Synthesis of a selective inhibitor of a fucose binding bacterial lectin from Burkholderia ambifaria

Article abstract of DOI:10.1039/c3ob40520f

Burkholderia ambifaria is a bacterium member of the Burkholderia cepacia complex (BCC), a closely related group of Gram-negative bacteria responsible for "cepacia syndrome" in immunocompromised patients. B. ambifaria produces BambL, a fucose-binding lecti

Full text of DOI:10.1039/c3ob40520f

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Biomolecular Chemistry
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Synthesis of a selective inhibitor of a fucose binding
bacterial lectin from Burkholderia ambifaria†
Cite this: Org. Biomol. Chem., 2013, 11,
4086
Barbara Richichi,*a Anne Imberty,b Emilie Gillon,b Rosa Bosco,a Ieva Sutkeviciute,c,d,e
Franck Fieschic,d,e and Cristina Nativia
Burkholderia ambifaria is a bacterium member of the Burkholderia cepacia complex (BCC), a closely
related group of Gram-negative bacteria responsible for “cepacia syndrome” in immunocompromised
patients. B. ambifaria produces BambL, a fucose-binding lectin that displays fine specificity to human
fucosylated epitopes. Here, we report the first example of a synthetic ligand able to selectively bind, in
the micromolar range, the pathogen-lectin BambL. The synthetic routes for the preparation of the α con-
formationally constrained fucoside are described, focusing on a totally diastereoselective inverse electron
demand [4 + 2] Diels–Alder reaction. Isothermal titration calorimetry (ITC) demonstrated that this com-
pound binds to the pathogen-associated lectin BambL with an affinity comparable to that of natural
fucose-containing oligosaccharides. No binding was observed by LecB, a fucose-binding lectin from Pseu-
domonas aeruginosa, and the differences in affinity between the two lectins could be rationalized by
modeling. Furthermore, SPR analyses showed that this fucomimetic does not bind to the human fucose-
binding lectin DC-SIGN, thus supporting the selective binding profile towards B. ambifaria lectin.
Received 14th March 2013,
Accepted 29th April 2013
DOI: 10.1039/c3ob40520f
www.rsc.org/obc
Influenza virus uses hemagglutinin3a to bind to sialic acid
containing glycolipids inducing infection.
Introduction
Lectins1 are sugar-binding proteins that play multifaceted
Similarly, toxins7 produced by some bacteria of the Clostri-
roles in a large number of physiological as well as pathological dium group are able to bind, through a lectin domain, to
recognition phenomena. More specifically, animal lectins have gangliosides present on neurons, resulting in tetanus and
been implicated in direct first-line defense against pathogens, botulism diseases. Pathogen-associated lectins have so far
cell trafficking, immune regulation and prevention of auto- been considered as an attracting target for the development of
immunity.2 They also mediate cell–cell adhesion, cell differen- anti-adhesive drugs,8 able to bind in a selective way to the CDR
tiation, and tissue remodeling, and are involved in the of lectins. Such drugs must competitively interfere with the
clearance of glycoproteins. In contrast, lectins are produced by recognition processes between carbohydrates, expressed on
pathogens3 as a means of anchoring on eukaryotic cell surface the host cell surface, and pathogen lectins, fighting infections.
during the first step of infection. The ability of lectins to dis- In this framework, multivalent glycoconjugates like glyconano-
tinguish among the oligosaccharides expressed on the cell particles, glycodendrimers and glycoclusters have been
surface is related to their specific carbohydrate-recognition exploited as antimicrobial agents and the positive influence of
domain (CDR). For example, LecA4 (PA-IL) and LecB5 (PA-IIL), multivalency on the affinity to lectins has been clearly demon-
respectively specific for galactose and fucose, are involved in strated.8 Furthermore, the influence of the glycoconjugate topo-
the early step of the infection process, virulence and biofilm logy on binding to lectins has been also studied by titration
formation of the opportunistic pathogen Pseudomonas aeruginosa.6 microcalorimetry and surface plasmon resonance.9
Conversely, the selectivity in the binding profile of glyco-
conjugates towards different lectins has been less investigated.
aDepartment of Chemistry, University of Florence, via della Lastruccia, 13,
In this context, in a recent paper, the binding ability of a series
50019 Sesto F.no (FI), Italy. E-mail: barbara.richichi@unifi.it; Fax: +390554573570;
of multivalent glycoconjugates vs. lectins, with different archi-
Tel: +390554573492
bCERMAV-CNRS (Affiliated to University of Grenoble and ICMG), BP 53,
tectures and binding-site topologies, has been exploited, focus-
ing on the influence of the topology of the multivalent systems
on the selectivity.10 Furthermore, the group of Ungaro11 has
also demonstrated that the conformation properties as well as
the shape and the valency of calyx[n]arene systems induce
selectivity in binding to different galectin subgroups.
