Synthesis and characterization of linker-armed fucose-based glycomimetics

Article abstract of DOI:10.1002/ejoc.201300236

Glycomimetic molecules can be used to antagonize the action of carbohydrate-binding proteins (lectins) involved in biological processes of high relevance for human and plant disease. In this paper we describe the derivatization with appropriate linkers of

Full text of DOI:10.1002/ejoc.201300236

FULL PAPER
DOI: 10.1002/ejoc.201300236
Synthesis and Characterization of Linker-Armed Fucose-Based Glycomimetics
Daniela Doknic,[a] Morena Abramo,[a] Ieva Sutkeviciute,[b,c,d] Anika Reinhardt,[e,f]
Cinzia Guzzi,[g] Mark K. Schlegel,[e,f] Donatella Potenza,[a] Pedro M. Nieto,[g]
Franck Fieschi,[b,c,d] Peter H. Seeberger,[e,f] and Anna Bernardi*[a]
Keywords: Carbohydrates / Glycoconjugates / Bioorganic chemistry / Dendrimers
Glycomimetic molecules can be used to antagonize the ac- cose-based anchor allows the mimic to interact efficiently
tion of carbohydrate-binding proteins (lectins) involved in
biological processes of high relevance for human and plant
disease. In this paper we describe the derivatization with ap-
propriate linkers of a previously described glycomimetic con-
taining an α-fucosyl amide anchor that is known to act as
antagonist of the dendritic cell lectin DC-SIGN. Key steps of
the functionalization were the stereoselective epoxidation of
with fucose-binding lectins, the linker could also be ex-
ploited for the synthesis of glycodendrimers, as well as for
the study of ligand interactions with commercially available
lectins by array technology. In particular, a tetravalent fucos-
ylated dendrimer was obtained that displayed a low-micro-
molar range of activity against DC-SIGN. Additionally,
screening of the ligand against lectins with common fucose
an intermediate β-amino-cyclohexene-carboxylic acid deriv- specificity in an array format allowed the lectin from the bac-
ative, followed by regioselective opening with 2-chloro-
ethanol. Introduction of the linker does not alter the DC-
SIGN binding properties of the molecule, as shown by Sur-
face Plasmon Resonance and NMR studies. Whereas the fu-
terium Ralstonia solanacearum (RSL) to be identified as a po-
tential target protein and suggested that even simple glyco-
mimetic structures can attain a significant amount of selec-
tivity among lectins with analogous specificity.
achieved with small molecules mimicking the 3D structure
of oligosaccharide epitopes.[3] In 2008 the Bernardi group
introduced a mimic of the blood group antigen Lewis X
[Fucα-1,4-(Galβ-1,3-)GlcNAc, Figure 1].[4] The fucose
moiety of this branched trisaccharide binds to the Ca2+
present in the binding site of DC-SIGN,[5] a C-type lectin
of dendritic cells that participates in infection processes of
pathogens such as HIV or Ebola.[6] Blocking DC-SIGN af-
fords a way of contrasting the initial phases of the infection
and of preventing sexual transmission of AIDS.[7] With the
aim of blocking that protein, compound 1 (Figure 1) was
synthesized by employing fucose as an anchor and intro-
ducing 2-amino-cyclohexanecarboxylic acid as a central
building block. The galactose fragment of Lewis X, on the
other hand, was substituted by a mimic that helps the mo-
lecular shape to be consistent with the overall conformation
of the trisaccharide. The IC50 value for mimic 1 was deter-
mined by means of surface plasmon resonance (SPR) and
showed a slightly higher affinity for DC-SIGN when com-
pared with Lewis X. Based on docking studies that sug-
gested the interaction of the galactose mimic in 1 with lipo-
philic areas of the protein, a library of fucosyl amides with
the general structure 2 was generated.[8] Apart from the ex-
change of the galactose mimic with predominantly aromatic
systems, the central building block was also varied by ex-
ploiting all possible configurations of 2-amino-cyclohex-
anecarboxylic acid and by introducing the acyclic, commer-
cially available β-alanine. A set of 40 compounds was pre-
Introduction
As one of the major constituents of the biomass, carbo-
hydrates display a broad range of functions in biological
systems. Beside their most prominent role as a source and
storage of energy, they are also involved in cellular com-
munication, proliferation, adhesion, and apoptosis.[1] More-
over, numerous human and plant diseases are promoted by
the interaction of carbohydrate-binding proteins (lectins)
with carbohydrate structures expressed on cell surfaces.[2]
Antagonism of lectin binding can lead to the control of
many important biological processes. Such control can be
[a] Università degli Studi di Milano, Dipartimento di Chimica,
Via Golgi 19, 20133 Milano, Italy
E-mail: anna.bernardi@unimi.it
Homepage: www.unimi.it
[b] Univers. Grenoble Alpes, Institut de Biologie Structurale (IBS),
38000 Grenoble, France
[c] CEA, IBS,
38000 Grenoble, France
[d] CNRS, IBS,
38000 Grenoble, France
[e] Department of Biomolecular Systems, Max Planck Institute of
Colloids and Interfaces,
Am Mühlenberg 1, 14476 Potsdam, Germany
[f] Institute for Chemistry and Biochemistry, Freie Universität
Berlin,
Arnimallee 22, 14195 Berlin, Germany
[g] Instituto de Investigaciones Químicas CSIC-Universidad de
Sevilla,
Américo Vespucio 49, 41092 Seville, Spain
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300236.
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Figure 1. Structure of Lewis X, its mimic 1 and core 2 of a library of molecules.
pared and tested to establish their affinity for the target valent presentation require the presence of appropriate
protein DC-SIGN, but no major improvement over Lewis functional groups at the monomeric unit,[10] which allows
X was achieved. Compound 3 (Figure 2), with an IC50 of the active ligand to be tethered to a polyvalent scaffold.
470 μm, was identified as the most potent inhibitor, and the Herein, we report on the functionalization of 3 and 4 to
synthetically more accessible compound 4 was found to be afford linker-armed molecules that could be used for the
as active as Lewis X (IC50 800 μm).
synthesis of glycodendrimers, as well as for the study of
ligand interactions with commercially available lectins by
glycan array technology (Scheme 1).
Results and Discussion
As previously described,[8] the central building block of
ligand 3 originates from the desymmetrization of tetra-
hydrophthalic anhydride by quinine-promoted meth-
anolysis using quinine as a chiral additive (Scheme 2). In
the following step, carboxylic acid 5 was transformed into
the N-Cbz-protected β-amino acid 6 by the Curtius re-
arrangement in the presence of benzyl alcohol, followed by
hydrolysis of the methyl ester. Coupling of 6 to tri-O-acetyl-
fucosyl azide 8 by using DeShong’s[11] procedure led to the
formation of α-fucosyl amide 9.
The double bond of the cyclohexene scaffold was elabo-
rated at this level because functionalization of synthetic pre-
cursors was detrimental to the DeShong reaction. As de-
scribed by Reina et al. for a model system,[12] epoxidation
of the double bond followed by nucleophilic addition of
chloroethanol is a viable strategy for the introduction of a
linker on β-amino-cyclohexene-carboxylic acid derivatives.
For the formation of the epoxide, m-chloroperoxybenzoic
acid (mCPBA) was used as the oxygen source. The reaction
of 9 was stereoselective, due to the directing property of the
carbamate group;[13] a single isomer was formed in moder-
ate yield and its structure assigned as the cis isomer 10
(Scheme 3). In fact, the product configuration could not be
confirmed on the basis of its spectroscopic data at this
stage, but was determined after epoxide opening (see be-
low). Nucleophilic opening of the epoxide was performed
with 2-chloroethanol, using Cu(OTf)2 as catalyst, and af-
forded a 3:1 mixture of regioisomers 11 and 12 (Scheme 3).
The major compound was assigned as the product of C4
opening 11 based on the chemical shifts in the 13C NMR
spectrum. The C5-peak (δ = 67.4 ppm) appears more up-
field than the C4-peak (δ = 78.8 ppm), which is typical of
an alcohol in comparison with an ether. In the case of prod-
uct 12, which is derived from C5 opening, the opposite
Figure 2. IC50 values (μm) obtained by SPR competition assay for
fucose-based ligands inhibiting DC-SIGN binding to Man-BSA
surface.
A general approach that can be used to compensate for
weak protein-carbohydrate interaction is the synthesis of
multivalent ligands that present several copies of the mono-
meric epitope.[9] Many of the available methods for a multi-
Scheme 1. Functionalization of monomeric ligands as a basis for
multivalent presentation.
