCl2, and 0.005% of P20 surfactant. All the samples were
prepared in the running buffer.
The stability of the surface during a campaign was evaluated
by DC-SIGN ECD binding capacity as a function of the number
of cycles. The chip surfaces demonstrated a strong stability with
negligible decrease of the binding capacity of only 0.06% per cycle
(see data in the Supplementary Information).
˚
0.34 A of the crystallographic positions. Docking calculations
Modeling
were performed in Standard Precision mode with standard
OPLS-AA(2001)30 force field; non-planar conformations of amide
bonds were penalized, Van der Waals radii were scaled by 0.80
and the partial charge cut off was fixed to 0.15. The shape
and properties of the binding site were mapped onto grids with
dimensions of 36 A (enclosing box) and 14 A (ligand diameter
midpoint box), centered on the ligand in the X-ray structure of
the DC-SIGN-LeX complex. Docking was constrained in the Ca2+
binding site by specifying a reference core corresponding to the
C1–C6 carbon and O3–O4–O5 oxygen atoms of the fucose residue
of the reference ligand in the X-ray structure of the DC-SIGN-LeX
complex: ligands that feature the same core moiety as the reference
ligand are subject to the constraint. The RMSD tolerance for the
Grid analysis. The properties of the protein surface in the
vicinity of the Ca2+ site were determined using the protein crystal
structure derived from the complex DC-SIGN-LeX (pdb code
1SL512) and the program GRID17 (version 22). In particular
the DRY probe and the WATER probe were used to identify
hydrophilic and lipophilic regions of the binding site. The accom-
panying program GREAT was used to check the crystal structure
file and to prepare the file of coordinates in standard PDB format,
which is used as an input for the program GRIN. This program,
which prepares the input for the main program GRID, was used
to automatically assign atom types and charges for every atom
of the protein, using provided standard parameters. Calculations
of the interaction energy between the probe and each atom of the
˚
˚
˚
position of the core was set to 3.5 A. This parameter enforces the
˚
˚
˚
˚
protein were performed on a box (36.6 A ¥ 23.0 A ¥ 16.6 A per side)
fucose moiety to be located within 3.5 A of the fucose residue in the
˚
centered on the protein, with a grid spacing of 0.2 A (NPLA = 5)
reference ligand: ligand poses that do not match this constraint are
screened out. These constraints allow coordination of the Ca2+ ion
by the hydroxy groups OH-2 and OH-3 of fucose, as observed in
several crystal structure of C-type lectins complexed with fucose-
and its value was evaluated at each grid point. A dielectric constant
of 80 was used to simulate a bulk aqueous phase, while a dielectric
constant of 4 was assigned to the interior of the protein.
˚
The output, which consists of an array of interaction energies,
can be visualized as contour surfaces at appropriate energy
levels together with the protein structure. Contours at negative
energy levels delineate regions of attraction between probe and
protein, whereas positive energy levels define the surface of the
protein. Visual inspection of the contour surfaces superimposed
on the active site of DC-SIGN enabled the identification of the
most favored hydrophilic and lipophilic regions, facilitating the
interpretation of protein–ligand interaction. Moreover interaction
energy values between the WATER probe and the protein were
used to identify the important structural water molecules out of
all the crystallized water molecules found in the X-ray structure.
An arbitrary cut-off of -10.42 kcal mol-1 (the most negative
energy value was -14.97 kcal mol-1) was chosen to detect the most
favorable hot spots for a water molecule. This cut off allowed us to
identify two important structural water molecules, corresponding
to W13 and W36 in the crystal structure of DC-SIGN-LeX
complex (1SL5). The DC-SIGN crystal structure including these
two water molecules was subsequently used for additional docking
runs.
containing ligands. The RMSD tolerance of 3.5 A was selected
to allow docking poses to explore both possible binding modes
of the vicinal diol. Indeed, crystallographic and NMR data on
DC-SIGN show that the monosaccharide moiety of ligands can
coordinate the Ca2+ ion with the vicinal hydroxyl groups in two
possible orientations, differing by a 180◦ rotation. The quality of
this docking protocol was validated by re-docking the LeX ligand
in the DC-SIGN-LeX complex, which yielded a ligand pose that
could be superimposed with crystalline Le with RMSD of 1.71 A
X
˚
˚
(0.31 A for the fucose residue).