38041 Grenoble, France
cUniv. Grenoble Alpes, Institut de Biologie Structurale (IBS), 38000 Grenoble, France
dCNRS, IBS, 38000 Grenoble, France
eCEA, IBS, 38000 Grenoble, France
†Electronic supplementary information (ESI) available: 1D and 2D NMR of
compounds 7 and 1. See DOI: 10.1039/c3ob40520f
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Fucose is a key monosaccharide on human tissue since it is
exposed on human oligosaccharide epitopes such as ABO and
Results and discussion
Lewis antigens.12 Fucose-binding bacterial lectins have been The synthesis of α-O-fucosyl derivative 1 relies on a totally dia-
identified in P. aeruginosa and bacteria from the Burkholderia stereoselective inverse electron demand [4 + 2] Diels–Alder
cepacia complex (BCC). LecB from P. aeruginosa is a tetrameric reaction between the O-thioquinone 2 and the protected glycal
protein that displays an unusually strong micromolar affinity 3 (Scheme 1).
to L-fucose in a tight binding site which requires two Ca2+
The O-thioquinone 219 (Scheme 1) is a versatile compound
ions.5a BambL13 is a fucose-specific lectin from Burkholderia that belongs to a class of electron poor heterodienes, which
ambifaria.14 The BCC species are a closely related group of have been widely investigated as useful intermediates for the
Gram-negative bacteria with dual action as they are able to stereoselective synthesis of O-aryl-2-deoxy glycosides.20
protect plant against fungal infections but also cause severe
Thione 2 can be easily prepared on the basis of the known
respiratory infections in patients with cystic fibrosis (CF). reactivity of phthalimidesulfenyl chloride 4 (Scheme 2) with
BambL associates as a trimer with two fucose-binding sites per electron rich aromatic substrates.21 Indeed, the activated
monomer, generating therefore a hexavalent β-propeller struc- hydroxyl arene 5 (Scheme 2) was treated with phthalimidesul-
ture. It is able to recognize a wide spectrum of human fucosy- fenyl chloride 4 in chloroform at 45 °C for 24 h to give the
lated epitopes with high affinity for small fucosylated glycans, corresponding monosulfenyl derivative 6 (50%). Base treat-
as demonstrated by microcalorimetry (Kd < 1 μM) data.13 In ment of the sulfenyl derivative 6 (pyridine, rt) generated the
addition, fucose is recognized by the dendritic cell specific highly reactive O-thioquinone 2 which was trapped in situ with
ICAM-3 grabbing non-integrin, more commonly known as glycal 3 to form the corresponding cycloadduct 7 diastereo-
DC-SIGN lectin.15 DC-SIGN belongs to the class of C-type selectively (Scheme 1).
lectins, a large family of Ca2+ dependent lectins that play rele-
As reported, glycals have successfully been employed as
vant roles in recognition phenomena. DC-SIGN lectin is electron-rich dienophiles in highly selective Diels–Alder reac-
involved as a receptor in specific pathogens uptake16 and is tions.22 As a matter of fact, this reaction, which takes place
able to bind to human-mimicking fucosylated structures such under mild conditions (room temperature or 40–60 °C), has
as the Lewis antigens expressed at the surface of Schisto- unique features and afforded a large class of diastereomeri-
soma17 or synthetic Lewis X glycomimetics.18
In this context, in view of the development of potent inhibi- vant biological properties.23
cally pure glyco-condensed derivatives, some of them with rele-
tors, it is of great importance to design sugar-based derivatives
The electron-rich dienophile 3 (Scheme 3) is a fucal deriva-
able to selectively bind to specific pathogen-associated lectins tive easily prepared starting from the commercially available
with the aim of fighting bacterial infections without interfer- peracetyl fucal 824 which was deprotected under mild basic
ing with human lectins involved in physiological processes.
We present here the stereoselective synthesis of the new
fucose-based glycomimetic 1 (Fig. 1) selectively recognized by
BambL and not by other lectins. The synthetic route we develo-
ped capitalizes on an unconventional strategy to obtain, in few
steps, the α-O-fucosyl derivative 1 as a single diastereoisomer.
The binding affinity of 1 to BambL was evaluated in a
hemagglutination inhibition assay (HIA) monitoring the inhi-
bition properties of BambL-induced agglutination of rabbit
erythrocytes in comparison to α-O-methyl-fucopyranoside
(αMeFuc) used as a positive control. The corresponding dis-
sociation constant and thermodynamic parameters were
measured by isothermal titration calorimetry (ITC). The selecti-
vity of ligand 1 was evaluated by assessing the binding
affinities of 1 vs. different fucose binding lectins, and in par-
ticular, we report here binding studies of 1 vs. the pathogen
LecB,5 from P. aeruginosa, and vs. the human DC-SIGN,15 an
Scheme 1 Synthesis of α-O-fucosyl derivative 1.
example of widespread lectin.
Fig. 1 Structure of fucosyl derivative 1.
Scheme 2 Synthesis of sulfenyl derivative 6.
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ligand αMeFuc (positive control) were in the micromolar
range for both lectins (Table 1).
To confirm the data obtained with HIA, isothermal titration
calorimetry (ITC) measurements were run. Dissociation con-
stants and thermodynamic parameters were determined by
adding the ligand to the solution of lectin (see the Experi-
mental section). The ITC plots obtained from the titrations of
BambL with 1 and with the control αMeFuc are displayed in
Fig. 2. The stoichiometry of BambL/αMeFuc (Table 1 and
Fig. 2A) was closed to 2 (n = 1.7, i.e. 15% of inactive protein),
which is consistent with the presence of two fucose binding
sites per monomer in the crystal structure.13 The measured
dissociation constants (Kd) confirmed the submicromolar
binding constant of αMeFuc13 for BambL (Table 1).