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Linker-Armed Fucose-Based Glycomimetics
Scheme 2. Synthesis of the central building block 6 and coupling to fucosyl azide 8 to afford 9.
Scheme 3. Introduction of a linker by epoxidation of 9 and nucleophilic addition to 10.
pattern could be observed in the 13C NMR spectrum (δC4 illustrated in Scheme 4b. Analysis of the minor epoxide
= 69.2 ppm and δC5 = 78.0 ppm).
opening product 12 was also consistent with C5 opening of
After chromatographic separation, the stereochemistry the cis epoxide 10 (see the Supporting Information Fig-
of the epoxidation step was confirmed by NMR analysis of ures SI-1 and SI-2).
11 (Scheme 4). The NOESY spectrum of 11 (CDCl3,
The CbzN-protecting group of the major product 11 was
400 MHz) showed two cross-peaks for proton H5 of the then removed (H2/Pd, Scheme 5) and the resulting amine
cyclohexane ring with protons H1 and H3. These must arise was coupled with 3-acetoxybenzoic acid by using O-benzo-
from conformer 11a, for which the three protons are in a triazole-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophos-
1,3-diaxial relationship and would not have been observed phate (HBTU) as the coupling agent to afford 15, which
if the starting material had been the trans epoxide 13, as was fully deprotected with Zemplen’s procedure. In the final
Scheme 4. Possible conformations of the cyclohexane ring after nucleophilic C4-opening of (a) cis epoxide 10 and (b) the hypothetical
trans epoxide 13.
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Scheme 5. Coupling of 3-acetoxybenzoic acid, deprotection, and substitution of the chloride by an azide affords functionalized ligand
16.
step of the synthesis, the chloride at the linker was replaced the scaffold. Indeed, coupling constant analysis of 16 in
by an azide to afford 16 as a functionalized version of 3.
D2O solution showed that, as in compound 3,[8] the carb-
For comparison (see below), an azido-functionalized ver- oxyl group of the β-amino amide is equatorial on the cyclo-
sion of 4 was prepared by reaction of 4b with the PEG- hexane ring, whereas the amino functionality is axial (Fig-
1
like linker 17 (see the Supporting Information), followed by ure 3, a). In the H NMR spectrum, H1 appears as a dt
3
3
deprotection to afford 18 (Scheme 6).
with J1,6ax = 12.4 Hz and J1,6eq = 5.4 Hz. The lack of
cross peaks in the NOESY spectrum between the protons of
fucose and the aromatic ring, along with a conformational
analysis by means of OPLSA force field, suggested that the
structure of 16 can be described by the extended conforma-
tion shown in Figure 3b.
Scheme 6. Functionalisation of 4b to afford 18.
Binding of 16 to DC-SIGN was tested by employing an
SPR competition assay, according to a previously described
protocol.[8] The assay allows the relative affinity of ligands
to be compared on the basis of their ability to inhibit DC-
SIGN binding to mannosylated bovine serum albumin
(Man-BSA) immobilized on a carboxymethyl dextran-func-
tionalized gold SPR sensor chip (CM4). Inhibition studies
were performed by using the extracellular domain (ECD)
of DC-SIGN (20 μm) injected either alone or in the pres-
ence of increasing concentrations of the ligands. The 50%
inhibition concentration (IC50) of 16 was determined and
compared to that of unfunctionalized ligand 3, the β-alanyl
fucosyl azide 4, its linker-armed variant 18, Lewis X, and
Fucose (Figure 2; sensorgrams and inhibition curves are
collected in the Supporting Information, Figure SI–3).
Figure 3. Preferred conformation of 16 in water solution (a) as de-
termined by NMR coupling constant analysis, and (b) calculated
by conformational analysis (OPLSA force field).
TR-NOESY spectra in the presence of DC-SIGN extra-
The results show that functionalization of the scaffold cellular domain indicate that the conformation found in the
does not reduce the activity of 3 (Figure 2). Both ligands 3 free state is also the conformation that interacts with the
and 16 were found to be more active than Lewis X and 4 protein. To obtain the ligand binding epitope, we performed
and very similar to one another. Functionalization of 4 to STD NMR experiments at different saturation times by
yield 18 also did not modify its activity significantly.
using the initial slope approach (STD0) (Figure 4).[14] As
These results appear to imply that functionalization of expected, the fucose ring was the main ligand recognition
the cyclohexane ring does not perturb the conformation of element, coordinating the calcium atom as found for other
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Linker-Armed Fucose-Based Glycomimetics
Figure 4. (a) Reference 1H NMR spectrum (above) and STD spectrum (below) of 16 (1.6 mm) in the presence of DC-SIGN ECD (19 μm).
Some key proton signals are labeled. (b) STD growth curves of the same sample (STD vs. the saturation time). The symbols represent
experimental data, whereas lines show the mathematical fit, in blue for protons of fucose and in red for the aromatic ring. (c) Relative
STD values (ligand epitope mapping) for 16 (same sample). The ratio of the intensities ISTD/I0 was normalized by using the most intense
STD value of the anomeric proton H1 of fucose residue (100%) as a reference.
fucose-based ligands of DC-SIGN.[4,8,15] Compared with
The relative affinity of 21 and 22 for DC-SIGN was ex-
previously examined ligands that contain the same elements amined in the SPR competition assay (Figure 2). The tetra-
as 3 but use a stereoisomeric β-amino acid scaffold,[15] the valent presentation of 18, compound 22, resulted in an im-
intensities of the STD signals for the aromatic ring of 16 proved activity by a factor of five over the monomeric
are higher, which implies a closer proximity of this fragment structure (β = 1.3). For the tetravalent presentation of 16,
to the protein. On the other hand, the cyclohexane scaffold compound 21, an improvement of one order of magnitude
displayed reduced contacts (Figure 4).
was achieved (β = 2). Most likely, the amplification ob-
The presence of a functional linker in 16 and 18 allows served occur through statistical rebinding[19] because, with
polyvalent presentations of the ligands to be generated. We a distance of 35 to 38 Å in the DC-SIGN tetramer, the den-
focused our attention on the tetravalent dendrimer 20 drimer is too small to bridge two binding sites.[20] Com-
(Scheme 7), which is based on tetraerythritol 19.[16] Com- pound 21 was the most effective DC-SIGN blocker in the
pounds 16 and 18 were connected through 1,3-cycload- tested series, with an IC50 value of 39 μm.
dition to 20 by using the Sharpless protocol.[17,18] Due to
Functionalization of the monovalent ligands could also
a challenging chromatographic purification, the tetravalent be exploited for solid-phase support in an array format.
dendrimers 21 and 22 were obtained in low to moderate Glycan arrays have been extensively used as a tool for rapid
yields.
analysis of the interaction of different carbohydrates with a
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Scheme 7. Synthesis of dendrimers 21 and 22 obtained through 1,3-cycloaddition.
wide variety of biological targets, including lectins.[21] This rimetry and SPR experiments have shown that RSL binds
approach can be used to begin examining the question of to α-Me-fucoside with a dissociation constant (Kd) of
selectivity for glycomimetic lectin ligands, which has very 0.7 μm and to Lewis X with an affinity 15 times lower.[25]
rarely been addressed.[8,22] Thus, in a preliminary experi- Our array data suggest that N-fucosyl amides also bind
ment, the interactions of 16 and 18 with several commer- strongly to RSL. The observation is consistent with the X-
cially available fucose-binding lectins were tested by means ray structure of the RSL/O-Me-fucoside complex, which
of glass-supported arrays. The functionalized Lewis X de- shows that the anomeric oxygen of the sugar is in a rela-
rivative 23 and the mimics 24 and 25, which were obtained tively open area and is not involved in interactions with the
after reduction of azides 16 and 18 by H2/Lindlar-Pd (Fig- protein.[25] Thus, O/NH replacement should not be de-
ure 5), were immobilized on N-hydroxysuccinimide (NHS) trimental for the formation of the complex. In our experi-
activated glass slides. Additionally, fucosyl amide 26, ment, the response of both 24 and 25 was already maximal
equipped with just a short β-alanine linker, was conjugated at 0.001 mg/mL concentration of RSL (Figure 6, a) and no
to the surface, together with the negative controls mannose increase in fluorescence was observed upon increasing the
and glucose. All compounds were spotted on the slide by protein concentration by one order of magnitude (0.01 mg/
using a printing robot. The interaction of the glycomimetics mL, Figure 6, b), likely because saturation of the binding
with FITC-labeled fucose-binding lectins was tested at dif- sites was already reached. Compound 26 was slightly less
ferent protein concentrations [Figure 6a (0.001 mg/mL of active, which suggests that the length of the linker may in-
protein) and Figure 6b (0.01 mg/mL of protein)]. After in- fluence the accessibility of the surface-bound epitope to the
cubation with plant lectin from Ulex europaeus[23] as well lectin. Lewis X was identified as the weakest binder of RSL
as bacterial lectins LecB (PA-IIL),[24] RSL,[25] and BC2LC- among those tested.
