Synthesis and binding properties of divalent and trivalent clusters of the Lewis a disaccharide moiety to Pseudomonas aeruginosa lectin PA-IIL

Article abstract of DOI:10.1039/b708227d

The synthesis of oligomeric glycocomimetics has been performed for targeting the Pseudomonas aeruginosa PA-IIL lectin, which is of therapeutical interest for anti-adhesive treatment. The disaccharide α-l-Fucp-(1→4) -β-d-GlcNAc, which is a high-affinity li

Full text of DOI:10.1039/b708227d

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and binding properties of divalent and trivalent clusters of the Lewis
a disaccharide moiety to Pseudomonas aeruginosa lectin PA-IIL
Karine Marotte,a Cathy Pre´ville,a Charles Sabin,b Myriame Moume´-Pymbock,a
Anne Imberty*b and Rene´ Roy*a
Received 30th May 2007, Accepted 20th July 2007
First published as an Advance Article on the web 7th August 2007
DOI: 10.1039/b708227d
The synthesis of oligomeric glycocomimetics has been performed for targeting the Pseudomonas
aeruginosa PA-IIL lectin, which is of therapeutical interest for anti-adhesive treatment. The
disaccharide a-L-Fucp-(1→4)-b-D-GlcNAc, which is a high-affinity ligand of the lectin, has been
coupled to dimeric and trimeric linkers with various lengths and geometries. A series of linear dimers
displayed an efficient clustering effect and a very strong affinity, with a lower dissociation constant of
90 nM. The trimeric compound was less efficient in inhibition assays but displayed high affinity in
solution. Titration microcalorimetry and molecular modeling allowed in-depth analysis and
rationalization of the binding data. These glycoclusters could act by crosslinking the lectins present on
the surface of bacteria and therefore interfere with host recognition or biofilm formation.
and it has been proposed to play a role in host recognition,
Introduction
adhesion and biofilm formation.13 The isolated protein displays
Many pathogens exploit host cell-surface glycoconjugates as
receptors for attachment, tissue colonization and/or invasion.1
micromolar affinity for L-fucose (Fuc) and ten times higher
affinity when this residue is attached to position 4 of N-D-
acetylglucosamine (GlcNAc), as in the case of the Lewis a (Lea)
antigen.14 The lectin quaternary structure consists of a ball-
shaped tetramer.15 It is unlikely that multivalent glycoconjugates
possessing short interglycosidic linkers can bind to different sites
of the same tetrameric proteins, but it is expected that dimeric or
trimeric ligands would be very efficient in the formation of often
insoluble cross-linked complexes, resulting in higher avidity.
At the cell surface, the glycan structures offer a wide range
of diversity for encoding information, a fact directly tied to
the variability of possible isomeric configurations of monosac-
charides, increased even further when taking into account the
possible linkages between the sugar units.2 As a counterpart of this
variety of carbohydrate structures, pathogenic bacteria use a panel
of carbohydrate-binding proteins such as toxins, soluble lectins
or fimbrial adhesins.3,4
While plant and animal lectins display weak affinity for their
carbohydrate ligands (millimolar range), a very different behavior
is observed for bacterial lectins that interact with high affinity with
glycoconjugates5. As a consequence, the design of glycomimetics
that could interfere in host recognition and adhesion is an
attractive antibacterial strategy.6 In addition, glycoclusters and
glycodendrimers can be constructed in order to increase the
affinity of the ligands for the lectins by a “glycoside cluster effect”.7
Di-, tri- or multimeric glycoclusters, either in the form of polymeric
or dendrimeric materials, have been used for a variety of biological
applications.7–9 Such multivalent analogs strongly enhanced the
binding of glycoconjugates to microbial proteins with an avidity
well above that expected from monomeric glycomimetics.9
We focused our work on PA-IIL, a fucose-binding lectin from
Pseudomonas aeruginosa, an opportunistic bacteria responsible for
nosocomial infection that can cause in particular life-threatening
damage to cystic fibrosis patients. The lectin has been widely
characterized by both biochemical and structural approaches.10–12
This soluble lectin is located on the outer membrane of the bacteria
Results and discussion
Synthesis of dimeric and trimeric ligands
For the synthesis of both flexible and more rigid dimeric
glycoclusters, oligoethylene glycols and methyl 3,5-bis(prop-2-
ynyloxy)benzoate were used as linkers, respectively.16 Different
sizes of oligomeric ethylene glycol linkers were initially evaluated
with the aim of optimizing the distance between two disaccharidic
residues.
The key disaccharide 3, bearing an azide aglycone, was prepared
according to Scheme 1 following recently described optimized
conditions.17 The corresponding linkers were functionalized with
aDe´partement de Chimie et Biochimie, Universite´ du Que´bec a` Montre´al,
Case Postale 8888, Succ. Centre-Ville, Montre´al, Que´bec, Canada
H3C 3P8. E-mail: roy.rene@uqam.ca; Fax: +1 (514)987-4054; Tel: +1
(514)987-3000#2546
bCERMAV, BP53, 38041 Grenoble cedex 9, France. E-mail: Anne.Imberty@
cermav.cnrs.fr; Fax: +33 476 547203; Tel: +33 476 037636
Scheme 1 Synthesis of the key a-L-Fucp-(1→4)-b-D-GlcNAc azide
derivative.
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alkyne end groups to be coupled to glycosyl azide 3 by a copper(I)-
catalyzed 1,3-dipolar cycloaddition (“click chemistry”).18,19
First, disaccharide 3 was coupled to dimeric linker 416 using
the original CuSO4/sodium ascorbate conditions.18 At room tem-
perature (24 h), the yield of dimers 10 was only 48%, presumably
because of the poor solubility of the reactants under the conditions
employed (t-BuOH–H2O). However, the yield could be raised to
hydrogen catalysed by palladium hydroxide on carbon. The final
yields are reported in Table 1.
Interaction with PA-IIL
Dimeric and trimeric ligands were then evaluated for their relative
capacity to inhibit the binding of biotinylated polymeric L-fucose
to immobilized PA-IIL on the surface of microtitre plates, as pre-
viously described for the assays using milk oligosaccharides.14 Two
different strategies were used: the first one involved immobilized
lectin following detection by biotinylated polyacrylamide–fucose
and the second one with polyacrylamide–fucose immobilized in
the wells and detection of binding by biotinylated PA-IIL. Since
at the present time it is not clear if the lectin acts as a mobile
entity in the extracellar medium or as one fixed on the bacterial
cell surface, these two methodologies allowed both situations to be
simulated.
◦
83% when the reaction was run at 55 C for 40 minutes. Hence,
for the synthesis of the remaining dimers and trimers, the more
organic-like conditions using soluble copper(I) salt were chosen
(CuI, THF, DIPEA) (Scheme 2).19
All dimeric glycoclusters displayed competition power two or
three times better than for the entire Lewis a trisaccharide. The
differences due to both coating methods used were generally
negligible. For the linear molecules, the IC50 did not seem to depend
on the length of the linkers, but rather on its geometry in space,
since 16, having a more rigid methyl 3,5-dihydroxybenzoate core,
was the most potent inhibitor in the ELLA tests, with an IC50
value of 0.24 lM. The flexibility of the clusters also seemed to
play an important role, since the trimeric construct 21, with rather
rigid branches, did not perform significantly better than the Lewis
a trisaccharide in the ELLA assays.