Scheme 3 Synthesis of electron-rich dienophile 3.
conditions (K2CO3) to afford the corresponding L-fucal 9 in
high yield (95%).
The protection of the hydroxyl groups at C-3 and C-4 of 9
(Scheme 3) was accomplished in good yield (62%) reacting 9
with di-tert-butylsilyl bis(trifluoro-methanesulfonate) under
very mild conditions (0 °C, 1 h).20c,25 The cycloaddition reac-
tion was performed in chloroform at 60 °C in the presence of
pyridine (10 eq.) and, as expected,22 the reaction was totally
chemo- and regioselective. Cycloadduct 7 (Scheme 1), formed
from the attack of the O-thioquinone 2 on the top face of
glycal 3, was formed as a pure α diastereoisomer (76%).
After the removal of the silylidene protective group on 7,
with a freshly prepared solution of TBAHF (1 M in THF, 8
eq.),26 the cycloadduct was acetylated under standard con-
ditions (Ac2O, Py, rt) to obtain 10 in 64% yield (calculated over
two steps) (Scheme 1). Deacetylation of 10 under Zemplen’s
conditions (11, 91%), followed by deprotection of the car-
boxylic residue of 11 with lithium hydroxide (1 M in H2O,
4 eq.) in THF as a solvent, provided the desired α-O-fucosyl
derivative 1 (Scheme 1) in high yield (94%).
The titration curve obtained with compound 1 showed
some differences with the one obtained with αMeFuc. While
the stoichiometry is still close to 2, it was not possible to fit
the titration curve with a one site model. The inclusion of an
The binding affinity of 1 towards the fucose-binding lectins
BambL13 and LecB5 was assessed using two different tech-
niques: the hemagglutination inhibition assay (HIA) and iso-
thermal titration calorimetry (ITC). HIA is a simple, sensitive
and versatile screening tool usually used for detecting lectin
activity. It easily reproduces the mode of binding of glyco-
conjugates to lectins in biological systems and measures the
minimum inhibitory concentration (MIC) of drugs able to
inhibit lectin-induced agglutination of rabbit erythrocytes.
The HIA measurements, as shown in Table 1, displayed a
selective binding of 1 for BambL. Indeed, the minimum
inhibitory concentration of 1 is in the low millimolar range
towards BambL, while no inhibition vs. LecB-induced aggluti-
nation was obtained under the same experimental conditions
(Table 1). In contrast, the MIC values measured for the known
Fig. 2 Thermograms (top) and titration curves (bottom) obtained from the
titration of BambL (48.6 μM) by (A) αMeFuc and (B) compound 1 (both 1 mM).
Fitting procedure was performed using a one site model (solid line for LecB and
BambL) or with a two-site model (dotted line for BambL). (C) Absence of titra-
tion for LecB (46.4 μM) with the injection of compound 1 (0.5 mM).
Table 1 Hemagglutination and titration calorimetry data for 1 and αMeFuc
HIA
ITC
Ligands
MIC (mM)
N
ΔH (kJ mol−1
)
−TΔS (kJ mol−1
)
ΔG (kJ mol−1
)
Kd (μM)
BambL
1a
αMeFuc
LecB
2.9
0.05
1.71
1.71 0.02
−28.9 0.1
−50.1 0.1
−4.3
−33.2
−34.3
1.54 0.18
0.99 0.07
15.8
1
nbc
0.01
nb
1.1
nb
−41.3
nb
4.9
nb
−36.4
nb
0.43
αMeFucb
a Fitted with a second weak affinity binding site (Kd = 0.4 mM; ΔH = −50 kJ mol−1) with the stoichiometry of both sites fixed at 1.71. b From ref.
27. c nb: no binding.
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additional site with low affinity (Kd = 0.5 mM) was included in using surface plasmon resonance (SPR) competition assays fol-
the fitting model. Such an additional site could be due to lowing an optimized protocol widely used for weak ligands.18a,b
contact of the aromatic ring with hydrophobic patches on the This protocol allows the affinity evaluation of a ligand with
protein surface. Under these conditions, the affinity of BambL respect to another on the basis of an IC50 determination. In
for 1 (Kd = 1.5 μM) is in the same range as for αMeFuc (Kd
=
our case, we determined the ability of the compounds 1 and
1 μM). Some differences are observed for the thermodynamics L-fucose to inhibit the DC-SIGN binding to mannosylated
contribution. The enthalpy of binding of 1 is twice smaller bovine serum albumin (Man-BSA).
than that of αMeFuc, which is counterbalanced by a favorable
entropy of binding, most likely correlated to the rigidity due to average of 12 glycosylation sites displaying the Manα1–3
the additional ring.