Nt,[26] intense binding-signals for the interaction of 24 and
The array data also show that amides 24–26 bind to
25 with RSL were detected at the lowest tested protein con- LecB (Figure 6), although a strong response was detected
centration of 0.001 mg/mL (Figure 6, a). Isothermal calo- only at high protein concentration (0.01 mg/mL). Lewis X
Figure 5. Compounds tested on the array.
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Linker-Armed Fucose-Based Glycomimetics
Figure 6. Array interaction studies. Fluorescence intensities for ligand-lectin interactions at (a) 0.001 mg/mL and (b) 0.01 mg/mL protein
concentration. Each ligand was printed as a solution at the same concentration (0.5 m) in three replicates. The black bars represent the
standard deviations obtained from the average of three values. Selectivity RSL ϾϾ LecB ϾϾ BC2LC-Nt Ͼ Ulex.
was also the weakest binder. This is consistent with pre- 0.01 mg/mL protein concentration only weak binding of 24
vious ITC experiments that established the affinity of α- to Ulex was registered.
Me-fucoside for Lec-B to be about twofold higher than that
of 26 and other α-fucosylamides.[27] On the other hand,
Lewis X was reported to bind to Lec-B one order of magni-
Conclusions
tude less effectively than fucose itself.[28] Analysis of the
Lec-B fucose binding site shows that the NH group at the
In this work we have described the functionalization of
anomeric position of α-fucosylamides perturbs a crystallo- previously reported functional Lewis X mimics, which are
graphically observed water molecule, which is highly con- synthetically more accessible and biologically more effective
served in all structures of Lec-B complexes and participates than the natural ligand of DC-SIGN. Functionalization of
in a H-bonding network that involves the protein and the these monovalent ligands allowed polyvalent constructs to
ligands. Thus, compared with simple O-fucosides, N-fucos- be generated, which increased the statistical binding to DC-
ylamides are weaker ligands of Lec-B.
SIGN and therefore resulted in higher activity. The tetra-
Ulex europaeus I agglutinin is a lectin that is specific for valent construct 21 allowed a low-micromolar range of ac-
the H blood group antigen (αFuc1–2Galβ1–4GlcNAc), tivity to be reached, which makes this approach useful for
which is recognized mainly through its α-l-fucose resi- use in cellular tests.
due.[23] BC2L-C has a strict specificity for fucosylated oligo-
Addition of a functionalized tether to the ligands could
saccharides but it was noted that short fragments such as also be exploited to support the molecules on solid phase
fucose itself and disaccharides (αFuc1–2Gal, αFuc1– in an array format. By using these arrays as analytical tools
3GlcNAc, αFuc1–4GlcNAc) as well as the Lewis X trisac- for screening the mimics with commercially available lectins
charide are not efficient ligands.[26a] The tested products with common fucose specificity, we have identified the lec-
showed no affinity for Ulex europaeus or BCIILC-Nt. At tin from the bacterium Ralstonia solanacearum (RSL) as
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Fabrication of Glycan Arrays: The carbohydrate samples were dis-
solved in disodium hydrogen phosphate buffer (50 mm, pH 8.5) at
three different concentrations (0.25, 0.50, and 1.00 mm; data shown
in Figure 6 were obtained for a ligand concentration of 0.5 mm).
Each solution (0.4 nL) was printed in three replicates by means of
a piezoelectric spotting device (S3, Scienion, Berlin, Germany) onto
NHS-coated CodeLink slides (SurModics). The slides were stored
for 16 h in a moisture chamber. Before further treatment, the slides
were washed with water, the unreacted NHS-groups were quenched
with 100 mm ethanolamine in disodium hydrogen phosphate buffer
a potential target protein of α-fucosylamide ligands. This
bacterium is responsible for lethal wilt of more than 200
plants and thereby causes substantial economic damage to
farmers in tropical regions. LecB (PA-IIL) is highly homol-
ogous to RSL and has similar affinity for fucose.[29] Thus,
this experiment suggests that even simple glycomimetic
structures can attain a significant amount of selectivity
among lectins with analogous specificity. Work is in pro-
gress in our group to exploit array analysis of a larger group
of glycomimetic structures towards the identification of lec- (50 mm Na2HPO4, pH 9) for 1 h at 50 °C. After removal of the
quencher, the slides were washed three times with water. In the final
preparation step, the slides were treated with 1% BSA in HBS
buffer (pH 7.4) for 1 h at room temp. and washed three times with
HBS buffer (10 mm HEPES, 150 mm NaCl, pH 7.4).
tin-selective glycomimetic entities.
Experimental Section
The prepared microarrays were incubated with FITC-labeled lec-
tins (Ulex, RSL, LecB, BCIILC-Nt) at different concentrations
(0.001 mg/mL, 0.01 mg/mL) in lectin-binding buffer (10mm
HEPES, 1 mm MgCl2, 1 mm CaCl2, pH 7.4) with 1% BSA for
40 min at room temp. During that time the slides were kept in the
dark. After incubation, the slides were washed three times with
HBS buffer, dried by centrifugation and read out by a fluorescence
micro array scanner (Genepix 4300A, Molecular Devices). For the
evaluation and interpretation of the results, the average of three
values, obtained for the same sugar concentration, were considered
as the resulting signal intensity.
Glycan Arrays
General Considerations: FITC-labeled LecB, RSL, and BCIILC-Nt
were purchased from Elicityl, OligoTech, France. FITC-labeled
Ulex europaeus was purchased from Sigma–Aldrich, Germany. DC-
SIGN ECD protein (residues 66–404) was overexpressed and puri-
fied as described previously.[20]
Surface Plasmon Resonance: SPR experiments were performed with
a Biacore 3000 at 25 °C using a CM4 chip. During functionaliza-
tion steps, HBS-P running buffer was used at 5 μL/min flow rate.
Flow cells (Fc) 1 and 2 were activated with a 0.2 m EDC/0.05 m
NHS mixture (50 μL); after this step Fc2 was functionalized with
mannosylated bovine serum albumin (Man α1–3[Manα1–6]Man
BSA (Man-BSA), Dextra Laboratories) by injecting 60 μg/mL of
protein prepared in 10 mm sodium acetate pH 4, and finally the
remaining activated groups of both flow cells were blocked with
1 m ethanolamine (30 μL). After blocking, both flow cells were
treated with 10 mm HCl (10 μL) to remove unspecific bound pro-
tein and 50 mm EDTA (20 μL) to expose the surface to the regener-
ation protocol. After these steps, 1600 RU of Man-BSA was immo-
bilized on Fc2.
Synthesis
General: All reactions were performed in an N2 atmosphere using
anhydrous solvents. Deuterated solvents as well as anhydrous di-
methylformamide (DMF), dichloromethane (CH2Cl2), methanol
and N,N-diisopropylethylamine (DIPEA) were purchased from
Sigma–Aldrich. THF was dried by the application of standard pro-
cedures. 1H NMR, 13C NMR and HSQC spectra were recorded
with a Bruker Avance 400 at 298 K using CDCl3 (δ = 7.24 ppm),
CD3OD (δ = 3.31 ppm), or D2O (δ = 4.80 ppm) as solvent and
internal standard. Multiplicity of signals has been described as s
(singlet), d (doublet), dd (doublet of doublets), ddd (doublet of
doublets of doublets), dt (doublet of triplet), t (triplet), td (triplet
of doublets), q (quartet), m (multiplet), and br (broad). Chemical
shifts (δ) are reported in ppm scale, and coupling constants (J) are
stated in Hz. Mass spectra were obtained with a Bruker ion-trap
Esquire 3000 apparatus (ESI ionization) or an Autospec Fission
Instrument (FAB ionization). An Apex (FT-ICR) instrument from
the Centro Interdipartimentale Grandi Apparecchiature (Uni-
versità degli Studi di Milano) was used. HRMS (FT-ICR, ESI)
were obtained with an Apex II instrument. MALDI-TOF mass
spectra were recorded with an Ultra Flex III MALDI TOF-TOF
instrument (Bruker Daltonics). Optical rotation values were
measured with a Perkin–Elmer 241 polarimeter at 589 nm (sodium
lamp). Silica gel 60M (mesh 230–400) from Macherey–Nagel was
used for purification by flash-chromatography. For thin-layer
chromatography (TLC), silica gel coated aluminum foils were used
(Sigma–Aldrich). The synthesis of linker 17 and amines 4b, and
For inhibition studies, DC-SIGN ECD (20 μm) mixed with increas-
ing concentrations of inhibiting compounds were prepared in a
running buffer composed of 25 mm Tris (pH 8), 150 mm NaCl,
4 mm CaCl2, 0.005% P20 surfactant, and each sample (13 μL) was
injected onto the surfaces at a 5 μL/min flow rate. Concentrations
of inhibiting compounds ranged from 0.67 to 4367 μm, except for l-
fucose (1.33–8733 μm) and dendrimers (0.27–1747 μm). The bound
lectin was washed off by a 1 min injection of 50 mm EDTA (pH 8).