It was previously demonstrated that the whole Lewis a
trisaccharide is not necessary to obtain high affinity and that
several a-L-Fucp-(1→4)-b-D-GlcNAc derivatives displayed similar
IC50 values.17 A series of triazole derivatives have been recently
synthesized and assayed, and we could check that the presence of
the triazole ring does not hamper the interaction with the bacterial
Scheme 2 Synthesis of oligomeric ligands. Reagents and conditions:
(a) for coupling conditions and linkers, see Table 1; (b) MeONa, MeOH;
(c) H2, Pd(OH)2/C, MeOH
An excess of disaccharide 3 and one equivalent of the ethylene
glycol linkers 5,20 6,20 7,21 or 821 were dissolved in tetrahydrofuran
and treated at room temperature with copper iodide (0.6 eq.) and
DIPEA (2.0 eq.). Dimers 11–14, as single 1,4-regioisomers, were
obtained in very good yields (Table 1). The coupling reaction
between 3.1 equivalents of 3 and the trifunctionalized linker 922
in THF, catalysed by CuI and DIPEA, gave trimer 15 in excellent
yield (85%).
The deprotection steps of the carbohydrate moieties occurred
following a sequence of debenzoylation using a catalytic amount
of sodium methanolate in methanol and debenzylation with
Table 1 Dimer and trimer synthesis
Azide
Multivalent linker
Coupling conditions
Product
Yield (%)
83
Deprotected compounda
Yield (%)b
96
3
4
CuSO4, ascorbic acid, tBuOH–H2O
10
16
(1 : 1), 55 ◦C, 40 min
3
3
5
6
CuI, DIPEA, THF, rt, overnight
CuI, DIPEA, THF, rt, overnight
11
12
Quant.
Quant.
17
18
95
55
3
3
3
7
8
9
CuI, DIPEA, THF, rt, overnight
CuI, DIPEA, THF, rt, overnight
CuI, DIPEA, THF, rt, overnight
13
14
15
81
19
20
21
79
56
65
Quant.
85
a For structural formulae, see Scheme 3. b Yields are for two steps: debenzoylation and debenzylation.
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Scheme 3 Glycoclusters tested for the interaction with PA-IIL.
lectin.17 The inhibition potency of the monovalent compound 22
is displayed in Fig. 1 for comparison.
geometry to cross-link the PA-IIL protein tetramer, resulting in
an insoluble three-dimensional network. The microcalorimetry
assays were therefore performed at low concentration of protein,
and only limited precipitation was observed. Fig. 2 displays
a typical ITC curve obtained when titrating PA-IIL with a
dimeric ligand, with a very sharp decrease in the amplitude
of the exothermic peaks, correlated with the high affinity of
binding.
ITC experiments have been performed on selected compounds,
i.e. trimeric ligand 21, dimeric ligand 16 and dimeric compound
20 (which has the longest linker in the series 17–20). As displayed
in Table 2, the two divalent compounds 16 and 20 displayed
stoichiometries of 0.6 to 0.7, indicating that the disaccharides at
both extremities bound efficiently in solution. The longest linear
dimer 20 had the highest affinity for PA-IIL, with a dissociation
constant of 90 nM.
Fig. 1 Inhibition potency (relative to Lewis a trisaccharide with IC50 of
0.65 lM) of several compounds towards PA-IIL–fucose interaction. (Data
for compound 22 are taken from ref. 17).
Surprisingly, the order of affinities obtained in solution were
almost opposite to those measured with PA-IIL attached to a
plastic surface. The flexible dimer 20 behaved similarly in both
situations, with affinities twice as high as that for Lea. The
inhibitory activity, when divided by the number of ligands on 20,
was therefore identical to the one obtained for Lea. In contrast,
16, which is a powerful inhibitor in the surface assay, had half
Binding affinity with PA-IIL
In all binding assays, the addition of dimeric or trimeric gly-
coclusters to PA-IIL resulted in precipitation of the protein.
Consequently, all of the tested multivalent ligands have the proper
1
2
the inhibitory power in solution, although the n value close to
indicated its ability to cross-link PA-IIL. Trimer 21 behaved better
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Table 2 Microcalorimetry data for the interaction of PA-IIL with Lewis a and multimeric compounds
a
Ka × 10−4/M−1
Kd/nM
n
−DG/kJ mol−1
−DH/kJ mol−1
−TDS/kJ mol−1
b
Lea
16
470
576
1104
968
210
170
90
100
310
1.08
0.62
0.66
1.11
0.98
38.1
38.6
40.2
39.9
37.1
35.0
73.9
69.5
37.2
43.4
−3.1
35.3
29.3
−2.7
6.3
20
21
22c
320
a Experimental data are averaged from three independent experiments and standard deviations are lower than 10%. b Data from ref. 14. c Data from
ref. 17.
Molecular modeling
In order to evaluate the potential cross-linking abilities of the
various glycoclusters to form insoluble cross-linked lattices be-
tween different lectins, modeling experiments were conducted. The
molecular modeling study was performed in two steps. All ligands
were built using the disaccharide conformation observed in the
crystal structure of PA-IIL complexed with a aFuc14GlcNAc
derivative,17 that corresponds closely to the main low energy
conformation in solution.23
All linkers were generated in their most extended conformation,
˚
yielding fucose–fucose distances of 26 A for 16, and distances
˚
˚
increasing from 30 A to 41 A for compounds 17–20. For the
trimeric compound 21, the three arms could be arranged on the
same side of the central ring, yielding a bowl-shaped molecule with
˚
distances of 22 A between terminal fucose residues, or they could
be arranged with one arm pointing to the other sides, resulting in
˚
a longer distance between two of the fucose residues (30 A).
The complexes with PA-IIL were built by fitting the disaccharide
at each of the extremities in the position that it occupies in
coordination with two calcium ions, in one binding site of a PA-
IIL tetramer. The models could be built with no steric conflict
(Fig. 3), except for trimer 21 when the three arms were oriented
on the same face of the ring. The model displayed in Fig. 3C
corresponds therefore to the other conformation of 21 with two
fucose residues far apart.
Fig. 2 ITC analysis of the interaction of PA-IIL (18 lM) with compound
20 (0.21 mM). Upper panel: data obtained from 18 injections (10 lL
each) of 20 in the PA-IIL-containing cell. Lower panel: plot of the total
heat released as a function of total ligand concentration. The solid line
represents the best fit obtained with a one-site model.
Discussion and conclusion
Dimeric and trimeric linkers were coupled to disaccharide 3 and
then deprotected in good yields. This resulted in the synthesis
of five dimeric clusters and one trimeric cluster bearing the
aFuc14GlcNAc epitopes with various geometries. There have been
a limited number of fucosylated dendrimer syntheses reported
in the literature recently,24–26 but the present work describes
the first synthesis of glycoclusters bearing the aFuc14GlcNAc
disaccharide.
Molecular modeling was performed in order to rationalize the
experimental binding data obtained in the presence of the bacterial
lectin PA-IIL. Indeed, the series 17–20 allowed for easy and inde-
pendent binding of two tetramers. Even the shorter linker resulted
in solution, with a high affinity, but its stoichiometry was close to
1, indicating that, when the ligand was not strongly concentrated,
it had a tendency to bind to only one PA-IIL tetramer.
Analysis of the thermodynamical contribution (Table 2) demon-
strated that both dimeric molecules paid a high entropic price for
binding, compensated for by the very strong enthalpy of binding.