The commercially available Man-BSA used contains an
[Manα1–6]Man branched trisaccharide. Man-BSA was co-
The reported affinity of LecB for αMeFuc (Kd = 0.55 mM)27 valently attached to a carboxymethyl dextran functionalized
is in the micromolar range; a similar affinity was observed for gold SPR sensor chip S-CM4 following a standard protocol (see
BambL. Conversely, no binding was observed when compound the Experimental section). Inhibition studies were performed
1 was titrating vs. LecB (Fig. 2C). Therefore, both HIA and using the extracellular domain (ECD) of DC-SIGN (20 μM)
ITC data indicated selectivity in the binding of the fucose injected in the presence of increasing concentration of the
mimetic 1.
ligands. The sensorgrams obtained from the competition
In order to rationalize the observed difference, a three- assay of DC-SIGN with 1 and with L-fucose are displayed in
dimensional model of 1 was built using the Tripos package. Fig. 4. The data obtained confirmed the binding of L-fucose
Due to cyclization constraints, only one main conformer was (IC50 = 1.77 mM) vs. DC-SIGN ECD and clearly showed that the
obtained after minimization, with the exception of the
CH2COOH group that can adopt several orientations. When
docking 1 in the BambL binding site by superimposition with
the fucose residue, the aglycon fit in the crevasse-shaped site,
with no steric conflict with the protein surface (Fig. 3A). The
additional contacts are limited to interaction between the
sulfur atoms and the CH2 group of Gly37 and the NH group of
Ala38. When comparing the location of the aromatic aglycone
with fucosylated oligosaccharides in available crystal structures
of BambL, the aglycon occupies the position of the αGal1–3Gal
disaccharide in the BambL–blood group B complex (Fig. 3B).
In contrast, when 1 is superimposed on fucose in the binding
site of LecB, non-favorable contacts are observed. On the one
hand, the sulfur atom, which is positively charged, would be
sitting too close to one of the two calcium ions. On the other
hand, the CH2COOH group is creating a steric conflict with a
loop encompassing residues 96 to 98.
Capitalizing on these results, we tried to better highlight
the binding profile of 1 extending our study to a human lectin,
DC-SIGN that is also able to bind to fucose. Monovalent
L-fucose has a weak affinity for DC-SIGN’s Ca2+ binding site;
indeed the reported dissociation constant is in the millimolar
range (6 mM).28 Therefore, in order to avoid higher concen-
trations, necessary for ITC measurements, the binding affinity
of 1 with respect to the monovalent L-fucose was evaluated
Fig. 4 Inhibition of DC-SIGN ECD binding to the mannosylated-BSA surface
with (A) L-fucose and (B) 1. DC-SIGN ECD (20 μM) was incubated with (A)
Fig. 3 (A) Model of 1 in the fucose binding pocket of BambL; (B) superimposi-
tion of 1 (salmon: model) and blood group B tetrasaccharide (green: crystal
structure PDB code 3ZW0); (C) model of 1 in the LecB binding site.
L-fucose at 9 increasing concentrations from 2.8 μM to 18.33 μM or (B) compound
1 at 8 increasing concentrations from 3.2 μM to 6937 μM, and co-injected onto
the mannosylated-BSA functionalized surface (1850 RU immobilized).
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fucose mimetic 1 did not hamper the DC-SIGN ECD binding and a resolution of 100 000. Optical rotation measurements
to Man-BSA when the concentration used for 1 reached were carried out with a Jasco DIP-370 polarimeter. Complete
2.32 mM.
signal assignments from 1D and 2D NMR were based on
COSY, HSQC correlations. Elementary analysis experiments on
purified products were performed with an Elementary Analyzer
2400 Series II from Perkin-Elmer.
Conclusion
Material
Here, we have reported on the stereoselective synthesis of the
fucose containing mimetic 1. The mimetic 1 was efficiently Recombinant BambL,13 LecB31 and the extracellular domain of
prepared in a few steps following a powerful diastereoselective DC-SIGN32 were produced from E. coli as described previously.
[4 + 2] Diels–Alder reaction. The binding profile of 1 vs. bio- αMeFuc was purchased from Sigma-Aldrich.
logically relevant lectins was evaluated in isothermal titration
Haemagglutination inhibition assays
calorimetry, hemagglutination inhibition and surface plasmon
resonance assays in comparison to the standard ligand Hemagglutination inhibition assays (HIA) were performed in
L-fucose. HIA and ITC tests demonstrated that 1 binds to the U-shaped 96-well microtitre plates (Nunc). Rabbit erythrocytes
pathogen-associated lectin BambL with an affinity (1, Kd
=
were purchased from Biomérieux and used without further
1.5 μM) comparable to that of the natural ligand (αMeFuc, washing. Erythrocytes were diluted to 4% in a solution of NaCl
Kd = 1 μM). Unexpectedly, 1 did not bind to the fucose binding (150 mM). Lectin solutions of 50 μM were prepared in Tris-HCl
lectin LecB, showing a selective binding profile with respect to 20 mM, NaCl 150 mM and CaCl2 100 μM. The hemagglutina-
the standard ligand (Kd = 0.43 μM for αMeFuc).27
tion unit (HU) was first obtained by the addition of 50 μL of a
The possible binding of 1 for the human lectin DC-SIGN 4% erythrocyte solution to 50 μL aliquots of sequential (two-
was investigated by surface plasmon resonance. SPR analyses fold) lectin dilutions. The mixture was incubated at 25 °C for
showed that the fucose-based mimetic 1 does not inhibit the 60 minutes. The HU was measured as the minimum lectin
DC-SIGN ECD binding to Man-BSA, thus supporting a selective concentration required to observe hemagglutination. For the
binding profile of 1.