Synthesis
Solvents were dried by standard procedures: dichloromethane,
methanol, N,N-diisopropylethylamine and triethylamine were
dried over calcium hydride, chloroform and pyridine were dried
over activated molecular sieves. Reactions requiring anhydrous
conditions were performed under nitrogen. 1H, 13C and 31P-
NMR spectra were recorded at 400 MHz on a Bruker AVANCE-
400 instrument. Chemical shifts (d) for H and 13C spectra are
1
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expressed in ppm relative to internal Me4Si as standard. Signals
are abbreviated as s, singlet; bs, broad singlet; d, doublet; t, triplet;
q, quartet; m, multiplet. Mass spectra were obtained with a Bruker
ion-trap Esquire 3000 apparatus (ESI ionization) or an Autospec
Fission Instrument (FAB ionization). HRMS (FT-ICR, ESI) were
obtained with an Apex II instrument. Thin layer chromatography
(TLC) was carried out with pre-coated Merck F254 silica gel plates.
Flash chromatography (FC) was carried out with Macherey-
Nagel silica gel 60 (230–400 mesh). The libraries were synthesized
through the common approach shown in Scheme 1.14 All synthetic
schemes and procedures, the synthesis and characterization of new
intermediates, the synthesis and characterization of the full library
23.8 (CCy4 or CCy5), 24.2 (CCy4 or CCy5), 27.8 (CCy6), 30.4 (CCy3), 46.2
(CCy1), 50.2 (CCy2), 68.2 (CF2), 68.8 (CF5), 71.7 (CF3), 73.3 (CF4),
78.4 (CF1), 115.3 (Ar), 119.4 (Ar), 119.7 (Ar), 130.8 (Ar), 137.5
(Cquart.Ar.), 158.9 (Cquart.Ar.), 170.0 (NHCO), 178.0 (NHCO). HRMS
(FT-ICR, ESI): m/z calcd for C20H28N2O7: 431.17887 [M + Na]+;
found: 431.17948. [a]D -53.1 (c 0.25, MeOH)
N-[(1R,2S)-2-(3,5-Dihydroxybenzamido)cyclohexanecarboxyl]-a-
L-fucopyranosylamine (10c)
The crude hydrogenation product of 28 (see Supple-
mentary Information–SI-Scheme 3) was coupled with 3,5-
dihydroxybenzoic acid using the HBTU general procedure (see
Supplementary Information) and the product was purified by
flash chromatography (7 : 3 ethyl acetate : n-hexane, Rf 0.43). Yield:
17 mg (37%). Zemplen deprotection and flash chromatography
(85 : 15 chloroform : methanol, Rf 0.14) afforded 10c. Yield: 10 mg
1
of ligands (3, 9, 11 and 12) as well as their H- and 13C-NMR
spectra are collected in the Supplementary Information. Below we
report the characterization of compounds 10a–d (from (1R,2S)-
2-aminocyclohexanecarboxylic acid). Compounds 3a and 5 were
previously described.14
1
(91%). H-NMR (400 MHz, CD3OD): d (ppm) = 1.19 (d, 3H,
N-((1R,2S)-2-Acetamido-cyclohexanecarboxyl)-a-L-fucopyrano-
sylamine (10a)
J5-6 = 6.5 Hz, HF6), 1.42–1.55 (m, 2H, HCy4ax and HCy5ax), 1.61–1.73
(m, 4H, HC6ax, HCy3ax, HCy4eq and HCyeq), 1.92–2.02 (m, 1H, HCy6eq),
2.16–2.22 (m, 1H, HCy3eq), 2.85–2.90 (m, 1H, HCy1), 3.64–3.66 (m,
1H, HF4), 3.77 (dd, J3-4 = 3.3 Hz, J2-3 = 10.3 Hz, 1H, HF3), 3.79
The crude hydrogenation product of 28 (see Supplementary
Information–SI-Scheme 3) was used in the general acetylation
method (see Supplementary Information). The product was puri-
fied by flash chromatography on silica gel (AcOEt, Rf 0.29). Yield:
27 mg (82%). Zemplen deprotection and flash chromatography
(85 : 15 CHCl3 : MeOH, Rf 0.18) afforded 10a. Yield: 13 mg (65%).