Interestingly, trimer 21, which also displayed high affinity, had a
very different behavior in solution. Once one disaccharide extrem-
ity was bound, the remaining two were not readily accessible to
other proteins.
˚
The rigidity of 21 resulted in a slightly favorable entropy of
binding. Nevertheless, precipitation of the protein solution was
observed when the ligand concentration reached a two-fold excess,
indicating that it was also capable of bridging the binding sites of
several proteins.
in the terminal fucosides being 30 A apart (Fig. 3B), explaining the
observed stoichiometry and cross-linking properties. The enthalpy
is doubled when compared to monomeric Lewis a, since both
disaccharides can bind efficiently. As described above, the entropy
term does not follow in direct proportion to the valency.27 In the
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17–20 displayed an efficient cluster effect while keeping the
natural high affinity of PA-IIL for the aFuc14GlcNAc ligand.
The dissociation constant of 90 nM obtained for 20 made it the
highest affinity ligand ever reported for this lectin. Since PA-IIL
is associated to the outer membrane of Pseudomonas aeruginosa,
we expect that high affinity cross-linking compounds will either
precipitate the lectins away from the bacteria, or agglutinate
bacteria themselves. The effect on biofilm formation and infection
in animal models is currently under investigation.
Experimental
General methods
When needed, reactions were run under an atmosphere of dry
nitrogen using oven-dried glassware and freshly distilled and
dried solvents. THF was distilled from sodium benzophenone
ketyl. All reagents were purchased from commercial suppliers
and used without further purification. Analytical thin-layer chro-
matography (TLC) was performed using silica gel 60F254 precoated
plates (0.2 mm thick) with a fluorescent indicator from Merck
(Germany). Detection was done with molybdate solution or 5%
H2SO4 in EtOH. Flash chromatography was performed using silica
˚
gel 60 A (40–63 lm) from Silicycle Chemical division, Quebec.
Chromatographic eluents are given as volume-to-volume ratios. 1H
and 13C NMR spectra were recorded on a Varian Gemini300 NMR
spectrometer at 300 and 75.5 MHz respectively. Routine spectra
were referenced to TMS or to the residual proton or carbon signals
of the solvent. Chemical shifts are reported in ppm, and coupling
constants are reported in Hz. Multiplicities are abbreviated as
follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m), and broadened (b). Some assignments were supported by
2D homonuclear chemical-shift correlation spectroscopy (COSY).
Melting points are uncorrected. Optical rotations were measured
at room temperature in quartz cells using a Perkin–Elmer JASCO
P-1010 instrument in CHCl3 or in the solvent indicated. ESI-
MS analyses were carried out on a MICROMASS Quattro LC
instrument.
Fig. 3 Models of compounds (a) 16, (b) 17 and (c) 21 interacting with
two tetramers of PA-IIL. Polypeptide chains are represented by ribbons,
calcium ions by purple spheres and synthetic ligands by sticks. Hydrogen
atoms are not displayed for sake of clarity.
present case, a large entropic cost arises when two Lewis a moieties
are joined by a linker.
The more rigid dimeric 16, having a shorter inter-fucosidic
˚
distance of 26 A, had the same overall behavior to that of the
slightly more elongated dimer 17 possessing an inter-fucosidic
distance of 30 A. However, the entropy cost for binding is higher,
General procedure for debenzoylation
˚
resulting in a lower affinity constant in solution. In the present
state, it is difficult to associate this phenomenon to flexibility
difference or to an effect on solvent. This compound had the
higher inhibitory power in ELLA assays, indicating that it may be
more efficient on more concentrated proteins.
Trimeric glycocluster 21 could not cross-link PA-IIL in dilute
solution. Nevertheless, this more rigid compound did not have any
entropic cost upon binding, and the resulting affinity determined
by ITC was almost as strong as that for the longest linear dimer 20.
The weak inhibitory potency observed in the ELLA test confirmed
that the cross-linking was not efficient.
The multivalent glycoclusters studied here have not shown the
ability to simultaneously bind to two different sites of one PA-
IIL tetramer, and therefore no huge increase in affinity could be
observed. Different strategies may be used in the future, such as
chemically modifying the high affinity disaccharide or producing
highly multivalent clusters. Nevertheless, the series of linear dimers
The protected compound was dissolved in methanol at room
temperature (or in a methanol–THF mixture, to allow complete
solubilisation of the product). A catalytic amount of sodium
methoxide (0.1 eq.) was added to this solution. The mixture was
stirred at room temperature until completion of the reaction and
neutralized with H+ resin (amberlyst IR120). After filtration and
concentration, the product was purified by column chromatogra-
phy on silica gel.
General procedure for debenzylation
Benzyl ether was dissolved in methanol (0.02–0.05 M) and
hydrogenolyzed on 20 wt% palladium hydroxide on carbon at
room temperature. After completion of the reaction, filtration
through Celite, evaporation and, if necessary, chromatography or
filtration through a microfilter, gave the desired product.
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Synthesis of dimeric and trimeric compounds 16–21
44H, H-Ar, 2 × H-c), 7.90 (s, 2H, H-triazole), 7.98–8.06 (m, 8H,
◦
H-Ar) ppm. 13C NMR (75 MHz, CDCl3, 25 C): d = 15.9, 22.6,
Tri-O-benzyl-a-L-fucopyranosyl-(1→4)-2-acetamido-3,6-di-O-
benzoyl-2-deoxy-b-D-glucopyranosyl azide 3. Thioglycoside ac-
ceptor 228 (500 mg, 1.1 mmol) and donor 129 (695.0 mg, 1.3 mmol,
1.2 eq.) were dissolved in dry chloroform (16.5 mL, 0.1 M) and
29.7, 52.2, 53.8, 61.9, 62.5, 67.8, 72.3, 74.4, 75.0, 75.8, 77.3, 79.3,
86.2, 100.9, 106.0, 106.7, 108.8, 127.4, 127.5, 127.8, 128.1, 128.2,
128.4, 128.5, 128.6, 128.7, 128.9, 129.6, 129.7, 129.9, 132.0, 133.2,
133.8, 137.8, 138.3, 138.5, 159.1, 165.8, 166.5, 167.3, 170.5 ppm.
ESI-MS: m/z = 2008.8 [M + Na]+.
˚
stirred for 30 minutes in the presence of 4 A molecular sieves
at room temperature under a nitrogen atmosphere. The solution
was then cooled to −15 ◦C before addition of N-iodosuccinimide
(322 mg, 1.4 mmol, 1.3 eq.) and triflic acid (78 lL, 0.88 mmol,
0.8 eq.). After 1 h at −15 ◦C, the reaction mixture was neutralized
with a few drops of triethylamine, filtered through Celite, and
extracted with dichloromethane. The mixture was washed with
10% sodium thiosulfate solution and water. The aqueous layer
was extracted twice with dichloromethane. The combined organic
phases were dried over sodium sulfate and concentrated. The crude
product was purified by column chromatography on silica gel
(hexane–ethyl acetate 1 : 1) to give the disaccharide 3 (900.2 mg,
94%) as a white solid. Precipitation of the product in diethyl
Protected dimer 11. To a solution of the disaccharide 3
(100.0 mg, 0.11 mmol, 2.0 eq.) and the bis-propargylated linker
5 (10.3 mg, 0.056 mmol, 1.0 eq.) in tetrahydrofuran (1.15 mL),
were added copper iodide (6.6 mg, 0.035 mmol, 0.6 eq.) and
diisopropylethylamine (20 lL, 0.11 mmol, 2.0 eq.). The reaction
was stirred overnight at room temperature and then concentrated.