following lectin-inhibition assays, lectin concentrations of
In summary, we have reported here the first example of a 16 HU were used. This concentration was 24 μM for BambL
synthetic ligand able to selectively bind, in the micromolar and 50 μM for LecB.
range, the pathogen-lectin BambL. The fucose-based mimetic
Subsequent inhibition assays were then carried out by the
1 is thus a promising candidate for the development of anti- addition of a 12.5 μL lectin solution (at the required concen-
bacterial agents against infection by B. ambifaria. For this tration) to 25 μL of sequential dilutions of glyco-compounds
reason, the synthesis of multivalent systems containing the dissolved in buffer (Tris-HCl 20 mM, NaCl 150 mM, CaCl2
fucose mimetic 1 is actually in progress in order to improve 100 μM buffer containing 2.5% DMSO). These solutions were
the affinity vs. B. ambifaria’s lectin.
then incubated at 25 °C for 2 h and then 12.5 μL of a 4% ery-
Furthermore, BambL and the related lectin from the phyto- throcyte solution was added followed by an additional incu-
pathogenic bacterium Ralstonia solanacearum29 have strong bation at 25 °C for 30 minutes. The minimum inhibitory
sequence and structure similarities with the fucose-binding concentration for each molecule was determined for each
lectin from Aleuria aurantia mushroom, indicating a evolution- duplicate.
ary relationship.30 This sequence family also includes AOL
from Aspergillus oryzae and AFL from Aspergillus fumigatus;
Isothermal titration calorimetry (ITC)
BambL can therefore be considered as a good model for devel- Purified and lyophilized BambL and LecB were dissolved in
oping compounds against infections by Aspergillus fumigatus, buffer (Tris-HCl 20 mM, NaCl 150 mM, CaCl2 100 μM buffer
an opportunistic lung pathogen responsible of severe air-borne containing 2.5% DMSO) and degassed. Carbohydrate ligands
infection in immunocompromised patients.
were dissolved directly into the same buffer degassed, and
placed in the injection syringe. Isothermal titration calori-
metry was performed with a VP-ITC MicroCalorimeter from
MicroCal Incorporated. BambL and LecB were placed in the
1.4478 mL sample cell, at 25 °C, at concentrations of 48.6 μM
Experimental
All solvents were of reagent grade quality and were purchased and 46.4 μM, respectively. Glyco-compounds (1 mM vs. BambL
commercially. All starting materials were purchased commer- and 0.5 mM vs. LecB) were injected by 10 μL steps every
cially and used without further purification. All NMR spectra 300 s. Carbohydrate ligands were also titrated alone against
were recorded on Varian instruments (200 or 400 MHz) and the buffer. Data were fitted with MicroCal Origin 7 software,
Mercury 400 instruments. The NMR spectra were referenced to according to standard procedures. Fitted data yielded the stoi-
a solvent. Mass spectra were recorded on a LCQ-FLEET ion chiometry (n), the association constant (Ka) and the enthalpy
trap from Thermo Fischer. ESI-MS analysis was performed of binding (ΔH). Other thermodynamic parameters (i.e.
both in positive and negative ion modes. HRMS were per- changes in free energy, ΔG, and entropy, ΔS) were calculated
formed on a LTQ-IT-Orbitrap with a spray voltage of 2.10 kV from the equation ΔG = ΔH − TΔS = −RT ln Ka where T is the
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absolute temperature and R = 8.314 J mol−1 K−1. Two or three mp: 52–55 °C; 1H NMR (400 MHz, CDCl3): δ 6.38 (dd, J1,2
=
=
independent titrations were performed for each ligand tested.
6.4 Hz, J1,3 = 2.0 Hz, 1H, H-1), 4.68 (dt, J2,1 = 6.0 Hz, J2,3 = J2,4
2.0 Hz, 1H, H-2), 4.38 (bs, 1H, H-3–H-4), 4.03 (qd, J5,6 = 6.8 Hz,
J5,4 = 0.4 Hz, 1H, H-5), 2.59 (d, J = 8.0 Hz, 1H, OH), 2.29 (d, J =
7.6 Hz, 1H, OH), 1.37 (d, J6,5 = 6.8 Hz, 3H, H-6); 13C NMR
(100 MHz, CDCl3): δ 144.7 (C-1), 103.0 (C-2), 73.2 (C-5), 68.2
(C-3–C-4), 64.9 (C-3–C-4), 16.8 (C-6); ESI-MS m/z: 153.0 [M +
Na]+; elemental analysis found: C, 55.50; H, 7.76. Calcd for
C6H10O3: C, 55.37; H, 7.74%.
Surface plasmon resonance (SPR)
SPR experiments were performed on a Biacore T200 using a
Series S CM4 sensorchip, functionalized using a HBS-P
running buffer at a 5 μL min−1 flow rate. Flow cells (Fc) 1 and
2 were activated as recommended by the manufacturer, and
then Fc2 was functionalized with 60 μg mL−1 of Manα1–3
[Manα1–6]Man-BSA (Dextra) prepared in 10 mM sodium
acetate, pH 4. The remaining activated groups of both flow
cells were blocked with 30 μL of 1 M ethanolamine. The two
flow cells were then treated with 10 μL of 10 mM HCl and
20 μL of 50 mM EDTA to expose the surface to the regeneration
protocol. After these steps 1850 RU of Man-BSA was immobi-
lized on Fc2.