DC-SIGN ECD equilibrium binding responses (Req) for each sam-
ple were obtained from the reference surface corrected sensorgrams
150 s after the start of the injection.
The DC-SIGN ECD binding responses were extracted from the
sensorgrams, converted into percent residual activity values (y) with
respect to lectin alone binding, and plotted against the correspond-
ing compound concentration. The four-parameter logistic model
[Equation (1)] was fitted to the plots, and the IC50 values were cal-
culated by using the values of fitted parameters (Rhi, Rlo, A1, and
A2) and Equation (2).
1
24–26 as well as H and 13C NMR spectra of the new compounds
are collected in the Supporting Information. Below we report the
procedures and characterization of compounds 10–16 and the den-
drimers 21 and 22. Compounds 3, 4, 9,[8] and 20[16] as well as 23[30]
were previously described.
(1)
(2)
N-[(1R,2S,4R,5S)-2-(Benzyloxycarbonylamino)-4,5-epoxycyclohex-
ane]-2,3,4-tri-O-acetyl-α-L-fucopyranosylamine (10): Compound 9
(550 mg, 1.00 mmol, 1.0 mol equiv.) and m-CPBA (275 mg,
1.59 mmol, 1.6 mol equiv.) were dissolved in CH2Cl2 (7.3 mL) and
5310
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Eur. J. Org. Chem. 2013, 5303–5314

Linker-Armed Fucose-Based Glycomimetics
the reaction mixture was stirred for 4 h at room temperature. The
mixture was diluted in CH2Cl2 and extracted with a satd. aq
NaHCO3. The organic phase was dried with Na2SO4 and the sol-
vent was evaporated. The product was purified by flash-chromatog-
and -CH2-O), 4.01 (q, JF5,F6 = 7.1 Hz, 1 H, HF5), 4.11–4.24 (m, 1
H, HCy2), 4.99 (d, JipsoH = 12.2 Hz, 1 H, Cbz), 5.14 (d, JipsoH
12.2 Hz, 1 H, Cbz), 5.28 (m, 1 H, HF4), 5.32–5.41 (m, 2 H, HF2
=
and HF3), 5.94 (dd, JF1,F2 = 3.0, JF1,NH = 8.9 Hz, 1 H, HF1), 6.03
raphy (CHCl3/MeOH, 98:2; Rf = 0.33). Product 10 was obtained (br. s, 1 H, NH-Cy), 7.28–7.37 (m, 6 H, NH-F1 and Cbz) ppm. 13
C
as a white foam, yield 288 mg (51 %); [α]D = –50.8 (c = 0.50,
MeOH). 1H NMR (400 MHz, CDCl3): δ = 1.15 (d, JF5,F6 = 6.4 Hz,
3 H, HF6), 1.93 (s, 3 H, CH3COO), 1.95 (s, 3 H, CH3COO), 2.05–
NMR (100 MHz, CDCl3): δ = 16.5 (CF6), 20.8 (CH3COO), 20.9
(CH3COO), 33.3 (CCy3), 34.5 (CCy6), 42.8 (CCy1), 43.7 (-CH2-Cl),
47.7 (CCy2), 66.3 (CF5), 66.4 (CF2 or CF3), 67.5 (CH2-Cbz), 68.2
2.12 (m, 1 H, HCy6ax), 2.15 (s, 3 H, CH3COO), 2.16–2.25 (m, 2 H, (CF2 or CF3), 69.2 (CCy4), 69.6 (-CH2-O), 70.8 (CF4), 74.6 (CF1),
H
Cy3), 2.40–2.51 (m, 1 H, HCy1), 2.51–2.61 (m, 1 H, HCy6eq), 3.20–
78.0 (CCy5), 128.4 (Cbz), 128.5 (CAr), 128.8 (CAr), 136.4 (CAr), 157.2
(-CO-), 169.8 (-CO-), 169.8 (-CO-), 170.3 (-CO-), 170.9 (-CO-
3.31 (m, 1 H, HCy4), 3.31–3.39 (m, 1 H, HCy5), 4.09 (q, JF5,F6
=
6.3 Hz, 1 H, HF5), 4.20 (m, 1 H, HCy2), 5.04 (d, J = 12.3 Hz, 1 H, ) ppm. ESI-MS: m/z (%) = 665 (100) [M + Na]+.
Cbz), 5.17 (d, J = 12.3 Hz, 1 H, Cbz), 5.33 (m, 1 H, HF4), 5.34 (dd,
N-[(1R,2S,4S,5S)-2-(3-Acetoxybenzamido)-4-(2-chloroethoxy)-5-
hydroxycyclohexane]-2,3,4-tri-O-acetyl-α-L-fucopyranosylamine
JF1,F2 = 5.6, JF2,F3 = 11.0 Hz, 1 H, HF2), 5.43 (dd, JF3,F4 = 3.1,
JF2,F3 = 11.0 Hz, 1 H, HF3), 5.94 (dd, JF1,F2 = 5.6, JF1,NH = 8.4 Hz,
1 H, HF1), 6.03 (d, JNH,Cy2 = 9.3 Hz, 1 H, NH-Cy2), 7.28–7.36 (m,
5 H, Cbz), 8.06 (d, JNH,F1 = 8.3 Hz, 1 H, NH-F1) ppm. 13C NMR
(100 MHz, CDCl3): δ = 16.5 (CF6), 20.8 (CH3COO), 20.89
(CH3COO), 24.2 (CCy6), 31.0 (CCy3), 43.3 (CCy1), 45.2 (CCy2), 51.8
(CCy5), 52.5 (CCy4), 66.3 (CF5), 66.5 (CF2), 67.7 (Cbz), 68.3 (CF3),
70.9 (CF4), 74.63 (CF1), 128.3 (Cbz), 128.5 (Cbz), 128.7 (Cbz), 136.3
(CAr), 157.47 (-CO-), 169.80 (-CO-), 170.16 (-CO-), 170.90 (-CO-),
172.54 (-CO-) ppm. ESI-MS: m/z (%) = 585 (100) [M + Na]+.
(15): Pd/C (10wt.-%, 10%, Degussa type) was added to a solution
of 11 in DMF/methanol (4:1, 0.05 m) and the reaction mixture was
stirred under H2 (1 bar) at room temperature until full conversion
was observed by TLC. The catalyst was filtered through Celite and
the solvent was evaporated to obtain the crude amine.
In the following step, 3-acetoxybenzoic acid (2.0 mol equiv.),
HBTU (2.0 mol equiv.), and DIPEA (3.7 mol equiv.) were added to
a solution of the crude amine in DMF (0.03 m). The reaction mix-
ture was stirred for 15 h at room temperature, then the solvent was
evaporated, the crude material was diluted in EtOAc and extracted
with HCl (1 m), saturated Na2CO3 and water, and the organic
phase was dried with Na2SO4. Product 15 was purified by flash-
chromatography (n-hexane/ethyl acetate, 7:3; Rf = 0.21), yield
90 mg (40%). [α]D = –33.4 (c = 0.75, MeOH). 1H NMR (400 MHz,
CDCl3): δ = 1.07 (d, JF5,F6 = 6.4 Hz, 3 H, HF6), 1.67 (s, 3 H,
CH3COO), 1.73–1.77 (m, 1 H, HCy3ax), 1.91 (s, 3 H, CH3COO),
1.92–1.97 (m, 1 H, HCy6ax), 2.09 (s, 3 H, CH3COO), 2.15–2.24 (m,
1 H, HCy6eq), 2.25 (s, 3 H, CH3COO), 2.27–2.35 (m, 1 H, HCy3eq),
2.86–2.94 (dd, Jeq/ax = 5.2, Jax/ax = 11.2 Hz, 1 H, HCy1), 3.45–3.52
(m, 1 H, HCy4), 3.52–3.60 (m, 2 H, -CH2-Cl), 3.60–3.66 (m, 1 H,
-CH2-O), 3.67–3.76 (m, 1 H, HCy5), 3.83–3.90 (m, 1 H, -CH2-O),
3.95 (q, JF5,F6 = 6.3 Hz, 1 H, HF5), 4.40–4.49 (m, 1 H, HCy2), 5.14
(dd, JF3,F4 = 3.3, JF3,F2 = 10.4 Hz, 1 H, HF3), 5.15–5.18 (m, 1 H,
Synthesis of 11 and 12 by Ring Opening of Epoxide 10: Cu(OTf)2
(37 mg, 0.10 mmol) was added to a solution of epoxide 10 (280 mg,
0.51 mmol) in 2-chloroethanol (1.7 mL) and the mixture was stirred
for 16 h at 40 °C. After evaporation of the solvent, the crude mate-
rial was dissolved in EtOAc and extracted with NH4Cl/NH3 solu-
tion (1:1). The organic phase was dried with Na2SO4 and the sol-
vent was evaporated. Purification by flash-chromatography on SiO2
(Et2O) afforded 12 (62 mg, 19%). Further column chromatography
(CHCl3/MeOH, 9:1) gave 11 (220 mg, 67%).