The crude product was purified by column chromatography on
silica gel (CH2Cl2–MeOH 30 : 1) to give the dimer 11 (120.7 mg,
quant.) as a slightly yellow oil. [a]2D1 = −37.5 (c 0.65, chloroform).
◦
3
1H NMR (300 MHz, CDCl3, 25 C): d = 0.66 (d, 6H, J6b–5b
=
6.4 Hz, 6 × H-6b), 1.55 (s, 6H, 2 × CH3CO), 3.52–3.61 (m, 10H,
2 × H-4b, 8 × CH2O), 3.80 (q, 2H, 2 × H-5b), 3.92 (dd, 2H,
◦
ether gave a white solid. m.p.: 164–166 C. [a]2D2 = −68.3 (c 1.0,
chloroform). H NMR (300 MHz, CDCl3, 25 ◦C): d = 0.64 (d,
1
3J3b–4b = 2.5 Hz, 3J3b–2b = 10.3 Hz, 2 × H-3b), 4.01 (dd, 2H, 3J2b–1b
=
3
3H, J6b–5b = 6.6 Hz, H-6b), 1.78 (s, 3H, CH3CO), 3.48 (b, 1H,
3.3 Hz, 2 × H-2b), 4.18 (m, 2H, 2 × H-5a), 4.28 (m, 2H, 2 × H-4a),
4.54 (d, 2H, 2J = 11.4 Hz, 2 × CH2Ph), 4.56–4.78 (m, 12H, 2 ×
H-6a, 6 × CH2Ph, 4 × O-CH2-triazole), 4.83 (m, 2H, 2 × H-2a),
4.83 (d, 2H, 2J = 11.8 Hz, 2 × CH2Ph), 4.87 (d, 2H, 2J = 11.3 Hz,
H-4b), 3.71 (m, 1H, H-5b), 3.85 (dd, 1H, 3J3b–4b = 2.5 Hz, 3J3b–2b
=
10.2 Hz, H-3b), 3.90 (m, 1H, H-5a), 3.95 (dd, 1H, 3J2b–1b = 3.3 Hz,
3
H-2b), 4.01 (t, 1H, 3J4a–3a = J4a–5a = 9.1 Hz, H-4a), 4.17 (m, 1H,
H-2a), 4.51 (d, 1H, 2J = 11.5 Hz, CH2Ph), 4.63–4.74 (m, 4H, 2 ×
CH2Ph, H-1a, H-6a), 4.78 (d, 1H, 2J = 11.5 Hz, CH2Ph), 4.81 (d,
3
2 × CH2Ph), 4.97 (d, 2H, J1b–2b = 3.4 Hz, 2 × H-1b), 5.06 (bd,
2H, 3J6a,6a = 11.1 Hz, 2 × H-6a), 5.80 (m, 2H, 2 × H-3a), 6.14 (d,
ꢀ
2
2
1H, J = 12.1 Hz, CH2Ph), 4.84 (d, 1H, J = 11.5 Hz, CH2Ph),
2H, 3J1a,2a = 10.0 Hz, 2 × H-1a), 6.74 (d, 2H, 3JNH,2a = 9.3 Hz, 2 ×
NHAc), 7.10–7.65 (m, 42H, H-Ar), 7.88 (s, 2H, 2 × H-triazole),
8.02–8.09 (m, 8H, H-Ar) ppm. 13C NMR (75 MHz, CDCl3, 25 ◦C):
d = 16.0, 22.6, 53.8, 62.8, 64.2, 67.8, 69.5, 70.4, 72.6, 74.3, 74.8,
74.9, 75.4, 76.0, 77.7, 79.3, 86.0, 100.7, 121.8, 127.5, 127.7, 128.1,
128.2, 128.3, 128.4, 128.5, 128.6, 129.3, 129.8, 129.8, 133.1, 133.6,
138.0, 138.5, 138.6, 145.3, 165.9, 167.0, 170.4 ppm.
4.87 (d, 1H, H-1b), 4.96 (dd, 1H, 3J6a–5a = 2.2 Hz, 2J = 12.4 Hz,
H-6a), 5.41 (dd, 1H, 3J3a–2a = 8.5 Hz, H-3a), 5.74 (d, 1H, 3JNH–2a
=
9.3 Hz, NHAc), 7.18–7.98 (m, 21H, H-Ar), 7.97 (2 × bd, 2 × 2H,
3J = 8.5 Hz, H-Ar) ppm. 13C NMR (75 MHz, CDCl3, 25 ◦C): d =
16.0, 23.1, 53.8, 62.8, 67.7, 72.7, 74.0, 74.3, 74.8, 75.2, 75.5, 75.5,
76.6, 79.2, 88.5, 100.00, 127.4, 127.5, 127.6, 127.8, 128.1, 128.3,
128.4, 128.4, 128.5, 129.0, 129.8, 129.9, 133.1, 133.6, 138.0, 138.3,
138.5, 166.0, 167.0, 170.3 ppm. ESI-MS: m/z = 893.4 [M + Na]+.
Protected dimer 12. To a solution of the disaccharide 3
(100.0 mg, 0.11 mmol, 2.1 eq.) and the bis-propargylated linker
6 (12.4 mg, 0.055 mmol, 1.0 eq.) in tetrahydrofuran (1.15 mL)
were added copper iodide (6.2 mg, 0.032 mmol, 0.6 eq.) and
diisopropylethylamine (19 lL, 0.11 mmol, 2.0 eq.). The reaction
was stirred overnight at room temperature and then concentrated.
The crude product was purified by column chromatography on
silica gel (CH2Cl2–MeOH 25 : 1) to give the protected dimer 12
(108.6 mg, quant.) as an amorphous solid. [a]2D1 = −41.6 (c 1.0,
Protected dimer 10. To a solution of the disaccharide 3
(150.0 mg, 0.17 mmol, 2.1 eq.) and bis-propargylated linker 416
(20.0 mg, 0.082 mmol, 1.0 eq.) in a tert-butanol–water mixture (1 :
1, 0.04 M) were added copper sulfate (17.2 mg, 0.069 mmol, 0.8 eq.)