(q, J5-6 = 6.5 Hz, 1H, HF5), 3.95 (dd, 1H, J1-2 = 5.6 Hz, J2-3
=
10.3 Hz, HF2), 4.25–4.30 (m, 1H, HCy2), 5.55 (d, 1H, J1-2 = 5.6 Hz,
HF1) 6.39 (bs, 1H, Ar), 6.68 (bs, 2H, Ar). 13C-NMR (100 MHz,
CD3OD): d (ppm) = 17.0 (CF6), 23.8 (CCy4 or CCy5), 24.1 (CCy4 or
CCy5), 27.9 (CCy6), 30.4 (CCy3), 46.2 (CCy1), 50.2 (CCy2), 68.2 (CF2),
68.8 (CF3 or CF5), 71.7 (CF3 or CF5), 73.3 (CF4), 78.4 (CF1), 106.8
(Ar), 106.8 (Ar), 138.1 (Cquart., Ar.), 160.0 (Cquart., Ar.), 170.0
(NHCO), 178.0 (NHCO). HRMS (FT-ICR, ESI): m/z calcd for
C20H28N2O8: 447.17379 [M + Na]+; found: 447.17407. [a]D -65.7
(c 0.20, MeOH)
1H-NMR (400 MHz, CD3OD): d (ppm) = 1.18 (d, 3H, J5-6
=
6.5 Hz, HF6), 1.32–1.50 (m, 2H, HCy4ax and HCy5ax), 1.55 –1.68 (m,
3H, HCy6ax, HC3ax and HC4eq or HC5eq), 1.73–1.78 (m, 1H, HCy4eq or
HCy5eq), 1.83–1.92 (m, 1H, HCy3eq and HC6eq), 1.94 (s, 3H, Ac-Me),
2.71–2.75 (m, 1H, HCy1), 3.63–3.66 (m, 1H, HF4), 3.74 (dd, J3-4
=
3.3 Hz, J3-2 = 10.3 Hz, 1H, HF3), 3.79 (q, J5-6 = 6.5 Hz, 1H, HF5),
3.94 (dd, J1-2 = 5.6 Hz, J2-3 = 10.2 Hz, 1H, HF2), 4.18–4.25 (m,
1H, HCy2), 5.51 (d, 1H, J1-2 = 5.6 Hz, HF1). 13C-NMR (100 MHz,
CD3OD): d (ppm) = 17.0 (CF6), 23.0 (Ac-Me), 23.6 (CCy4 or CCy5),
24.2 (CCy4 or CCy5), 26.8 (CCy6), 30.7 (CCy3), 40.3 (CCy1), 46.4 (CCy2),
68.3 (CF2), 68.8 (CF5), 71.8 (CF3), 73.3 (CF4), 78.3 (CF1), 173.1
(NHCO), 177.5 (NHCO). HRMS (FT-ICR, ESI): m/z calcd for
C15H26N2O6: 353.16831 [M + Na]+; found: 353.16838. [a]D -77.5
(c 0.35, EtOH)
N-[(1R,2S)-2-(3-Pyridinecarboxamido)cyclohexanecarboxyl]-a-
L-fucopyranosylamine (10d)
The crude hydrogenation product of 28 (see Supplementary
Information–SI-Scheme 3) was coupled with nicotinic acid us-
ing the HBTU general procedure (see Supplementary Infor-
mation) and the product was purified by flash chromatogra-
phy (97 : 3 ethyl acetate : triethyl amine, Rf 0.17). Yield: 19 mg
(50%) Zemplen deprotection and flash chromatography (85 : 15
chloroform : methanol, Rf 0.17) afforded 10d. Yield: 6 mg (43%).