N-[(1R,2S,4S,5S)-2-(Benzyloxycarbonylamino)-4-(2-chloroethoxy)-
5-hydroxycyclohexane]-2,3,4-tri-O-acetyl-α-L-fucopyranosylamine
(11): [α]D = –41.5 (c = 0.43, MeOH). 1H NMR (400 MHz, CDCl3):
δ = 1.13 (d, JF5,F6 = 6.4 Hz, 3 H, HF6), 1.67–1.72 (m, 1 H, HCy3),
1.83–1.90 (m, 1 H, HCy6), 1.91 (s, 3 H, CH3COO), 1.96 (s, 3 H,
CH3COO), 2.15 (s, 3 H, CH3COO), 2.16 (m, 1 H, HCy3), 2.19 (m,
1 H, HCy6), 2.86–3.01 (m, 1 H, HCy1), 3.48–3.55 (m, 1 H, HCy4),
3.59 (t, JCH2,CH2 = 5.5 Hz, 2 H, -CH2-Cl), 3.64–3.69 (m, 1 H,
-CH2-O), 3.70–3.76 (m, 1 H, HCy5), 3.76–3.86 (m, 1 H, -CH2-O),
3.93 (q, JF5,F6 = 6.3 Hz, 1 H, HF5), 4.12–4.20 (m, 1 H, HCy2), 4.97
(d, J = 12.2 Hz, 1 H, Cbz), 5.15 (d, J = 12.2 Hz, 1 H, Cbz), 6.03
(m, 1 H, NH-Cy), 5.20 (m, 1 H, HF4), 5.23 (m, 1 H, HF3), 5.36 (dd,
JF1,F2 = 5.3, JF2,F3 = 10.9 Hz, 1 H, HF2), 5.85 (dd, JF1,F2 = 5.6,
JF1,NH = 8.4 Hz, 1 H, HF1), 7.21 (br. s, 1 H, NH-F), 7.27–7.36 (m,
5 H, Cbz) ppm. 13C NMR (100 MHz, CDCl3): δ = 16.4 (CF6), 20.7
(CH3COO), 20.9 (CH3COO), 20.89 (CH3COO), 28.4 (CCy6), 39.9
(CCy3), 43.6 (-CH2-Cl), 44.6 (CCy1), 46.9 (CCy2), 66.2 (CF2), 66.4
(CF5), 67.3 (Cbz), 68.0 (CCy5), 68.1 (CF4), 69.7 (-CH2-O), 70.6 (CF3),
74.9 (CF1), 79.1 (CCy4), 128.4 (Cbz), 128.6 (Cbz), 128.8 (Cbz), 128.8
(Cbz), 136.2 (CAr), 152.7 (-CO-), 169.7 (-CO-), 170.5 (-CO-), 170.8
(-CO-) ppm. ESI-MS: m/z (%) = 665 (100) [M + Na]+.
HF4), 5.26 (dd, JF1,F2 = 5.3, JF2,F3 = 10.4 Hz, 1 H, HF2), 5.77 (dd,
JF1,F2 = 5.4, JF1,NH = 7.8 Hz, 1 H, HF1), 7.07 (d, JNH,Cy2 = 6.2 Hz,
1 H, NH-Cy), 7.17 (ddd, JCH3ta = 0.9, JCH3ta = 2.9, Jortho = 8.1 Hz,
1 H, HAr4), 7.37 (t, Jortho = 7.9 Hz, 1 H, HAr5), 7.45 (t, JCH3ta
=
1.9 Hz, 1 H, HAr2), 7.55 (d, Jortho = 7.9 Hz, 1 H, HAr6), 7.71 (br. d,
JNH,F1 = 6.2 Hz, 1 H, NH-F1) ppm. 13C NMR (400 MHz, CDCl3):
δ = 16.3 (CF6), 20.4 (CH3COO), 20.9 (CH3COO), 20.9 (CH3COO),
21.3 (CH3COO), 29.2 (CCy6), 29.9 (CCy3), 43.8 (-CH2-Cl), 43.9
(CCy1), 46.3 (CCy2), 66.1 (CF2), 66.1 (CF5), 68.3 (CF3), 69.6 (CCy5),
70.7 (-CH2-O), 75.1 (CF4), 75.1 (CF1), 79.1 (CCy4), 120.9 (CAr2),
124.5 (CAr6), 125.5 (CAr4), 130.0 (CAr5), 135.6 (CAr1), 151.2 (CAr3),
166.7 (-CO-), 169.4 (-CO-), 169.8 (-CO-), 170.7 (-CO-), 170.9 (-CO-
), 175.1 (-CO-) ppm. ESI-MS: m/z (%) = 693 (100) [M + Na]+.
N-[(1R,2S,4S,5S)-2-(3-Hydroxybenzamido)-4-(2-azidoethoxy)-5-
hydroxycyclohexane-carboxyl]-α-L-fucopyranosylamine (16): A
0.01 m solution of protected sugar 15 in MeOH was treated with a
1M solution of NaOMe in MeOH (1 mol equiv.). The reaction mix-
ture was stirred for 15 min at room temperature, then the pH was
N-[(1R,2S,4R,5R)-2-(Benzyloxycarbonylamino)-4-hydroxy-5-(2-
chloroethoxy)cyclohexane]-2,3,4-tri-O-acetyl-α-L
-fucopyranosyl- adjusted to pH 7 by addition of Amberlite IRA 120+, which was
1
amine (12): [α]D = –65.2 (c = 0.75, MeOH). H NMR (400 MHz,
then removed by filtration. The solvent was evaporated to afford
CDCl3): δ = 1.14 (d, JF5,F6 = 6.4 Hz, 3 H, HF6), 1.88 (m, 1 H, the deacetylated product, which was purified by flash-chromatog-
H
Cy3), 1.91 (s, 3 H, CH3COO), 1.95 (s, 3 H, CH3COO), 1.99–2.08
raphy (chloroform/methanol, 4:1; Rf = 0.31), yield 26 mg (81%).
[α]D = –42.8 (c = 1.0, MeOH). H NMR (400 MHz, CD3OD): δ =
1
(m, 3 H, HCy3 and HCy6), 2.15 (s, 3 H, CH3COO), 2.90 (dt, Jax/eq
= 5.3, Jax/ax = 10.9 Hz, 1 H, HCy1), 3.55–3.62 (m, 2 H, -CH2-Cl),
3.62–3.71 (m, 2 H, -CH2-O and HCy5), 3.80–3.92 (m, 2 H, HCy4
1.19 (d, JF5,F6 = 6.5 Hz, 3 H, HF6), 1.66 (m, 1 H, HCy3ax), 1.96–
2.10 (m, 2 H, HCy6ax), 2.46 (ddd, Jeq/ax = 3.7, Jeq/eq = 6.2, 2J =
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A. Bernardi et al.