and ascorbic acid (27.2 mg, 0.14 mmol, 1.6 eq.). The solution
was white and milky. After 40 minutes at 55 ◦C, the reaction
mixture, which now contained an orange precipitate, was extracted
with ethyl acetate. The organic layer was washed successively with
saturated sodium bicarbonate solution and with brine, dried over
sodium sulfate and concentrated. The crude product was purified
by column chromatography on silicagel (CH2Cl2–MeOH 30 : 1) to
give the desired triazole 10 (134.2 mg, 83%) as a white, amorphous
chloroform). H NMR (300 MHz, CDCl3, 25 ◦C): d = 0.65 (d,
1
6H, 3J6b–5b = 6.4 Hz, 6 × H-6b), 1.58 (s, 6H, 2 × CH3CO), 3.48–
3.56 (m, 14H, 2 × H-4b, 12 × CH2O), 3.76 (m, 2H, 2 × H-5b),
3.89 (dd, 2H, 3J3b–4b = 2.3 Hz, 3J3b–2b = 10.4 Hz, 2 × H-3b), 3.97
3
(dd, 2H, J2b–1b = 3.3 Hz, 2 × H-2b), 4.07 (m, 2H, 2 × H-5a),
1
solid. [a]2D2 = −42.3 (c 1.0, chloroform). H NMR (300 MHz,
4.14 (m, 2H, 2 × H-4a), 4.52 (d, 2H, 2J = 11.5 Hz, 2 × CH2Ph),
4.57–4.89 (m, 18H, 2 × H-6a, 10 × CH2Ph, 4 × OCH2-triazole,
◦
3
CDCl3, 25 C): d = 0.60 (d, 6H, J6b–5b = 6.6 Hz, 6 × H-6b),
1.52 (s, 6H, 2 × CH3CO), 3.57 (b, 2H, 2 × H-4b), 3.77 (m, 2H,
2 × H-5b), 3.81–3.83 (m, 5H, CH3O, 2 × H-3b), 3.97 (dd, 2H,
3J2b–1b = 3.0 Hz, 3J2b–3b = 10.4 Hz, 2 × H-2b), 4.16–4.28 (m, 4H,
2 × H-2a), 4.91 (m, 2H, 3J1b–2b = 3.3 Hz, 2 × H-1b), 4.97 (bd, 2H,
3
ꢀ
J6a,6a = 11.1 Hz, 2 × H-6a), 5.72 (m, 2H, 2 × H-3a), 6.10 (d, 2H,
3J1a,2a = 10.0 Hz, 2 × H-1a), 6.45 (d, 2H, JNH,2a = 9.3 Hz, 2 ×
3
2
2 × H-4a, 2 × H-5a), 4.48 (d, 2H, J = 11.3 Hz, CH2Ph), 4.54–
NHAc), 7.17–7.62 (m, 42H, H-Ar), 7.82 (s, 2H, 2 × H-triazole),
7.95–8.09 (m, 8H, H-Ar) ppm. 13C NMR (75 MHz, CDCl3, 25 ◦C):
d = 16.0, 22.6, 53.9, 62.7, 64.2, 67.8, 69.5, 70.4, 70.5, 72.5, 74.3,
74.7, 75.0, 75.2, 76.0, 76.9, 77.6, 79.3, 86.1, 100.8, 121.7, 127.5,
127.7, 128.1, 128.1, 128.3, 128.3, 128.5, 128.6, 129.2, 129.7, 129.9,
4.87 (m, 14H, 2 × H-2a, 2 × H-6a, 10 × CH2Ph), 4.93 (d, 2H,
2 × H-1b), 5.04 (bd, 2H, 2J = 11.8 Hz, 2 × H-6a), 5.10 (b, 4H, ×
H-e), 5.80 (m, 2H, 2 × H-3a), 6.07 (bd, 2H, 3J1a–2a = 9.1 Hz, 2 ×
H-1a), 6.39 (b, 2H, 2 × NHAc), 6.68 (b, 1H, H-d), 7.13–7.61 (m,
2958 | Org. Biomol. Chem., 2007, 5, 2953–2961
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133.1, 133.6, 138.0, 138.5, 138.6, 145.4, 165.8, 167.1, 170.4 ppm.
iodide (7.7 mg, 0.051 mmol) and diisopropylethylamine (46 lL,
0.263 mmol) were added. The mixture was stirred at room
temperature overnight. The solvent was evaporated under reduced
pressure, and the crude was purified by flash chromatography
(CH2Cl2–MeOH 20 : 1) to give the trimer 15 as a white solid
ESI-MS: m/z = 1991.4 [M + Na]+.
Protected dimer 13. To a solution of the disaccharide 3
(100.0 mg, 0.11 mmol, 2.1 eq.) and the bis-propargylated linker
7 (14.8 mg, 0.055 mmol, 1.0 eq.) in tetrahydrofuran (1.15 mL),
were added copper iodide (6.2 mg, 0.032 mmol, 0.6 eq.) and
diisopropylethylamine (19 lL, 0.11 mmol, 2.0 eq.). The reaction
was stirred overnight at room temperature and then concentrated.
The crude product was purified by column chromatography on
silica gel (CH2Cl2–MeOH 20 : 1) to give the protected dimer
13 (89.6 mg, 81%) as an amorphous solid. [a]2D1 = −41.1 (c 1.0,
◦
(110.3 mg, 85%). m.p.: 170–173 C. [a]2D1 = −37.8 (c 0.8, chlo-
◦
1
roform). H NMR (300 MHz, CDCl3, 25 C): d = 0.61 (d, 9H,
J6 –5 = 6.3 Hz, 9×H-6ꢀ), 1.25 (s, 9H, 3 × CH3CO), 3.49 (b, 3H,
3 × H-4ꢀ ), 3.76 (m, 3H, 3 × H-5ꢀ ), 3.93 (b, 6H, 3 × H-2ꢀ , 3 × H-3ꢀ ),
4.12 (b, 6H, 3 × H-2, 3 × H-5), 4.48–5.06 (m, 42H, 3 × H-1, 3 ×
H-3, 3 × H-4, 6 × H-6, 3 × H-1ꢀ , 18 × CH2Ph, 6 × CH2NH)
5.85, 6.20 (b, 6H, 3 × NHAc, 3 × CH2NH), 7.11–7.92 (m, 81H,
78 × H-Ar, 3 × H-triazole) ppm. 13C NMR (75 MHz, CDCl3,
25 ◦C): d = 16.0, 22.5, 35.4, 67.7, 72.6, 74.2, 74.9, 75.5, 77.2, 79.3,
100.6, 127.4, 127.5, 127.6, 128.1, 128.2, 128.2, 128.3, 128.4, 129.4,
129.7, 133.2, 138.0, 138.4, 138.6, 166.1, 166.3, 171.2 ppm. ESI-MS:
m/z = 1478.1 [M + H + Na]2+.
3
ꢀ
ꢀ
chloroform). H NMR (300 MHz, CDCl3, 25 ◦C): d = 0.64 (d,
1
6H, 3J6b–5b = 6.3 Hz, 6 × H-6b), 1.59 (s, 6H, 2 × CH3CO), 3.53–
3.69 (m, 18H, 2 × H-4b, 16 × CH2O), 3.82 (m, 2H, 2 × H-5b),
3.93 (bd, 2H, 3J3b–2b = 10.5 Hz, 2 × H-3b), 4.03 (dd, 2H, 3J2b–1b
=
3.3 Hz, 2 × H-2b), 4.25–4.38 (m, 4H, 2 × H-4a, 2 × H-5a), 4.54
(d, 2H, 2J = 11.4 Hz, 2 × CH2Ph), 4.56–4.92 (m, 18H, 2 × H-6a,
10 × CH2Ph, 4 × OCH2-triazole, 2 × H-2a), 5.00 (d, 2H, 3J1b–2b
=
Unprotected dimer 16. The debenzoylation step of dimer 10
with sodium methoxide (133.8 mg, 0.067 mmol) gave after column
chromatography on silica gel (CH2Cl2–MeOH 15 : 1) 16 (83.4 mg,
79%). The debenzylation step, using 78.7 mg (0.050 mmol) of this
product, by catalytic hydrogenation, gave the desired product as
a white, amorphous solid (45.6 mg, 96%). [a]2D2 = −115.9 (c 1.0,
methanol). 1H NMR (300 MHz, D2O–acetone 600 : 1, 25 ◦C): d =
1.17 (d, 6H, 3J6b–5b = 6.1 Hz, 6 × H-6b), 1.69 (s, 6H, 2 × CH3CO),
3.75–3.05 (m, 19H, 2 × H-3a, 2 × H-4a, 2 × H-5a, 4 × H-6a, 2 ×
H-2b, 2 × H-3b, 2 × H-4b, CH3O), 4.27 (m, 2H, 2 × H-2a), 4.38
(m, 2H, 2 × H-5b), 5.02 (d, 2H, 3J1b–2b = 3.2 Hz, 2 × H-1b), 5.29
(b, 4H, 4 × H-e), 5.85 (d, 2H, 3J1a–2a = 9.8 Hz, 2 × H-1a), 6.90 (b,
3
ꢀ
3.3 Hz, 2 × H-1b), 5.11 (bd, 2H, J6a,6a = 12.4 Hz, 2 × H-6a),
5.90 (m, 2H, 2 × H-3a), 6.17 (d, 2H, 3J1a,2a = 9.9 Hz, 2 × H-1a),
6.77 (d, 2H, 3JNH,2a = 9.9 Hz, 2 × NHAc), 7.12–6.68 (m, 42H, H-
Ar), 7.89 (s, 2H, 2 × H-triazole), 8.03–8.15 (m, 8H, H-Ar) ppm.