Synthesis of compound 3. To an ice-cooled solution of 9
(0.140 g, 1.08 mmol) in pyridine (5.0 mL) di-tert-butylsilyl bis-
(trifluoro-methanesulfonate) (0.521 mg, 0.383 mL, 1.18 mmol)
was added. The mixture was stirred for 1 h and then warmed
to rt, diluted with CH2Cl2 (100 mL) and washed with a satu-
rated solution of NH4Cl (2 × 10 mL), and brine (2 × 10 mL).
The organic phase was dried over Na2SO4 and concentrated to
dryness to give a crude which was purified by flash column
chromatography on silica gel (AcOEt–petroleum ether, 1 : 6) to
give 3 (0.180 g, 62%) as a yellow oil. [α]2D5 −12.37 (c 1.3 in
CH2Cl2); 1H NMR (400 MHz, CDCl3): δ 6.39 (d, J1,2 = 6.4 Hz,
1H, H-1), 4.79 (ddd, J2,1 = 6.0 Hz, J2,3 = 2.8 Hz, J2,4 = 1.2 Hz, 1H,
H-2) 4.75 (dd, J3,2 = 6.8 Hz, J3,4 = 2.8 Hz, 1H, H-3), 4.12–4.10
(m, 1H, H-4), 3.9 (qd, J5,6 = 6.4 Hz, J5,4 = 1.2 Hz, 1H, H-5), 1.41
(d, J6,5 = 6.8 Hz, 3H, H-6), 1.05 (s, 18H, (CH3)3C); 13C NMR
(100 MHz, CDCl3): δ 146.1 (C-1), 103.8 (C-2), 74.4 (C-4), 73.5
(C-5), 67.8 (C-3), 27.5 ((CH3)3C), 27.1 ((CH3)3C), 21.9 ((CH3)3C),
19.8 ((CH3)3C), 17.0 (C-6); ESI-MS m/z: 271.16 [M + H]+;
elemental analysis found: C, 62.16; H, 9.72. Calcd for
C16H26O3Si: C, 62.18; H, 9.69%.
Synthesis of compound 6. To an ice-cooled solution of 5
(0.518 g, 3.12 mmol) in CHCl3 (4.0 mL) PhtNSCl (0.731 mg,
3.43 mmol) was added. The mixture was warmed at 45 °C and
stirred for 24 h. After this, the reaction mixture was cooled to
0 °C, diluted with CHCl3 (10 mL) and filtered through a pad of
celite. Evaporation of the solvent gave a crude, which was puri-
fied by flash column chromatography on silica gel (CHCl3–
MeOH 100 : 1) to give 6 (0.500 g, 50%) as a white solid. mp:
144–146 °C; 1H NMR (200 MHz, CDCl3): δ 8.33 (s, 1H, OH),
7.93–7.90 (m, 2H, PhthN), 7.95–7.75 (m, 3H, PhthN, H-a),
7.33–7.27 (B part of an ABC system, JBC = 5.6 Hz, JBA = 1.2 Hz,
For inhibition studies, 20 μM of DC-SIGN ECD mixed with
increasing concentrations of inhibiting compounds were pre-
pared in a running buffer composed of 25 mM Tris, pH 8,
150 mM NaCl, 4 mM CaCl2, 0.005% P20 surfactant, and 13 μL
of each sample was injected onto the surfaces at 5 μL min−1
.
Concentrations of compound 1 ranged from 3.2 μM to
6937 μM, and from 2.8 μM to 18.33 mM for L-fucose. The
resulting sensorgrams were reference surface corrected.
Rhi ꢀ Rlo
1 þ
y ¼ Rhi
ꢀ
ð1Þ
ꢀ
ꢁ
A2
Conc
A1
ꢂ
ꢃ
1=A2
Rhi ꢀ Rlo
IC50 ¼ A1
ꢀ 1
ð2Þ
Rhi ꢀ 50
The DC-SIGN ECD binding responses were extracted from
the sensorgrams, converted to percent residual activity values
(y) with respect to lectin alone binding, and plotted against
the corresponding compound concentration. The 4-parameter
logistic model (eqn (1)) was fitted to the plots, and the IC50
value for fucose was calculated using the value of fitted para-
meters (Rhi, Rlo, A1, and A2) and eqn (2).