FULL PAPER
13.6 Hz, 1 H, HCy3eq), 2.93 (dt, Jeq/ax = 4.7, Jax/ax = 9.2 Hz, 1 H,
tion mixture was stirred for 15 min at room temperature, then the
H
H
Cy1), 3.53 (m, 1 H, HCy4), 3.61–3.67 (m, 4 H, HCy5, -CH2-Cl, pH was adjusted to pH 7 by addition of Amberlite IRA 120+,
F4), 3.73 (dd, JF3,F4 = 3.4, JF3,F2 = 10.2 Hz, 1 H, HF3), 3.77–3.88
which was removed by filtration. The solvent was evaporated to
(m, 3 H, -CH2-O and HF5), 3.93 (dd, JF1,F2 = 5.6, JF2,F3 = 10.2 Hz,
afford the deacetylated product 18, yield quantitative. [α]D = –39.5
1 H, HF2), 4.58 (dt, Jeq/ax = 3.9, Jax/ax = 7.5 Hz, 1 H, HCy2), 5.52 (c = 0.24, MeOH). 1H NMR (400 MHz, CD3OD): δ = 1.18 (d,
(d, JF1,F2 = 5.6 Hz, 1 H, HF1), 6.94 (dt, JCH3ta = 2.6, Jortho = 6.6 Hz, JF5,F6 = 6.5 Hz, 3 H, HF6), 2.44 (t, 3J = 6.2 Hz, 2 H, CH2), 2.51 (t,
1 H, HAr4), 7.22–7.28 (m, 3 H, HAr2,5,6) ppm. 13C NMR (100 MHz,
3J = 6.7 Hz, 2 H, CH2), 3.36–3.40 (m, 2 H, CH2), 3.46 (t, 3J =
CD3OD): δ = 17.0 (CF6), 31.3 (CCy6), 32.9 (CCy3), 44.4 (-CH2-Cl), 6.8 Hz, 2 H, CH2), 3.58–3.70 (m, 7 H, HF4 and 3ϫCH2), 3.73 (t,
45.2 (CCy1), 48.3 (CCy2), 68.2 (CF2), 69.0 (CF5), 71.4 (-CH2-O), 71.5
(CF4 or CCy5), 71.75 (CF3), 73.2 (CF4 or CCy5), 78.7 (CF1), 80.5
(CCy4), 115.5 (CAr), 119.6 (CAr), 119.8 (CAr), 130.8 (CAr), 137.5
(Cq u a t . , CA r ), 158.9 (Cq u a t . , CA r), 170.6 (NHCO), 177.0
(NHCO) ppm. ESI-MS: m/z (%) = 525 (100) [M + Na]+. The prod-
uct isolated from the Zemplen deprotection (26 mg, 0.052 mmol)
was dissolved in DMF (500 μL). After addition of NaN3 (25 mg,
0.448 mmol), the reaction mixture was stirred for ten days at 50 °C,
then the solvent was evaporated and product 16 was purified by
flash-chromatography (chloroform/methanol, 9:1; Rf = 0.32), yield
26 mg (81%). [α]D = –42.4 (c = 0.95, MeOH). 1H NMR (400 MHz,
D2O): δ = 1.09 (d, JF5,F6 = 6.5 Hz, 3 H, HF6), 1.61–1.73 (m, 1 H,
HCy3ax), 1.87–2.00 (m, 1 H, HCy6ax), 2.04 (m, 1 H, HCy6eq), 2.37
(m, 1 H, HCy3eq), 2.96 (dt, J1,6ax = 12.4, J1,6eq = 5.4 Hz, HCy1),
3.36–3.46 (m, 2 H, -CH2-Cl), 3.46–3.55 (m, 1 H, HCy4), 3.77–3.85
(m, 2 H, HF5 and HF3), 3.85–3.62 (m, 4 H, -CH2-O, HCy5 and HF4),
3.90 (dd, JF1,F2 = 5.6, JF2,F3 = 10.5 Hz, 1 H, HF2), 4.66 (q, 3J =
3.5 Hz, 1 H, HCy2), 5.41 (d, JF1,F2 = 5.6 Hz, 1 H, HF1), 7.02 (ddd,
JCH3ta = 0.9, JCH3ta = 2.6, Jortho = 8.2 Hz, 1 H, HAr4), 7.06–7.11
(m, 1 H, HAr2), 7.16 (ddd, JCH3ta = 0.9, JCH3ta = 2.6, Jortho = 8.2 Hz,
1 H, HAr6), 7.32 (t, Jortho = 7.9 Hz, 1 H, HAr5) ppm. 13C NMR
(100 MHz, D2O): δ = 15.5 (CF6), 29.2 (CCy6), 32.9 (CCy3), 43.7
(CCy1), 47.5 (CCy2), 50.7 (-CH2-Cl), 65.9 (CF2), 67.5 (CF5), 68.2 (-
CH2-O), 69.4 (CF3), 71.1 (CF4 or CCy5), 71.3 (CF4 or CCy5), 77.0
(CF1), 78.7 (CCy4), 114.1 (CAr2), 119.11 (CAr4 and CAr6), 130.10
(CAr5), 135.31 (CAr1), 155.8 (CAr3), 170.7 (NHCO), 175.6
(NHCO) ppm. ESI-MS: m/z = 508 (100) [M – 1 H]–. HRMS (FT-
ICR, ESI): m/z calcd. for C22H31N5O9Na [M + Na]+ 532.20140;
found 532.20055.
3J = 6.2 Hz, 2 H, CH2), 3.75–3.82 (m, 2 H, HF3 and HF5), 3.96 (dd,
JF1,F2 = 5.6, JF2,F3 = 10.3 Hz, 1 H, HF2), 5.56 (d, JF1,F2 = 5.6 Hz,
1 H, HF1) ppm. 13C NMR (100 MHz, CD3OD): δ = 16.9 (CF6),
36.6 (CH2), 36.7 (CH2), 37.6 (CH2), 51.8 (CH2), 68.0 (CF2), 68.3
(CH2), 68.6 (CF3), 71.1 (CH2), 71.4 (CH2), 71.4 (CH2), 71.4 (CF5),
73.2 (CF4), 78.3 (CF1), 174.0 (-CONH-), 175.1 (-CONH-) ppm.
HRMS (FT-ICR, ESI): m/z calcd. for C22H33N3O9Na [M + Na]+
442.19083; found 442.19061. ESI-MS: m/z (%) = 442 (100) [M +
Na]+.
Dendrimer 21: A solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)-
methyl]amine (TBTA) (2.28 mg, 0.0043 mmol, 1.0 mol equiv.) in
THF (325 μL), a solution of CuSO4·5H2O (10 mol-%) in degassed
water (135 μL) and a solution of sodium ascorbate (0.376 mg,
0.4 mol equiv.) in degassed water (87 μL) were added to a solution
of core 20 (1.24 mg, 0.0043 mmol, 1.0 mol equiv.) in freshly dis-
tilled THF (155 μL) under a N2 atmosphere. Addition of 16
(6 mol equiv.) in water (260 μL) led to a water/THF (1:1 mixture)
and the reaction was stirred for 3 d at room temperature (progress
monitored by MALDI-MS analysis). Sodium ascorbate
(0.1 mol equiv.) was added at 24 h intervals. After 3 d, full conver-
sion was observed by MALDI mass spectrometry (DHB; CH3CN/
H2O, 1:1). The product was purified by flash-chromatography on
SiO2 (CHCl3/MeOH, 1:1) to give the product as a white solid, yield
3
3
4 mg (35%). 1H NMR (400 MHz, D2O): δ = 1.10 (d, JF1,F2
=
6.5 Hz, 12 H, HF6), 1.40–1.51 (m, 4 H, HCy3ax), 1.85–2.09 (m, 12
H, HCy3eq and HCy6), 2.91 (dt, Jeq/ax = 4.1, Jax/ax = 11.9 Hz, 4 H,
HCy1), 3.23 (s, 8 H, CH2 core), 3.36–3.45 (m, 4 H, HCy4), 3.56–3.62
(m, 4 H, HCy5), 3.68–3.73 (m, 4 H, HF4), 3.77–3.83 (m, 8 H, HF3
and HF5), 3.92 (dd, JF1,F2 = 5.6, JHF2,HF3 = 10.5 Hz, HF2), 3.99–
N-{3-[2-(2-Azidoethoxy)ethoxy]propanecarboxyl}-α-
L-fucopyranosyl- 4.02 (m, 8 H, CH2-N-), 4.37 (s, 8 H, CH2 core), 4.46–4.53 (m, 8 H,
amine (18): Compound 17 (17.0 mg, 0.0837 mmol) and HATU CH2-O-), 4.53–4.54 (m, 4 H, HCy2), 5.42 (d, JF1,F2 = 5.6 Hz, 4 H,
(33.0 mg, 0.0868 mmol) were dissolved in DMF (450 μL) and the
solution was treated with DIPEA (40.0 μL, 0.229 mmol) and
stirred for 15 min. After addition of a solution of 4b (30 mg,
0.0768 mmol) in DMF (300 μL), the reaction was stirred at room
temperature for 16 h. The solvent was evaporated and the product
was purified by flash-chromatography on SiO2 [MeOH/EtOAc, 0–
H
F1), 6.99 (dd, JCH3ta = 2.5, Jortho = 8.1 Hz, 4 H, HAr4), 7.02–7.08
(m, 4 H, HAr2), 7.13 (d, Jortho = 7.8 Hz, 4 H, HAr6), 7.27 (t, Jortho
=
7.9 Hz, 4 H, HAr5), 7.89 (s, 4 H, triazole) ppm. 13C NMR (HSQC,
100 MHz, D2O): δ = 15.3 (CF6), 28.6 (CCy3), 32.4 (CCy6), 43.3
(CCy1), 47.3 (CCy2), 50.3 (CH2-O-), 63.4 (CH2 core), 65.6 (CF2),
67.2 (CF3 or CF5), 67.7 (CH2 core), 67.9 (CH2-N-), 69.1 (CF3 or
3%; Rf = 0.36 (MeOH/CHCl3, 3:97)], yield 20.0 mg (50%). [α]D
=
C
F5), 70.6 (CCy5), 71.2 (CF4), 76.7 (CF1), 78.8 (CCy4), 114.0 (CAr2),
–76.0 (c = 0.60, MeOH). H NMR (400 MHz, CD3OD): δ = 1.11 119.0 (CAr4), 119.2 (CAr6), 130.0 (CAr5) ppm. HRMS (FT-ICR,
(d, JF5,F6 = 6.4 Hz, 3 H, HF6), 1.97 (s, 3 H, CH3COO), 2.02 (s, 3 ESI): m/z calcd. for C105H144N20O40Na2 (+2): 2370.96331 observed
1
3
H, CH3COO), 2.16 (s, 3 H, CH3COO), 2.44 (t, J = 6.1 Hz, 1 H, at 1185.48166, found 1185.48429.