13C NMR (75 MHz, CDCl3, 25 ◦C): d = 16.0, 22.6, 53.9, 62.6,
64.2, 67.8, 69.5, 70.4, 70.5, 72.4, 74.3, 74.7, 75.0, 76.0, 76.9, 77.6,
79.4, 86.1, 100.9, 121.5, 127.4, 127.5, 127.7, 128.1, 128.1, 128.3,
128.3, 128.4, 128.4, 128.5, 128.6, 129.1, 129.7, 129.8, 129.8, 129.9,
133.1, 133.6, 137.9, 138.4, 138.5, 145.4, 165.8, 167.2, 170.4 ppm.
ESI-MS: m/z = 2035.5 [M + Na]+.
Protected dimer 14. To a solution of the disaccharide 3
(100.0 mg, 0.11 mmol, 2.1 eq.) and the bis-propargylated linker
8 (17.1 mg, 0.054 mmol, 1.0 eq.) in tetrahydrofuran (1.15 mL)
were added copper iodide (6.2 mg, 0.032 mmol, 0.6 eq.) and
diisopropylethylamine (19 lL, 0.11 mmol, 2.0 eq.). The reaction
was stirred overnight at room temperature and then concentrated.
The crude product was purified by column chromatography on
silica gel (CH2Cl2–MeOH 20 : 1) to give the protected dimer 14
(115.6 mg, quant.) as an amorphous solid. [a]2D1 = −41.9 (c 1.0,
1H, H-d), 7.29 (m, 2H, 2 × H-c), 8.29 (s, 2H, H-triazole) ppm. 13
C
NMR (75 MHz, D2O–acetone 600 : 1, 25 ◦C): d = 15.9, 22.1, 53.5,
56.3, 60.3, 61.8, 61.9, 67.7, 68.7, 70.1, 72.6, 73.0, 77.2, 79.0, 86.9,
100.4, 108.8, 110.1, 124.6, 132.5, 143.9, 159.1, 169.0, 174.5 ppm.
ESI-MS: m/z = 1051.1 [M + Na]+.
Unprotected dimer 17. The debenzoylation step of the dimer
11 (66.0 mg, 0.034 mmol) with sodium methoxide gave after
column chromatography on silica gel (CH2Cl2–MeOH 10 : 1)
the disaccharide (50.8 mg, 98%). The debenzylation step, using
48.0 mg (0.032 mmol) of this product, by catalytic hydrogenation,
gave the desired product 17 as a white, amorphous solid (29.9 mg,
97%). [a]2D2 = −107.1 (c 1.0, methanol). 1H NMR (300 MHz, D2O–
acetone 600 : 1, 25 ◦C): d = 1.18 (d, 6H, 3J6b–5b = 6.6 Hz, 6 × H-6b),
1.81 (s, 6H, 2 × CH3CO), 3.67 (b, 8H, 8 × CH2O), 3.77–4.03 (m,
16H, 2 × H-3a, 2 × H-4a, 2 × H-5a, 4 × H-6a, 2 × H-2b, 2 ×
H-3b, 2 × H-4b), 4.28 (m, 2H, 2 × H-2a), 4.40 (m, 2H, 2 × H-5b),
chloroform). H NMR (300 MHz, CDCl3, 25 ◦C): d = 0.54 (d,
1
6H, 3J6b–5b = 6.3 Hz, 6 × H-6b), 1.50 (s, 6H, 2 × CH3CO), 3.47–
3.58 (m, 22H, 2 × H-4b, 20 × CH2O), 3.73 (m, 2H, 2 × H-5b),
3.83 (bd, 2H, 3J3b–2b = 10.4 Hz, 2 × H-3b), 3.93 (dd, 2H, 3J2b–1b
=
3.3 Hz, 2 × H-2b), 4.27–4.38 (m, 4H, 2 × H-4a, 2 × H-5a), 4.53
2
(d, 2H, J = 11.4 Hz, 2 × CH2Ph), 4.56–4.92 (m, 18H, 2 × H-
6a, 10 × CH2Ph, 4 × OCH2-triazole, 2 × H-2a), 5.00 (d, 2H,
3
3
ꢀ
J1b–2b = 3.4 Hz, 2 × H-1b), 5.12 (bd, 2H, J6a,6a = 12.2 Hz, 2 ×
H-6a), 5.91 (m, 2H, 2 × H-3a), 6.17 (d, 2H, 3J1a,2a = 10.0 Hz, 2 ×
4.69 (m, 4H, 4 × OCH2-triazole), 5.02 (d, 2H, J1b–2b = 3.3 Hz,
3
3
3
H-1a), 6.82 (d, 2H, JNH,2a = 9.6 Hz, 2 × NHAc), 7.16–6.69 (m,
2 × H-1b), 5.86 (d, 2H, J1a–2a = 9.7 Hz, 2 × H-1a), 8.26 (s, 2H,
42H, H-Ar), 7.89 (s, 2H, 2 × H-triazole), 8.04–8.13 (m, 8H, H-Ar)
ppm. 13C NMR (75 MHz, CDCl3, 25 ◦C): d = 15.9, 22.6, 53.7,
62.5, 64.1, 67.7, 69.4, 70.3, 70.4, 70.4, 72.2, 74.3, 74.6, 74.8, 74.9,
75.8, 76.8, 77.3, 79.3, 86.1, 100.8, 121.5, 127.4, 127.5, 127.7, 128.1,
128.2, 128.3, 128.3, 128.4, 128.5, 128.5, 128.6, 129.0, 129.7, 129.9,
133.2, 133.7, 137.8, 138.3, 138.4, 145.3, 165.8, 167.2, 170.5 ppm.
ESI-MS: m/z = 2079.4 [M + Na]+.
2 × H-triazole) ppm. 13C NMR (75 MHz, D2O–acetone 600 :
◦
1, 25 C): d = 15.9, 22.3, 56.5, 60.3, 63.5, 67.7, 68.7, 69.4, 70.1,
70.1, 72.6, 72.9, 77.2, 79.0, 86.9, 100.4, 124.4, 144.7, 174.7 ppm.
ESI-MS: m/z = 989.4 [M + NH4]+.