1H-NMR (400 MHz, CD3OD): d (ppm) = 1.19 (d, 3H, J5-6 = 6.5 Hz,
N-[(1R,2S)-2-(3-Hydroxybenzamido)cyclohexanecarboxyl]-a-L-
fucopyranosylamine (10b)
The crude hydrogenation product of 28 (see Supplementary
Information–SI-Scheme 3) was coupled with 3-hydroxybenzoic
acid using the HBTU general procedure (see Supplementary
Information) and the product was purified by flash chromatog-
raphy (6 : 4 AcOEt : petroleum ether, Rf 0.38). Yield: 13 mg
(31%). Zemplen deprotection and flash chromatography (85 : 15
chloroform : methanol, Rf 0.17) afforded 10b. Yield: 8 mg (89%).
1H-NMR (400 MHz, CD3OD): d (ppm) = 1.18 (d, 3H, J5-6 = 6.5 Hz,
HF6), 1.43–1.55 (m, 2H, HCy4ax and HCy5ax), 1.67–1.78 (m, 4H, HCy6ax,
HCy3ax, HCy4eq and HC5eq), 1.94–2.04 (m, 1H, HCy3eq), 2.14–2.21 (m,
1H, HCy6eq), 2.88–2.92 (m, 1H, HCy1), 3.63–3.66 (m, 1H, HF4), 3.74
(dd, J3-4 = 3.4 Hz, J2-3 = 10.3 Hz, 1H, HF3), 3.78 (q, J5-6 = 6.5 Hz,
1H, HF5), 3.93 (dd, 1H, J1-2 = 5.6 Hz, J2-3 = 10.2 Hz, HF2), 4.37–4.42
(m, 1H, HCy2), 5.53 (d, 1H, J1-2 = 5.6 Hz, HF1), 7.50–7.53 (m, 1H,
HAr5), 8.20 (d, 1H, JAr5-JAr6 = 8.0 Hz, HAr6), 8.65 (d, 1H, JAr4-JAr5
=
4.5 Hz, HAr4), 8.92 (s, 1H, HAr2). 13C-NMR (100 MHz, CD3OD):
d (ppm) = 17.0 (CF6), 23.7 (CCy4 and CCy5), 24.1 (CCy3), 27.4 (CCy6),
30.4 (CCy1), 50.4 (CCy2), 68.2 (CF2), 68.8 (CF5), 71.7 (CF3), 73.3 (CF4),
78.3 (CF1), 125.1 (CAr5), 132.7 (Cquart.Ar), 137.4 (CAr6), 149.5 (CAr2),
152.5 (CAr4), 167.95 (NHCO), 177.76 (NHCO). HRMS (FT-ICR,
ESI): m/z calcd for C19H27N3O6: 416.17921 [M + Na]+; found:
416.17934. [a]D -81.9 (c 0.15, MeOH)
HF6), 1.42–1.55 (m, 2H, HCy4ax and HCy5ax), 1.61–1.76 (m, 4H, HCy6ax
,
HCy3ax, HCy4eq and HCy5eq), 1.94–2.02 (m, 1H, HCy6eq), 2.16–2.24 (m,
1H, HCy3eq), 2.86–2.91 (m, 1H, HCy1), 3.63–3.66 (m, 1H, HF4), 3.74–
3.80 (m, 2H, HF5 and HF3), 3.91–3.95 (m, 1H, HF2), 4.28–4.33 (m,
1H, HCy2), 5.52–5.54 (m, 1H, HF1) 6.90–6.95 (m, 1H, Ar), 7.18–7.28
(m, 3H, Ar). 13C-NMR (100 MHz, CD3OD): d (ppm) = 17.0 (CF6),
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Acknowledgements
This work was supported by the FIRB program CHEM-
PROFARMANET (RBPR05NWWC), the Marie Curie ITN
FP7 project CARMUSYS (PITN-GA-2008-213592) and Comune
di Milano (Convenzione 55/2008). Exact mass were obtained
from CIGA (Centro interdipartimentale grandi apparecchiature,
Universita` degli Studi di Milano).
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©