3
CH2), 2.52 (t, J = 6.8 Hz, 1 H, CH2), 3.34–3.40 (m, 2 H, CH2),
Dendrimer 22:
A solution of TBTA (2.9 mg, 0.0054 mmol,
3.45 (td, 3J = 3.7, 3J = 6.7 Hz, 1 H, CH2), 3.56–3.69 (m, 6 H, CH2),
3.73 (d, 3J = 6.1 Hz, 2 H, CH2), 4.03 (q, JF5,F6 = 6.3 Hz, 1 H, HF5),
5.23 (dd, JF1,F2 = 5.6, JF2,F3 = 11.1 Hz, 1 H, HF2), 5.29 (m, 1 H,
1.0 mol equiv.) in THF (250 μL), a solution of CuSO4·5H2O
(10 mol-%) in degassed water (170 μL) and a solution of sodium
ascorbate (0.4 mg, 0.4 mol equiv.) in degassed water (100 μL) were
added to a solution of the core 20 (1.6 mg, 0.0054 mmol, 1.0 equiv.)
in freshly distilled THF (195 μL) under a N2 atmosphere. Addition
of 18 (10 mg, 0.024 mmol, 4.4 mol equiv.) in water (175 μL) led to
a water/THF (1:1) mixture that was stirred under N2 and moni-
tored by ESI-MS. After ca. 2 h, additional sodium ascorbate
(0.4 mg, 0.4 mol equiv. in 100 μL degassed water) was added. After
24 h, ESI-MS showed that the reaction had reached completion.
The reaction mixture was then loaded on a Sephadex LH20 column
and eluted with MeOH to give product 22 (6.6 mg, 62) as a white
H
F4), 5.48 (dd, JF3,F4 = 3.5, JF2,F3 = 11.2 Hz, 1 H, HF3), 5.84 (d,
JF1,F2 = 5.6 Hz, 1 H, HF1) ppm. 13C NMR (100 MHz, CDCl3): δ
= 16.7 (CF6), 20.6 (CH3COO), 20.7 (CH3COO), 20.7 (CH3COO),
36.5 (CH2), 36.8 (CH2), 37.7 (CH2), 51.9 (CH2), 67.0 (CF5), 67.8
(CF2), 68.4 (CH2), 69.5 (CF3), 71.3 (CH2), 71.6 (CH2), 71.7 (CH2),
72.3 (CF4), 75.6 (CF1), 171.6 (-CO), 171.7 (-CO-), 172.5 (-CO-),
174.5 (-CO-), 174.8 (-CO-) ppm. ESI-MS: m/z = 568 (100) [M +
Na]+.
A 0.01 m solution of the coupling product in MeOH was treated
with a 1M solution of NaOMe in MeOH (1 mol equiv.). The reac-
foamy solid. 1H NMR (400 MHz, D2O): δ = 1.15 (d, JF5,F6
=
5312
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Eur. J. Org. Chem. 2013, 5303–5314

Linker-Armed Fucose-Based Glycomimetics
3
[5] M. E. Taylor, K. Drickamer, Curr. Opin. Cell Biol. 2007, 19,
6.5 Hz, 12 H, HF6), 2.46 (t, J = 6.1 Hz, 8 H, CH2-linker), 2.57 (t,
3J = 6.7 Hz, 8 H, CH2-β-Ala), 3.40 (s, 8 H, CH2 core), 3.46 (t, J
= 6.5 Hz, 8 H, CH2-β-Ala), 3.54–3.63 (m, 16 H, CH2-linker), 3.67
(t, 3J = 6.1 Hz, 8 H, CH2-linker), 3.79–3.82 (m, 4 H, HF4), 3.85
(dd, JF3,F4 = 3.4, JF2,F3 = 10.6 Hz, 4 H, HF3), 3.85–3.92 (m, 4 H,
3
572–577.
[6]
a) T. B. H. Geijtenbeek, D. S. Kwon, R. Tonensma, S. J.
van Vliet, G. C. F. van Duijnhoven, J. Middel, I. L. M. H. A.
Cornelissen, H. S. L. M. Nottet, V. N. Kewal Ramani, D. R.
Littman, C. G. Figdor, Y. van Kooyk, Cell 2000, 100, 587–597;
b) C. P. Alvarez, F. Lasala, J. Carrillo, O. Muniz, A. L. Corbi,
R. Delgado, J. Virol. 2002, 76, 6841–6844.
H
F5), 3.92–3.98 (m, 8 H, CH2-linker), 4.03 (dd, JF1,F2 = 5.7, JF2,F3
= 10.5 Hz, 4 H, HF2), 4.52 (s, 8 H, CH2 core), 4.58–4.63 (m, 8
H, CH2-linker), 5.56 (d, JF1,F2 = 5.7 Hz, 4 H, HF1), 7.99 (s, 4 H,
triazole) ppm. 13C NMR (100 MHz, D2O): δ = 15.7 (CF6), 35.0
[7]
[8]
[9]
A. Berzi, J. J. Reina, R. Ottria, I. Sutkeviciute, P. Antonazzo,
M. Sánchez-Navarro, E. Chabrol, M. Biasin, D. Trabattoni, I.
Cetin, J. Rojo, F. Fieschi, A. Bernardi, M. Clerici, AIDS 2012,
26, 127–137.
M. Andreini, D. Doknic, I. Sutkeviciute, J. J. Reina, J. Duan,
E. Chabrol, M. Thepaut, E. Moroni, F. Doro, L. Belvisi, J.
Weiser, J. Rojo, F. Fieschi, A. Bernardi, Org. Biomol. Chem.
2011, 9, 5778–5786.
J. Luczkowiak, S. Sattin, I. Sutkeviciute, J. J. Reina, M.
Sánchez-Navarro, M. Thépaut, L. Martínez-Prats, A. Daghetti,
F. Fieschi, R. Delgado, A. Bernardi, J. Rojo, Bioconjugate
Chem. 2011, 22, 1354–1365.
(CH2-β-Ala), 35.6 (CH2-β-Ala), 35.9 (CH2-linker), 44.6 (Cquat.
–
core), 50.0 (CH2-linker), 63.5 (CH2, core), 66.0 (CF2), 66.6 (CH2-
linker), 67.5 (CF5), 68.1 (CH2, core), 68.8 (CH2-linker), 69.4 (CH2-
linker), 69.5 (CF3), 69.6 (CH2-linker), 71.4 (CF4), 76.7 (CF1), 125.3
(CH, triazole), 144.1 (Cquat. – triazole), 173.8 (Cquat., -C=O), 175.3
(Cquat., -C=O) ppm. ESI-MS: m/z = 1987.9 [M + Na]+. HRMS
(FT-ICR, ESI): m/z calcd. for C81H136N20O36 [M]+ 1964.94261;
found 1964.94286 (after deconvolution).