Molecular modeling
A model of 1 was built from fucose using the graphical editor 1H, H-b), 7.01–6.98 (C part of an ABC system, JCB = 5.6 Hz, 1H,
of Sybyl Software (Tripos, Saint Louis). Partial charges were cal- H-c), 3.68 (s, 3H, CO2CH3), 3.53 (s, 2H, CH2CO2CH3); 13C NMR
culated with MNDO and the geometry was optimized using (100 MHz, CDCl3): δ 171.7, 168.4, 158.1, 138.7, 135.8, 134.8,
Tripos software. The resulting molecules were placed in the 131.8, 126.1, 124.2, 118.2, 117.1, 52.0, 39.6; elemental analysis
binding site of BambL complexed with blood group B (PDB found: C, 59.50; H, 3.84; N 4.02. Calcd for C17H13NO5S: C,
code 3ZW0) tetrasaccharide by fitting the fucose residues. The 59.47; H, 3.82; N, 4.08%.
same fitting procedure was performed in the binding site of
Synthesis of compound 7. To an ice-cooled solution of 3
LecB complexed with fucose (PDB code 1GZT). All graphical (0.150 g, 0.55 mmol) in CHCl3 (4.0 mL), 6 (0.170 mg,
representations have been performed with PyMol software 0.495 mmol) and pyridine (0.561 mg, 0.515 mL, 5.50 mmol)
(Schrödinger).
were added. The mixture was warmed at 60 °C and stirred for
Synthesis of compound 9. To a stirred solution of 8 24 h. After this, 6 (0.151 mg, 0.44 mmol) was added and the
(0.350 g, 1.60 mmol) in MeOH (8.0 mL) K2CO3 (0.065 g, mixture was stirred at 60 °C for 24 h. The reaction mixture was
0.480 mmol) was added. The mixture was stirred at rt for 1 h cooled to 0 °C, diluted with CHCl3 (10 mL) and filtered
and then concentrated to dryness. The crude was purified by through a pad of celite. Evaporation of the solvent gave a
flash column chromatography on silica gel (AcOEt) to give 9 crude, which was purified by flash column chromatography on
(0.196 g, 95%) as a white solid. [α]2D5 +0.93 (c 0.48 in CH3OH); silica gel (AcOEt–petroleum ether, 1 : 6) to give 7 (0.194 g, 76%)
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as a white solid. [α]2D5 −70.42; mp: 51–54 °C; (c 1.15 in CH2Cl2). (bs 1H, H-4), 3.79 (B part of an ABC system, JBA = 10.0 Hz, JBC
=
1H NMR (400 MHz, CDCl3): δ 7.01–7.00 (A part of an ABC 3.2 Hz, 1H, H-3), 3.7 (s, 3H, CO2CH3), 3.51 (s, 2H,
system, JAB = 1.6 Hz, 1H, H-a), 6.97–6.95 (B part of an ABC, CH2CO2CH3), 3.45 (C part of an ABC system, JBC = 10.4 Hz,
JBC = 8.4 Hz, JBA = 2.0 Hz, 1H, H-b), 6.84–6.82 (C part of an J2,1 = 2.4 Hz, 1H, H-2), 1.4 (d, J6,5 = 6.8 Hz, 3H, H-6); 13C NMR
ABC system, JCB = 8.4 Hz, 1H, H-c), 5.67 (d, J1,2 = 2.4 Hz, 1H, (100 MHz, CDCl3): δ 151.0 (Cq), 127.9 (C-a), 127.7 (C-b), 118.3
H-1), 4.32–4.27 (m, 2H, H-4, H-5), 3.81 (dd, J3,2 = 11.4 Hz, J3,4
2.4 Hz, 1H, H-3), 3.69 (s, 3H, CO2CH3), 3.56 (dd, J2,3 = 11.2 Hz, 52.1 (CO2CH3), 40.1 (CH2CO2CH3), 39.8 (C-2), 16.3 (C-6);
J2,1 = 1.8 Hz, 1H, H-2), 3.51 (s, 2H, CH2CO2CH3), 1.39 (d, J6–5
ESI-MS m/z: 349.17 [M + Na]+; elemental analysis found: C,
6.4 Hz, 3H, H-6), 1.07 (s, 18H, (CH3)3C); 13C NMR (100 MHz, 55.30; H, 5.54. Calcd for C15H18O6S: C, 55.20; H, 5.56%.
CDCl3): δ 171.9 (Cq), 151.0 (Cq), 127.8 (C-a), 127.7 (Cq), 127.6 Synthesis of compound 1. To a stirred solution of 11
=
(C-c), 113.0 (Cq), 94.7 (C-1), 70.7 (C-4), 68.2 (C-5), 67.4 (C-3),
=
(C-b), 118.3 (C-c), 112.9 (Cq), 94.8 (C-1), 73.1 (C-4), 69.8 (C-5), (0.051 g, 0.156 mmol) in THF (7.0 mL), 607 μL of a 1 M solu-
68.1 (C-3), 52.1 (CO2CH3), 40.0 (CH2CO2CH3), 39.9 (C-2), 27.8 tion of LiOH in H2O were added. The mixture was stirred for
((CH3)3C), 27.6 ((CH3)3C), 21.3 ((CH3)3C), 20.7 ((CH3)3C), 17.2 2 h at rt and then a 1 M solution of H3PO4 was added to reach
(C-6); HRMS m/z: calcd for C23H35O6SSi [M + H]+ 467.19181, pH 6.0. Evaporation of the solvent under vacuum gave a crude
found 467.19150.