Unprotected dimer 18. The debenzoylation step of the dimer
12 (95.7 mg, 0.049 m mol) with sodium methoxide gave after
column chromatography on silica gel (CH2Cl2–MeOH 10 : 1)
the disaccharide (35.6 mg, 55%). The debenzylation step, using
32.0 mg (0.021 mmol) of this product, by catalytic hydrogenation,
gave the desired product 18 as a white, amorphous solid (21.1 mg,
Protected trimer 15. A solution of the linker 9 (14.2 mg,
0.044 mmol) and the disaccharide 3 (119.8 mg, 0.138 mmol)
in tetrahydrofuran (1.8 mL, 0.024 M) was prepared. Copper
This journal is
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1
quant.). [a]2D2 = −80.5 (c 1.0, methanol). H NMR (300 MHz,
the product by column chromatography (CH3CN–H2O 90 : 10 to
◦
3
70 : 30) afforded the fully deprotected trimer 21 as a white solid
D2O–acetone 600 : 1, 25 C): d = 1.18 (d, 6H, J6b–5b = 6.5 Hz,
6 × H-6b), 1.82 (s, 6H, 2 × CH3CO), 3.68 (b, 12H, 12 × CH2O),
3.75–4.03 (m, 16H, 2 × H-3a, 2 × H-4a, 2 × H-5a, 4 × H-6a, 2 ×
H-2b, 2 × H-3b, 2 × H-4b), 4.28 (m, 2H, 2 × H-2a), 4.39 (m, 2H,
(18.7 mg, 65%). [a]2D1 = −62.0 (c 0.6, DMSO). 1H NMR (300 MHz,
◦
D2O–acetone 600 : 1, 25 C): d = 1.15 (d, 9H, 3J6 –5 = 6.6 Hz, 9 ×
H-6ꢀ), 1.75 (s, 9H, 3 × CH3CO), 3.78–3.95 (m, 24H, 3 × H-3, 3 ×
H-4, 3 × H-5, 6 × H-6, 3 × H-2ꢀ , 3 × H-3ꢀ , 3 × H-4ꢀ ), 4.25 (app t,
3H, 3J2–1 = 9.6 Hz, 3 × H-2), 4.37 (m, 3H, 3 × H-5ꢀ ), 4.68 (s, 6H,
ꢀ
ꢀ
2 × H-5b), 4.69 (m, 4H, 4 × OCH2-triazole), 5.01 (d, 2H, 3J1b–2b
=
3.1 Hz, 2 × H-1b), 5.86 (d, 2H, 3J1a–2a = 9.7 Hz, 2 × H-1a), 8.25
(s, 2H, 2 × H-triazole) ppm. 13C NMR (75 MHz, D2O–acetone
3 × CH2N), 4.99 (d, 3H, 3J1 –2 = 3.3 Hz, 3 × H-1ꢀ ), 5.81 (d, 3H,
3J1–2 = 9.6 Hz, 3 × H-1), 8.18 (s, 3H, 3 × H-triazole), 8.31 (s, 3H,
H-Ar) ppm. 13C NMR (75 MHz, D2O–acetone 600 : 1, 25 ◦C):
d = 15.9, 22.2, 35.6, 56.4, 60.3, 67.7, 68.7, 70.0, 72.6, 72.9, 77.2,
79.0, 86.9, 100.4, 123.3, 129.9, 135.3, 169.2, 174.6 ppm. ESI-MS:
m/z = 1498.6 [M + H]+.
ꢀ
ꢀ
◦
600 : 1, 25 C): d = 15.9, 22.3, 56.5, 60.3, 63.5, 67.7, 68.7, 69.4,
70.1, 70.1, 70.2, 72.6, 72.9, 77.2, 79.0, 86.9, 100.4, 124.4, 144.7,
174.7 ppm. ESI-MS: m/z = 1033.4 [M + Na]+.
Unprotected dimer 19. The debenzoylation step of the dimer
13 (80.0 mg, 0.040 mmol) with sodium methoxide gave after
column chromatography on silica gel (CH2Cl2–MeOH 20 : 1)
the disaccharide (52.6 mg, 83%). The debenzylation step, using
47.0 mg (0.029 mmol) of this product, by catalytic hydrogenation,
gave the desired product 19 as a white, amorphous solid (29.6 mg,
95%). [a]2D2 = −80.3 (c 1.0, methanol). 1H NMR (300 MHz, D2O–
Preparation of PA-IIL protein
Recombinant PA-IIL was purified from Escherichia coli
BL21(DE3) containing the plasmid pET25pa2l as described
previously.30 Biotinylation of PA-IIL was performed as described
in the literature.31 Briefly, PA-IIL (200 lM) diluted in buffer
containing 0.1 M NaHCO3 and 0.2 M NaCl was mixed with
dimethyl formamide solution containing 4.5 mM biotin for 2 hours
at room temperature, with agitation. After dialysis against 0.15 M
of NaCl solution followed by water, the lectin was lyophilized.
◦
3
acetone 600 : 1, 25 C): d = 1.18 (d, 6H, J6b–5b = 6.5 Hz, 6 ×
H-6b), 1.82 (s, 6H, 2 × CH3CO), 3.65–3.70 (b, 16H, 16 × CH2O),
3.77–4.03 (m, 16H, 2 × H-3a, 2 × H-4a, 2 × H-5a, 4 × H-6a, 2 ×
H-2b, 2 × H-3b, 2 × H-4b), 4.28 (m, 2H, 2 × H-2a), 4.39 (m, 2H,
2 × H-5b), 4.69 (m, 4H, 4 × OCH2-triazole), 5.01 (d, 2H, 3J1b–2b
=
3.5 Hz, 2 × H-1b), 5.86 (d, 2H, 3J1a–2a = 9.8 Hz, 2 × H-1a), 8.26
(s, 2H, 2 × H-triazole) ppm. 13C NMR (75 MHz, D2O–acetone
Interaction studies with PA-IIL lectin
◦
600 : 1, 25 C): d = 15.9, 22.3, 56.5, 60.3, 63.5, 67.7, 68.7, 69.4,
ELLA (enzyme-linked lectin assay) experiments. First test
(plate coated with polyacrylamide–fucose): ELLA experiments
were conducted using 96-well microtitre plates (Nunc Maxisorb)
coated with polymeric a-L-fucose (5 lg mL−1; Lectinity Holding,
Inc., Moscow) diluted in carbonate buffer, pH 9.6 (100 lL) for
1 h at 37 ◦C. After blocking at 37 ◦C for 1 h with 100 lL per
well of 3% (w/v) BSA in PBS, plates were incubated at 37 ◦C
for 1 h with 100 lL of biotinylated PA-IIL at 0.1 lg mL−1 in
the presence of serial dilutions of inhibitors. After washing with
T-PBS (PBS containing 0.05% Tween), 100 lL of streptavidin–
peroxidase conjugate (dilution 1 : 10 000; Boehringer-Mannheim)
was added and left for 1 h at 37 ◦C. The color was developed using
100 lL per well of 0.05 M phosphate/citrate buffer containing
O-phenylenediamine dihydrochloride (0.4 mg mL−1) and urea
hydrogen peroxide (0.4 mg mL−1) (Sigma-Aldrich). The reaction
was stopped by the addition of 50 lL of 30% H2SO4. The
absorbance was read at 490 nm using a microtitre plate reader (Bio-
Rad; model 680). Second test (plate coated with PA-IIL): The same
protocol as above was used, but in this case, plates were coated
with PA-IIL (5 lg mL−1), and a biotinylated polymeric a-L-fucose
(5 lg mL−1; Lectinity Holding, Inc.) was used for competition with
serial dilutions of inhibitors.