STD Experiments: STD NMR experiments were performed at
25 °C with a Bruker Avance 600 instrument. Saturation was
achieved by using a train of Gaussian shaped pulses of 49 ms (field
strength of ca. 80 Hz), an inter-pulse delay of 1 ms[1] and 15 ms
spin-lock pulse (field strength of 3.7 kHz) prior to acquisition. The
on-resonance frequency was set to 0 ppm and the off-resonance
frequency was 40 ppm. Appropriate blank experiments were per-
formed to assure the lack of direct saturation of the ligand protons.
Several saturation times were used to obtain the STD build-up
curves. The binding epitope was characterized by analysis of initial
slopes of the STD intensities: the experimental (I0 – Isat/I0) curves
were fitted to an exponential function described by the equation:
STD (tsat) = STDmax (1 – e–ksat·tsat), which allows the STD at zero
saturation time (initial slopes) to be calculated from the product of
resulting parameters STDmax and ksatd.
[10]
[11]
L. L. Kiessling, J. E. Gestwicki, L. E. Strong, Angew. Chem.
2006, 118, 2408; Angew. Chem. Int. Ed. 2006, 45, 2348–2368.
F. Damkaci, P. DeShong, J. Am. Chem. Soc. 2003, 125, 4408–
4409.
J. J. Reina, A. Bernardi, Tetrahedron 2011, 67, 5770–5775.
A. Hoveyda, D. A. Evans, G. C. Fu, Chem. Rev. 1993, 93,
1307–1370.
M. Mayer, T. L. James, J. Am. Chem. Soc. 2004, 126, 4453.
C. Guzzi, J. Angulo, F. Doro, J. J. Reina, M. Thepaut, F. Fies-
chi, A. Bernardi, J. Rojo, P. M. Nieto, Org. Biomol. Chem.
2011, 9, 7705.
S. Fortier, M. Touaibia, S. Lord-Dufour, J. Galipeau, R. Roy,
B. Annabi, Glycobiology 2008, 18, 195–204.
[12]
[13]
[14]
[15]
[16]
[17]
V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, An-
gew. Chem. 2002, 114, 2708; Angew. Chem. Int. Ed. 2002, 41,
2596–2599.
[18]
[19]
J. E. Hein, V. V. Folk, Chem. Soc. Rev. 2010, 39, 1302–1315,
Supporting Information (see footnote on the first page of this arti-
1
and references cited therein.
cle): Synthesis of linker 17; synthesis of amines 4b and 24–26; H
and 13C NMR spectra of new compounds; NOESY spectra and
configuration assignment of 11 and 12; primary data of SPR ex-
periments (sensorgrams and inhibition curves).
V. M. Krishnamurthy, L. A. Estroff, G. M. Whitesides, Ap-
proaches in Drug Discovery (Eds.: W. Jahnke, D. A. Erlanson),
Wiley-VCH Verlag, Weinheim, Germany, 2006, p. 11–53.
G. Tabarani, M. Thepaut, D. Stroebel, C. Ebel, C. Vives, P.
Vachette, D. Durand, F. Fieschi, J. Biol. Chem. 2009, 32,
21229–21240.
a) S. Fukui, T. Feizi, C. Galustian, A. M. Lawson, W. Chai,
Nat. Biotechnol. 2002, 20, 1011–1017; b) M. D. Disney, P. H.
Seeberger, Chem. Eur. J. 2004, 10, 3308–3314; c) M. D. Disney,
P. H. Seeberger, Chem. Biol. 2004, 11, 1701–1707; d) J. L.
de Paz, C. Noti, P. H. Seeberger, J. Am. Chem. Soc. 2006, 128,
2766–2767; e) N. P. Pera, H. M. Branderhorst, R. Kooij, C.
Maierhofer, M. van der Kaaden, R. M. J. Liskamp, V.
Wittmann, R. Ruijtenbeek, R. J. Pieters, ChemBioChem 2010,
11, 1896–1904; f) Y.-H. Tsai, S. Götze, N. Azzouz, H. S. Hahm,
P. H. Seeberger, D. V. Silva, Angew. Chem. 2011, 123, 10136;
Angew. Chem. Int. Ed. 2011, 50, 9961–9964.
[20]
[21]
Acknowledgments
Financial support by the EU-ITN CARMUSYS (PITN-GA-2008-
213592) is gratefully acknowledged. F. F. thanks the Institut Uni-
versitaire de France for support. P. H. S. thanks the Max-Planck
Society for generous financial support. A doctoral fellowship of
the Studienstiftung des Deutschen Volkes (to A. R.) is gratefully
acknowledged.
[22]
[23]
a) M. Scharenberg, O. Schwardt, S. Rabbani, B. Ernst, J. Med.
Chem. 2012, 55, 9810–9816; b) N. Varga, I. Sutkeviciute, C.
Guzzi, J. McGeagh, I. Petit-Haertlein, S. Gugliotta, J. Weiser,
J. Angulo, F. Fieschi, A. Bernardi, Chem. Eur. J. 2013, 19,
4786–4797.
a) G. F. Audette, D. J. Olson, A. R. Ross, J. W. Quail, L. T.
Delbaere, Can. J. Chem. 2002, 80, 1010–1021; b) R. Loris, T.
Hamelryck, J. Bouckaert, L. Wyns, Biochim. Biophys. Acta
1998, 1383, 9–36.
D. Tielker, S. Hacker, R. Loris, M. Strathmann, J. Wingender,
S. Wilhelm, F. Rose, K.-E. Jaeger, Microbiology 2005, 151,
1313–1323.
N. Kostlanova, E. P. Mitchell, H. Lortat-Jacob, S. Oscarson,
M. Lahmann, N. Gilboa-Garber, G. Chambat, M. Wim-
merova, A. Imberty, J. Biol. Chem. 2005, 280, 27839–27849.
[1] A. Alavia, J. S. Axford, Rheumatology 2008, 47, 760–770.
[2] a) K. Chang, G. Chauduri, D. Fong, Annu. Rev. Microbiol.
1990, 44, 499–529; b) N. Maeda, J. Nigou, J. L. Herrmann, M.
Jackson, A. Amara, P. H. Lagrange, G. Puzo, B. Gicquel, O.
Neyrolles, J. Biol. Chem. 2003, 278, 5513–5516; c) Y.
van Kooyk, T. B. H. Geijtenbeek, Nat. Rev. Immunol. 2003, 3,
697–709 , and references cited therein. d) M. A. Schell, Annu.
Rev. Phythopathol. 2000, 38, 263–292.
[3] a) A. Bernardi, P. Cheshev, Chem. Eur. J. 2008, 14, 7434–7441;
b) B. Ernst, J. L. Magnani, Nature Rev. Drug Discov. 2009, 8,
661–677; c) M. Anderluh, G. Jug, U. Svajger, N. Obermajer,
Curr. Med. Chem. 2012, 19, 992–1007.
[24]
[25]
[4] G. Timpano, G. Tabarani, M. Anderluh, D. Invernizzi, F. Vas-
ile, D. Potenza, P. M. Nieto, J. Rojo, F. Fieschi, A. Bernardi,
ChemBioChem 2008, 9, 1921–1930.
Eur. J. Org. Chem. 2013, 5303–5314
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5313

A. Bernardi et al.
FULL PAPER
[26]
[28] E. Mitchell, C. Houles, D. Soudakevitz, M. Wimmerova, C.
Gautier, S. Pérez, A. M. Wu, N. Gilboa-Garber, A. Imberty,
Nat. Struct. Biol. 2002, 9, 918–921.
[29] J. Adam, M. Pokorna, C. Sabin, E. P. Mitchell, A. Imberty, M.
Wimmerova, BMC Struct. Biol. 2007, 7, 36.
[30] M. K. Schlegel, J. Hütter, M. Eriksson, B. Lepenies, P. H. See-
berger, ChemBioChem 2011, 12, 2791–2800.
a) O. Sulák, G. Cioci, M. Delia, M. Lahmann, A. Varrot, A.
Imberty, M. Wimmerová, Structure 2010, 18, 59–72; b) O. Su-
lak, G. Cioci, E. Lameignere, V. Balloy, A. Romd, I. Gutsche,
L. Malinkovska, M. Chignard, P. Kosma, D. F. Aubert, C. L.
Marolda, M. A. Valvano, M. Wimmerova, A. Imberty, PLoS
Pathog. 2011, 7, 1–14.
[27] M. Andreini, M. Anderluh, A. Audfray, A. Bernardi, A. Im-
Received: February 13, 2013
Published Online: July 9, 2013
berty, Carbohydr. Res. 2010, 345, 1400–1407.
5314
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