which was suspended in a mixture of AcOEt–MeOH 1 : 4 and
Synthesis of compound 10. A 1 M solution of TBAHF filtered. Evaporation of the solvent gave 1 (45 mg, 94%) as a
(2.1 mL) in dry THF was added to a flask containing com- white solid. [α]2D5 −93.54 (c 0.31 in CH3OH); mp: 188–191 °C;
pound 7 (0.130 g, 0.278 mmol) and the mixture was warmed at 1H NMR (400 MHz, CD3OD): δ 6.92–6.91 (A part of an ABC
40 °C for 8 h. The mixture was cooled to rt, diluted with AcOEt system, JAB = 2.4 Hz, 1H, H-a), 6.85–6.82 (B part of an ABC
(50 mL) and washed with H2O (2 × 5 mL). The organic phase system, JBC = 8.0 Hz, JBA = 2.0 Hz, 1H, H-b), 6.68–6.66 (C part
was dried over Na2SO4 and concentrated to dryness to give of an ABC system, JCB = 8.4 Hz, 1H, H-c), 5.49 (d, J1,2 = 2.8 Hz,
0.318 g of crude 11. The crude 11 was dissolved in CH2Cl2 1H, H-1), 4.12 (aq, J = 6.4 Hz, 1H, H-5), 3.59 (A part of an ABC
(4.0 mL), and then pyridine (0.212 mL, 2.62 mmol), acetic system, JAB = 3.2 Hz, J4,5 = 1.2 Hz, 1H, H-4), 3.54 (B part of an
anhydride (0.248 mL, 2.62 mmol) and DMAP (0.024 g, ABC system, JBC = 10.8 Hz, JBA = 3.2 Hz, 1H, H-3), 3.36 (s, 2H,
0.2 mmol) were added. The mixture was stirred for 1 h at rt CH2CO2CH3), 3.30 (C part of an ABC system, JCB = 11.2 Hz,
and then diluted with CH2Cl2 (100 mL) and washed with a 3% J2,1 = 3.2 Hz, 1H, H-2), 1.17 (d, J6,5 = 6.4 Hz, 3H, H-6); 13C NMR
solution of HCl (2 × 10 mL), brine (2 × 10 mL) and a saturated (100 MHz, CD3OD): δ 175.5(Cq), 152.5 (Cq), 129.7 (C-a), 128.8
solution of NaHCO3 (2 × 10 mL). The organic phase was dried (Cq), 128.2 (C-b), 118.7 (C-c), 115.9 (Cq), 96.9 (C-1), 73.0 (C-5),
over Na2SO4 and concentrated to dryness to give a crude, 70.0 (C-4), 68.6 (C-3), 41.2 (C-2), 40.9 (CH2COOH), 16.8 (C-6);
which was purified by flash column chromatography on silica HRMS m/z: calcd for C14H16O6NaS [M + Na]+ 335.05598, found
gel (CH2Cl2–acetone 10 : 1) to give 10 (0.073 g, 64% calculated 335.05606.
over two steps) as a glassy solid. [α]2D5 +160.94 (c 0.85 in
1
CH2Cl2); H NMR (400 MHz, CDCl3): δ 6.97–6.95 (m, 2H, H-a,
H-b), 6.85–6.83 (C part of an ABC system, JCB = 8.0 Hz, JCA = 0.8
Hz, 1H, H-c), 5.69 (d, J1,2 = 2.8 Hz, 1H, H-1), 5.30–5.29 (m, 1H,
Acknowledgements
H-4), 3.2 (dd, J3,2 = 12.0 Hz, J3,4 = 3.2 Hz, 1H, H-3), 4.5 (aq, J = B.R. and C.N. thank Ente Cassa di Risparmio di Firenze for
6.8 Hz, 1H, H-5), 3.68 (s, 3H, CO2CH3), 3.58 (dd, J2,3 = 11.8 Hz, financial support. A.I. and E.G. are members of CNRS. The
J2,1 = 2.8 Hz, 1H, H-2), 3.49 (s, 2H, CH2CO2CH3), 2.19 (s, 3H, COST actions CM1102 and BM1003 and the Labex ARCANE
CH3), 2.04 (s, 3H, CH3), 1.23 (d, J6,5 = 6.8 Hz, 3H, H-6); 13C (ANR-11-LABX-003) are thanked for support. F.F. thanks the
NMR (100 MHz, CDCl3): δ 171.9 (Cq), 170.3 (Cq), 169.8 (Cq), Institut Universitaire de France for financial support and the
150.2 (Cq), 128.1 (Cq), 127.3 (C-a–C-b), 127.2 (C-a–C-b), 118.4 Partnership for Structural Biology for access to the SPR plat-
(C-c), 113.9 (Cq), 94.7 (C-1), 70.4 (C-4), 68.1 (C-3), 66.9 (C-5), form. I.S. was supported by the EU ITN Marie Curie program
52.0 (CO2CH3), 40.2 (CH2CO2CH3), 37.1 (C-2), 20.6 (CH3), 16.1 (CARMUSYS grant number 213592).
(C-6); ESI-MS m/z: 433.25 [M + Na]+; elemental analysis found:
C, 55.62; H, 5.44. Calcd for C19H22O8S: C, 55.60; H, 5.40%.
Synthesis of compound 11. To a stirred solution of 10
(0.073 g, 0.178 mmol) in CH2Cl2–MeOH 1 : 2 (3.0 mL), 480 μL
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