70.1, 70.1, 70.2, 72.6, 72.9, 77.2, 79.0, 86.9, 100.4, 124.4, 144.7,
174.7 ppm. ESI-MS: m/z = 1077.4 [M + Na]+.
Unprotected dimer 20. The debenzoylation step of the dimer
14 (78.5 mg, 0.038 mmol) with sodium methoxide gave after
column chromatography on silica gel (CH2Cl2–MeOH 15 : 1)
the disaccharide (39.1 mg, 62%). The debenzylation step, using
32.6 mg (0.029 mmol) of this product, by catalytic hydrogenation,
gave the desired product 20 as a white, amorphous solid (20.6 mg,
95%). [a]2D2 = −96.1 (c 1.0, methanol). 1H NMR (300 MHz, D2O–
◦
3
acetone 600 : 1, 25 C): d = 1.18 (d, 6H, J6b–5b = 6.1 Hz, 6 ×
H-6b), 1.82 (s, 6H, 2 × CH3CO), 3.63–3.75 (b, 20H, 20 × CH2O),
3.77–4.03 (m, 16H, 2 × H-3a, 2 × H-4a, 2 × H-5a, 4 × H-6a, 2 ×
H-2b, 2 × H-3b, 2 × H-4b), 4.28 (m, 2H, 2 × H-2a), 4.38 (m, 2H,
2 × H-5b), 4.70 (m, 4H, 4 × OCH2-triazole), 5.02 (d, 2H, 3J1b–2b
=
3.5 Hz, 2 × H-1b), 5.87 (d, 2H, 3J1a–2a = 9.8 Hz, 2 × H-1a), 8.26
(s, 2H, 2 × H-triazole) ppm. 13C NMR (75 MHz, D2O–acetone
◦
600 : 1, 25 C): d = 15.9, 22.3, 56.5, 60.3, 63.5, 67.7, 68.7, 69.4,
70.1, 70.1, 70.2, 72.6, 72.9, 77.2, 79.0, 86.9, 100.4, 124.4, 144.7,
174.7 ppm. ESI-MS: m/z = 1121.4 [M + Na]+.
Unprotected trimer 21. Compound 15 (109.1 mg, 0.037 mmol)
was dissolved in MeOH–THF (0.01 M, 1 : 1) and the solution was
treated with a catalytic amount of 1 M NaOMe/MeOH. After
being stirred at rt for 2 days, the solution was neutralized
with Amberlite IR-120 (H+) resin, filtered and concentrated.
Methyl benzoate, resulting from the deprotection, was removed
by column chromatography (CH2Cl2–MeOH 9 : 1) to afford
the debenzoylated trimer (61.4 mg, 71%). The resulting trimer
(44.3 mg, 0.019 mmol) was dissolved in MeOH (5 mL) and the
debenzylation was accomplished with an excess of Pd(OH)2/C
(20 wt%). After one night under H2 atmosphere, the solution
was filtered through Celite and concentrated. The purification of
ITC (isothermal titration microcalorimetry) analysis. ITC ex-
periments were performed with a VP-ITC isothermal titration
calorimeter (Microcal). The experiments were carried out at
25 ◦C. Ligands and proteins were dissolved in the same buffer
(0.1 M Tris with 0.03 mM CaCl2) at pH 7.5. The protein
concentration in the microcalorimeter cell (1.4 mL) varied from
17.8 to 20 lM. A total of 30 injections of 13 lL of sugar solution
at concentrations varying from 0.08 to 0.21 mM were added at
intervals of 5 min whilst stirring at 310 rpm. Control experiments
performed by injection of buffer into the protein solution yielded
2960 | Org. Biomol. Chem., 2007, 5, 2953–2961
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insignificant heats of dilution. The experimental data were fitted to
a theoretical titration curve using software supplied by Microcal,
with DH (enthalpy change), Ka (association constant) and n
(number of binding sites per monomer) as adjustable parameters.
DG (free energy change) values and entropy contributions were
determined from the standard equation:
11 A. Imberty, M. Wimmerova, E. P. Mitchell and N. Gilboa-Garber,
Microb. Infect., 2004, 6, 222–229.
12 A. Imberty, M. Wimmerova, C. Sabin, and E. P. Mitchell, in Protein–
Carbohydrate Interactions in Infectious Disease, ed. C. Bewley, The
Royal Society of Chemistry, Cambridge, 2006, pp. 30–48.
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DG = DH − TDS
where T is the absolute temperature. All experiments were
performed with c values 100 < c < 200 (c = KaM, where M is
the initial concentration of the macromolecule).
Molecular modeling. All molecular editing was performed
with the Sybyl software (Tripos, St Louis). Linkers were generated
in their extended conformations, and geometry optimization was
performed with the Tripos force-field. Disaccharide conformation
was taken from the protein data bank,32 using the crystal structure
of the complex between PA-IIL and a derivative of aFuc14GlcNAc
disaccharide17 (PDB code 2JDK). Graphical representations were
drawn using PyMol software (Delano Scientific LCC, San Fran-
cisco).
16 D. T. S. Rijkers, G. W. van Esse, R. Merkx, A. J. Brouwer, H. J. F.
Jacobs, R. J. Pieters and R. M. J. Liskamp, Chem. Commun., 2005, 36,
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Wimmerova, E. P. Mitchell, A. Imberty and R. Roy, ChemMedChem,
DOI: 10.1002/cmdc.200700100.
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Chem., Int. Ed., 2002, 41, 2596–2599.
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20 Z.-J. Yao, H.-P. Wu and Y.-L. Wu, J. Med. Chem., 2000, 43, 2484–2487.
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22 D. Gigue`re, R. Patnam, M.-A. Bellefleur, S. St-Pierre, S. Satob and R.
Roy, Chem. Commun., 2006, 2379–2381.
23 T. Weimar, T. Peters, S. Pe´rez and A. Imberty, THEOCHEM, 1997,
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24 E. Fernandez-Megia, J. Correa, I. Rodriguez-Meizoso and R. Riguera,
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Acknowledgements
The work was supported by CNRS and by Vaincre la Mucovisci-
dose. We are also grateful to CRSNG (Canada) for a Canadian
Research Chair in Therapeutic Chemistry to R. R. We are thankful
to I. Deguise for the preparation of compound 4.
25 M. Kleinert, N. Ro¨ckendorf and T. K. Lindhorst, Eur. J. Org. Chem.,
2004, 3931–3940.
26 (a) E. Kolomiets, E. M. Johansson, O. Renaudet, T. Darbre and J. L.
Reymond, Org. Lett., 2007, 9, 1465–1468; (b) E. M. V. Johansson, E.
Kolomiets, F. Rosenau, K. E. Jaeger, T. Darbre and J. L. Reymond,
New J. Chem., 2007, 31, 1291–1299; (c) I. Deguise, D. Lagnoux
and R. Roy, New J. Chem., 2007, 31, 1321–1331; (d) F. Morvan,
A. Meyer, A. Jochum, C. Sabin, Y. Chevolot, A. Imberty, J. P.
Praly, J. J. Vasseur, E. Souteyrand and S. Vidal, Bioconjugate Chem.,
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